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https://globalizationandhealth.biomedcentral.com/track/pdf/10.1186/s12992-020-00563-6
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Globalization and health scholarship in a time of pandemic: from critical past to uncertain future
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Globalization and health
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© The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License,
which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give
appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if
changes were made. The images or other third party material in this article are included in the article's Creative Commons
licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons
licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain
permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the
data made available in this article, unless otherwise stated in a credit line to the data. Globalization and health scholarship in a
time of pandemic: from critical past to
uncertain future Ronald Labonté economy) was experiencing a rapid transition to high rates
of chronic disease (2008) [4]; and the second on how trade
liberalization, one of globalization’s defining features, was
doing the same in Central America (2009) [5]. The differ-
ential impacts on health of specific food items, in this case
palm oil consumption, suggested that globalization’s
health impacts were not equitably allocated (2011) [6]. Some of globalization’s unequal outcomes were manifest
in health systems themselves: from radiology services
(2010) [7], to biases that privilege research from rich
countries over poorer ones (2017) [8], to this year’s so-far
trending lead article on global surgery (2020) [9]. So many things in our lives became unhinged in a few
short weeks: lock-downs, quarantines, social distancing,
pandemic infection and death, an economic meltdown
to rival 2008, and shortages in everything from medical
supplies to food. Few nations or peoples in the world are
unaffected, and how SARS-CoV2 and its COVID-2019
disease will eventually play out remains a riveting ques-
tion mark for those of us with privileged lives, and an
imminent threat for those without. It is in this context that our journal finds itself cele-
brating its 15th year. Our publisher, BMC, has compiled
a short list of our critical past, highlighting the most ref-
erenced articles from each of our years. The list tells us
a story of how our journal has attempted to track the
trajectory of globalization and its many health pathways,
with embedded lessons for an uncertain future. A recurrent journal theme over the years has also been
development policy and health: initially pointing out how
partnerships between richer (donor) and poorer (recipi-
ent) countries can be mutually beneficial (2012) [10], thus
making the case for stronger international cooperation;
and later lauding the bold, expansive directions of the
Agenda 2030 and the Sustainable Development Goals
(2015) [11]. The following year (2016) the necessary role
of civil society to keep momentum moving captured
readers’ interests [12]. Unsurprisingly, our very first ‘big’ article (2005) did
precisely that: it mapped the many ways in which health
was already being, or could be, impacted by globalization
[1]. This macro-look was matched by a more focused as-
sessment of how unhealthy nutrition transitions and
chronic disease rates (2006) were globalization’s fellow
travellers [2]. Labonté Globalization and Health (2020) 16:31
https://doi.org/10.1186/s12992-020-00563-6 Labonté Globalization and Health (2020) 16:31
https://doi.org/10.1186/s12992-020-00563-6 Open Access Correspondence: rlabonte@uottawa.ca
School of Epidemiology and Public Health, Faculty of Medicine, University of
Ottawa, 600 Peter Morand Crescent, Ottawa, Ontario K1G 5Z3, Canada Globalization and health scholarship in a
time of pandemic: from critical past to
uncertain future A year later (2007), and as prelude to the
work of Globalization Knowledge Network of the World
Health Organization’s Commission on Social Determi-
nants of Health, globalization was defined as a ‘determin-
ant of social determinants’ trickling down in ways
sometimes healthy, but predominantly health-risky [3]. In the wake of the 2008 financial crisis, we were also
reminded of the importance of health investments in
sustaining both human and economic health (2013)
[13]. Economic health, however, is not necessarily good
for human health. Three recent articles specifically cau-
tioned us to be wary of globalization’s commercial
drivers: from dominating online health information
(2014) [14], to Big Tobacco’s game plans to sustain or
expand its global markets (2018) [15], and the use of
‘front groups’ to advance corporate interests under a
cloak of scientific neutrality (2019) [16]. The next two years featured more circumscribed top-
end papers: the first on how ‘modernizing’ China (code
for that country’s increased integration in the global Correspondence: rlabonte@uottawa.ca
School of Epidemiology and Public Health, Faculty of Medicine, University of
Ottawa, 600 Peter Morand Crescent, Ottawa, Ontario K1G 5Z3, Canada Some of these leading article themes led to the cre-
ation of special journal collections. The large number of Labonté Globalization and Health (2020) 16:31 Labonté Globalization and Health (2020) 16:31 Page 2 of 3 Labonté Globalization and Health Page 2 of 3 articles related to trade and health, for example, were
recently gathered in a collection, with new articles being
added as they are published [17]. Another collection fo-
cuses on health system innovations in low- and middle-
income countries, belying the notion that such countries
lack innovative ideas from which the world at large can
learn [18]. The enduring topic of global health partner-
ships has its own dedicated place in the journal [19],
while we recently hosted a special call for papers on
intersectoral actions for health in the SDG that gener-
ated a range of new contributions [20]. There is also a
new open collection, timely given the COVID-19 crisis,
examining governance and preparedness for infectious
disease threats [21]. The journal also published our first
supplement in 2019 on the political determinants of
health inequities and universal health coverage [22]. Many of these specially edited collections were pub-
lished in collaboration with partnering universities
and institutions. Climate change and environmental resource depletions
may be taking a recessionary breather but wait around
the corner. What new form(s) of globalization arise, and
with what health equity outcomes, will be the necessary
grist of our globalization and health scholarship of the
near future. Consent for publication
Not applicable Consent for publication
Not applicable Although such collections and the topics of our past
15 years of lead articles help to identify the parameters
of our field of study, the journal has sometimes veered
close to a more ‘international’ than ‘globalization and’
health perspective. A surge in single country health stud-
ies a few years ago had the editorial team reformulate
more precisely the journal’s scope to ensure its focus on
the importance of how globalization processes affected
health outcomes, whether in a given country, cross-
nationally interrogated, or examined at a global scale
[23]. The accompanying re-design in our sections and
editorial processes, and a revitalization of our Editorial
Board, has made the scholarship we publish more ger-
mane to globalization and health researchers and, we
hope, more useful to global health policy-makers and
advocates. Acknowledgments g
Mackenzie Shaheen, for present and past assistance in referencing and
formatting of articles. Competing interests The author is Editor-in-Chief of Globalization and Health. The author is Editor-in-Chief of Globalization and Health. References 1. Huynen MM, Martens P, Hilderink HB. The health impacts of globalisation: a
conceptual framework. Glob Health. 2005;1(1):14. 1. Huynen MM, Martens P, Hilderink HB. The health impacts of globalisation: a
conceptual framework. Glob Health. 2005;1(1):14. 2. Hawkes C. Uneven dietary development: linking the policies and processes
of globalization with the nutrition transition, obesity and diet-related
chronic diseases. Glob Health. 2006;2(1):4. 3. Labonté R, Schrecker T. Globalization and social determinants of health:
introduction and methodological background (part 1 of 3). Glob Health. 2007;3(1):5. 4. Ding EL, Malik VS. Convergence of obesity and high glycemic diet on
compounding diabetes and cardiovascular risks in modernizing China: an
emerging public health dilemma. Glob Health. 2008;4(1):4. 5. Thow AM, Hawkes C. The implications of trade liberalization for diet and
health: a case study from Central America. Glob Health. 2009;5(1):5. Events since the start of this year, however, present
the global health space with an oft-predicted but yet
unpredictable pandemic challenge. We would be re-
miss if we didn’t then re-assess somewhat our journal’s
future path. Although globalization (the increasing
interdependence of peoples across time and space) is
unlikely to cease, the economic rules that have largely
defined and been driving it for the past 40 years are
under ever-more critical questioning. The global sup-
ply chains that bound together our economies are
under threat, nativist sentiments are fraying inter-
national cooperation, the (still under-regulated) world
of financial speculation is once more haemorrhaging,
and it is again the poor and the vulnerable who will
bear the heaviest health burden. 6. Chen BK, Seligman B, Farquhar JW, Goldhaber-Fiebert JD. Multi-country
analysis of palm oil consumption and cardiovascular disease mortality for
countries at different stages of economic development: 1980-1997. Glob
Health. 2011;7(1):45. 6. Chen BK, Seligman B, Farquhar JW, Goldhaber-Fiebert JD. Multi-country
analysis of palm oil consumption and cardiovascular disease mortality for
countries at different stages of economic development: 1980-1997. Glob
Health. 2011;7(1):45. 7. Maru D, Schwarz R, Andrews J, et al. Turning a blind eye: the mobilization
of radiology services in resource-poor regions. Glob Health. 2010;6:18. 7. Maru D, Schwarz R, Andrews J, et al. Turning a blind eye: the mobilization
of radiology services in resource-poor regions. Glob Health. 2010;6:18. 8. Harris M, Macinko J, Jimenez G, Mullachery P. Measuring the bias against
low-income country research: an implicit association test. Glob Health. 2017;
13(1):80. 8. Harris M, Macinko J, Jimenez G, Mullachery P. Ethics approval and consent to participate
Not applicable Ethics approval and consent to participate
Not applicable 10.
Syed SB, Dadwal V, Rutter P, Storr J, Hightower JD, Gooden R, et al.
Developed-developing country partnerships: benefits to developed
countries? Glob Health. 2012;8(1):17. Author’s contributions I am the sole author. The author read and approved the final manuscript. Funding Not applicable Availability of data and materials Availability of data and materials
Not applicable Ethics approval and consent to participate Ethics approval and consent to participate References Measuring the bias against
low-income country research: an implicit association test. Glob Health. 2017;
13(1):80. 9. Truché P, Shoman H, Reddy CL, Jumbam DT, Ashby J, Mazhiqi A, et al. Globalization of national surgical, obstetric and anesthesia plans: the critical
link between health policy and action in global surgery. Glob Health. 2020;
16(1):1. 9. Truché P, Shoman H, Reddy CL, Jumbam DT, Ashby J, Mazhiqi A, et al. Globalization of national surgical, obstetric and anesthesia plans: the critical
link between health policy and action in global surgery. Glob Health. 2020;
16(1):1. 10. Syed SB, Dadwal V, Rutter P, Storr J, Hightower JD, Gooden R, et al. Developed-developing country partnerships: benefits to developed
countries? Glob Health. 2012;8(1):17. 11. Buse K, Hawkes S. Health in the sustainable development goals: ready for a
paradigm shift? Glob Health. 2015;11(1):13. 12. Smith J, Buse K, Gordon C. Civil society: the catalyst for ensuring health in
the age of sustainable development. Glob Health. 2016;12(1):40. How well our governments and our communitarian
ethos (gun purchases or uber-elite retreats to secure
boltholes notwithstanding) respond effectively to these
new existential challenges is the order of the day. 13. Reeves A, Basu S, McKee M, Meissner C, Stuckler D. Does investment in
the health sector promote or inhibit economic growth? Glob Health. 2013;9(1):43. 14. Mackey TK, Eysenbach G, Liang BA, Kohler JC, Geissbuhler A, Attaran A. A
call for a moratorium on the health generic top-level domain: preventing 14. Mackey TK, Eysenbach G, Liang BA, Kohler JC, Geissbuhler A, Attaran A. A
call for a moratorium on the health generic top-level domain: preventing Page 3 of 3 Page 3 of 3 Labonté Globalization and Health (2020) 16:31 Labonté Globalization and Health (2020) 16:31 Labonté Globalization and Health (2020) 16:31 the commercialization and exclusive control of online health information. Glob Health. 2014;10(1):62. the commercialization and exclusive control of online health information. Glob Health. 2014;10(1):62. the commercialization and exclusive control of online health information. Glob Health. 2014;10(1):62. 15. Gomis B, Lee K, Carrillo Botero N, Shepherd P, Iglesias RM. “We think
globally”: the rise of Paraguay’s Tabacalera del Este as a threat to global
tobacco control. Glob Health. 2018;14(1):110. 16. Steele S, Ruskin G, Sarcevic L, McKee M, Stuckler D. Are industry-funded
charities promoting “advocacy-led studies” or “evidence-based science”?: a
case study of the international Life Sciences Institute. Glob Health. 2019;
15(1):36. 17. Trade and Health (Series). References In: Ruckert A, Schram A, and Labonté R, editors. Globalization and Health [Internet]. London: BMC. Available from: https://
www.biomedcentral.com/collections/trade-health. Accessed 6 Apr 2020. 18. Innovation in health systems in low- and middle-income countries
(Collection). In: Balasubramanian P, Lakew D, and Bloom G, editors. Globalization and Health [Internet]. London: BMC. Available from: https://
www.biomedcentral.com/collections/innovationhs. Accessed 6 Apr 2020. 19. Health partnerships: an effective response to the global health agenda
(Collection). In: Pinder S, editor. Globalization and Health [Internet]. London:
BMC. Available from: https://www.biomedcentral.com/collections/
healthpartnerships. Accessed 6 Apr 2020. 20. Health in the SDGs: Intersectoral Action for Health (Collection). In: Hussain S.,
Javadi D., Andrey J., Labonté R., Ghaffar A., editors. Globalization and Health
[Internet]. London: BMC. Available from: https://www.biomedcentral.com/
collections/HealthinSDGs. Accessed 6 Apr 2020. 21. Cross border infectious disease threats: governance and preparedness
(Collection). In: Boland M. & Stoto M.A., editors. Globalization and Health
[Internet]. London: BMC. Available from: https://www.biomedcentral.com/
collections/cross-border-disease. Accessed 6 Apr 2020. 22. The political determinants of health inequities and universal health
coverage (Supplement 1). In: Fukuda-Parr S., Kittelsen S.K., Tagmatarchi
Storeng K., editors. Globalization and Health [Internet]. London: BMC; 2019. Available from: https://globalizationandhealth.biomedcentral.com/articles/
supplements/volume-15-supplement-1. Accessed 6 Apr 2020. 23. Labonté R. Reprising the globalization dimensions of international health. Glob Health. 2018;14(1):49. 23. Labonté R. Reprising the globalization dimensions of international health. Glob Health. 2018;14(1):49. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
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https://openalex.org/W4288276390
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https://zenodo.org/records/6461943/files/710-863-1-SM.pdf, https://zenodo.org/record/3688739/files/710-863-1-SM.pdf
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es
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SISTEMA EXPERTO DE REQUERIMIENTOS Y COMPATIBILIDAD SOBRE ENSAMBLAJE DE COMPUTADORAS
|
Zenodo (CERN European Organization for Nuclear Research)
| 2,019
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cc-by
| 4,052
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REVISTA INVESTIGACION OPERACIONAL
VOL. 40 , NO. 644-651, 2019
SISTEMA EXPERTO DE REQUERIMIENTOS Y
COMPATIBILIDAD SOBRE ENSAMBLAJE DE
COMPUTADORAS
Rita Azucena Díaz Vásquez * , Jorge Lenin Acosta Espinoza * , Glenda Cecibel Intriago Alcívar *
Universidad Regional Autónoma de los Andes, Riobamba, UNIANDES, Ecuador ** Universidad Técnica
Estatal de Quevedo, UTEQ, Ecuador
ABSTRACT
The purpose of this system is to satisfy the expectations and requirements of the customers of computer assembly, which is a
procedure that consists on correctly placing all the parts of a computer in order such that everything works efficiently. Then, it
will be able to install the operating system and other programs according to the user’s needs, thus a computer selection is based
on information on compatibility and assembly of computers. The information has been compiled from books, scientific papers,
and based on research tools such as: surveys, interviews, for the collection of information. The development of this system
serves as a tool that in the future may bring some comfort to users with little knowledge of hardware to make purchases
according to the needs and customer's economy, which is an important aspect in people's daily lives. The Expert System of
requirements and compatibility in assembly of computers realizes what an expert engineer in assembly of computers can do, but
the system does it in a fast way and it is always available.
KEYWORDS: Assembly, compatibility, expert system
MSC: 68T35
RESUMEN
El propósito de este sistema es cumplir con las expectativas y requerimientos de los clientes de ensamblaje de computadora que
es un procedimiento que consiste en colocar correctamente todas las partes de una computadora con la finalidad que todo
funcione eficientemente para luego poder instalar el sistema operativo y demás programas de acuerdo a las necesidades del
usuario, para que al momento de elegir una computadora se fundamente en información sobre compatibilidad y ensamblaje de
computadores. La información ha sido recopilada a través de libros, artículos científicos, con instrumentos de investigación
como: encuestas, entrevistas, para la recopilación de la información. El desarrollo de este sistema sirve como herramienta que en
el futuro pueda portar cierta comodidad a los usuarios con poco conocimiento de hardware para realizar compras acordes a las
necesidades y de acuerdo a la economía del cliente, aspecto importante en la vida diaria de las personas. El Sistema Experto de
requerimientos y compatibilidad en ensamblaje de computadoras realiza lo que un ingeniero experto en ensamblaje de
computadoras puede hacer, pero el sistema lo hace de una manera rápida y siempre está disponible.
PALABRAS CLAVES: Ensamblaje, compatibilidad, sistema experto
1.
INTRODUCCIÓN
La empresa MEGASYSTEM se dedica a la venta de computadoras además de todo tipo de dispositivos
tecnológicos. Esta empresa proporciona a sus clientes proformas manuales de los equipos que tiene a la venta,
por lo cual se pudo observar la falta de un servicio eficaz hacia los clientes, al no brindarles proformas con los
equipos de características precisas en cuanto a los requerimientos, funciones, presupuesto y necesidades de
cada cliente, razones por las cuales se vio en la necesidad de crear un sistema que ensamble un computador
con los componentes que reúnen las características que el cliente realmente necesita y si está en el
presupuesto con que el cliente cuenta.
Conociendo que los Sistemas Expertos (SE) son programas de computador que buscan simular los procesos
intelectuales de los expertos humanos como: la interpretación, el diagnóstico, la monitorización, el control, la
predicción, la planificación, el diseño y la enseñanza, entre otros. Una interesante orientación que caracteriza
los sistemas en Inteligencia Artificial (IA) es llamado Principio de Generalidad: Al tener una estructura lógica
básica necesaria para solucionar un problema, entonces, en principio, puede usarse la potencia del computador
para hallar la solución. Este principio acepta que hay un método general para expresar y solucionar problemas
con conocimiento común. Hoy día, los SE o Sistemas Basados en el Conocimiento (SBC) son base para
desarrollar sistemas inteligentes directamente asociados con la representación de conocimiento y procesos de
razonamiento, [1]. La idea de la construcción de los Sistemas Expertos se simplifica en dos aspectos:
a) La adquisición, la representación y el uso de conocimiento especializado.
b) El reconocimiento que los sistemas, además de resolver problemas, puedan poseer atributos propios de
expertos humanos como:
644
Capacidad para adquirir nuevo conocimiento.
Capacidad para justificar sus conclusiones.
Capacidad de hacer preguntas y explicar por qué las hacen.
Capacidad conversacional.
La utilización de software y materiales virtuales computarizados como un recurso para apoyar los procesos de
venta de las diversas áreas del conocimiento, se ha convertido en una necesidad y constituye una respuesta
ante la problemática que gira en torno de la comprensión de conceptos y nociones en un tema de escoger un
computador acorde a la necesidad del cliente.
En particular el surgimiento de la Inteligencia Artificial como una disciplina científica y el desarrollo
tecnológico que ha impulsado en el campo de los Sistemas Expertos ha abierto una gama de posibilidades al
público en general.
A pesar de que tanto el desarrollo como la adquisición de un Sistema Experto es relativamente caro, con la
adquisición de un sistema para el buen funcionamiento de determinada empresa, se puede tener un ahorro de
tiempo futuro, ya que el tratamiento y el coste marginal de su uso constante resulta ser más económico. De
igual forma, con el uso de Sistemas Expertos se obtienen ganancias muy altas en términos monetarios,
tiempo, y precisión, así como también la amortización resulta ser muy rápida. Aun así, es aconsejable realizar
un análisis de factibilidad y de costo-beneficio antes de adquirir o desarrollar un Sistema Experto, [5]. Existen
varias razones de peso que explican por qué usar Sistemas Expertos, esto tiene sus ventajas entre ellas
podemos mencionar:
1. Un Sistema Experto permite que un problema complejo, pueda ser resuelto por personas con poca
experiencia en el tema. Es esta una ventaja para la empresa MEGASYSTEM que cuenta con poco
personal experto en determinadas áreas. Por lo tanto, se puede deducir que con el uso de los Sistemas
Expertos aumenta el número de personas con acceso al conocimiento.
2. Es posible reunir y combinar el conocimiento de varios expertos humanos lo cual conlleva a tener
Sistemas Expertos confiables, puesto que usan la inteligencia y la sabiduría de varios humanos con
conocimientos del tema en vez de usar un solo criterio.
3. Una gran ventaja es que su tiempo de respuesta es menor al que tardaría una persona, aunque sea muy
experta en el tema. Es esta cualidad la que los hace más valiosos en circunstancias donde el tiempo de
respuesta es un factor crítico.
4. Dependiendo del grado de complejidad de determinado problema es posible o no resolverlo por un
experto humano. Es aquí donde se puede notar la importancia de los Sistemas Expertos, ya que,
aprovechando la evolución tecnológica, estos usan herramientas de cómputo para procesar gran cantidad
de operaciones complejas de forma rápida y acertada, suministrando respuestas confiables en situaciones
en las que los expertos humanos podrían tener dificultades.
5. Es posible usar Sistemas Expertos para la solución de problemas monótonos en medios en los cuales los
expertos humanos se sienten aburridos e incómodos. Un Sistema Experto es la única solución a
problemas donde la tarea a realizar desborda al ser humano.
6. Por medio de un Sistema Experto, es posible obtener un ahorro tanto económico como en el tiempo
requerido para la solución de determinado problema. El uso de los Sistemas Expertos es recomendado en
diversas situaciones, entre ellas están las siguientes:
Cuando el experto humano no cuenta con el conocimiento, o cuando dicho conocimiento es basado
en reglas las cuales requiere de la experiencia para poder ser aprendidas.
Cuando el problema depende de reglas que se encuentran en constante cambio.
Cuando la mejora continua del conocimiento es lo esencial para una organización.
Cuando no se cuenta con cantidad suficiente de expertos humanos.
Cuando contratar expertos humanos es demasiado costoso.
Cuando no se tiene el conocimiento suficiente sobre el problema que se pretende solucionar.
Este artículo tiene como objetivo mostrar el diseño del Sistema Experto desarrollado por la empresa
MEGASYSTEM para el requerimiento y compatibilidad sobre ensamblaje de computadoras. Se destacarán
las ventajas y utilidad del sistema para ser usado.
2.
MATERIALES Y MÉTODOS
Para el desarrollo de la aplicación se optó por la metodología de desarrollo de software. Se trabajó con
encuestas a clientes para conocer las necesidades como compradores de equipos de computación y así
645
implementar las funciones requeridas en el desarrollo del sistema. También se realizaron entrevistas para
verificar la factibilidad del sistema y los procesos que este debe cumplir.
Para la investigación se utilizó libros, internet, requerimientos en software y hardware, véase Tablas 1 y 2.
Software
Adobe Dreamweaver CS6
Para la programación PHP, Ajax, html5
Servidor Web IIS
Para ejecutar el sistema de manera local.
Gestor de Base de Datos: Mysql 5.0
Para guardar la información que contiene el
sistema.
Browser: Mozilla, Firefox, Google, Chrome, Para visualizar el sistema
Internet explorer
Tabla 1 Software necesario para el desarrollo del sistema.
Hardware
Computador HP coreI7
Para la elaboración del sistema experto
Impresora Epson TX730 Series
Impresión de proformas.
Tabla 2 Hardware necesario para el desarrollo del sistema
Fue necesario usar la Programación Extrema (eXtreme Programming en inglés) o Metodología XP para que el
sistema pueda estar orientado hacia las necesidades del cliente, haciendo del cliente parte fundamental del
equipo desarrollador del sistema quien estará presente desde el inicio hasta el final de su desarrollo y de esta
manera lograr satisfacer sus necesidades, [7, 11].
La metodología XP cuenta con las siguientes fases:
1. Planificación del proyecto. - Detalla las historias de usuarios en donde el cliente establece las entradas,
las salidas, y los procesos que debe tener el sistema para lograr los objetivos planteados. Estas historias
de usuario también se utilizan en la fase de pruebas, para verificar si el programa cumple con lo que
especifica la historia de usuario. Cuando llega la hora de implementar una historia de usuario, el cliente y
los desarrolladores se reúnen para concretar y detallar lo que tiene que hacer dicha historia, [11].Véase
Tabla 3.
HISTORIA DE USUARIO
HU 101 Creación de la interfaz de ingreso a la aplicación
Nombre de la historia:
Días asignados:
3
Prioridad:
Alta
Planificada
Estado:
Definida
Disposición:
Iteración asignada:
Iteración 1
Diseño de la interfaz para Autentificación de usuarios además del
Descripción:
formulario de registro de usuarios.
Observación:
2.
3.
La interfaz contendrá enlaces a otros formularios
Tabla 3 Historia de usuario
Diseño. - En esta fase se sugiere conseguir diseños simples y sencillos. Hay que procurar hacerlo todo lo
menos complicado posible para conseguir un diseño fácilmente entendible e implementable que a la larga
costará menos tiempo y esfuerzo desarrollar. De esta manera se da mayor importancia a la codificación
para que el sistema sea eficiente, confiable y pueda cumplir con las funciones requeridas por los usuarios,
[10]. La etapa de diseño de un Sistema Experto incluye el diseño de estructuras para archivar el
conocimiento, véase [6], al igual que el motor de inferencia, el subsistema de explicación, la interfaz de
usuario, entre otros.
Codificación. - Antes del desarrollo de cada historia de usuario el cliente debe especificar detalladamente
lo que esta hará y también tendrá que estar presente cuando se realicen los test que verifiquen que la
historia implementada cumple la funcionalidad especificada. La codificación debe hacerse ateniendo a
estándares de codificación ya creados. Programar bajo estándares mantiene el código consistente y
facilita su comprensión y escalabilidad. XP opta por la programación en pareja ya que permite un código
646
más eficiente y con una gran calidad. Esta metodología sugiere un modelo de trabajo usando repositorios
de código donde las parejas de programadores publican cada pocas horas sus códigos implementados y
corregidos junto a los test que deben pasar, [2].
4. Pruebas. - Uno de los pilares de la metodología XP es el uso de pruebas para comprobar el
funcionamiento de los códigos que se van implementando. Las pruebas ayudan a saber qué es
exactamente lo que tiene que hacer el sistema a implementar y se sabrá que una vez implementado pasará
dichas pruebas sin problemas. El construir y probar es necesario en esta etapa, [8], si las pruebas no
arrojan los resultados esperados, se hace necesario realizar modificaciones a las etapas anteriores, y
ejecutar nuevas pruebas tantas veces como sea necesario hasta obtener un sistema satisfactorio. El
refinamiento y generalización es en esta etapa donde aparte de corregir los errores encontrados, se
incorporan nuevas opciones las cuales no habían sido incluidas en el diseño inicial, [9].
El mantenimiento y puesta al día presenta el primer contacto del usuario final con el Sistema Experto, lo cual
permitirá al usuario plantear problemas o defectos de la primera versión obtenida del Sistema Experto, y estas
observaciones hechas por el usuario permitirá realizar correcciones y actualizar el producto con nuevos
avances. Este conjunto de etapas, enmarcan la calidad del Sistema Experto obtenido, [4], el cual debe ser
evaluado constantemente en función de los aportes entregados por los usuarios finales, véase [8].
La modelación matemática del SE se basa en relaciones n-arias, ver [3], en este caso con n = 8.
Definición 1. Una relación n-aria sobre
se define como
.
También se utilizará un árbol, que formalmente se define según la Definición 2.
Definición 2 Un árbol es un grafo conexo acíclico, véase [11].
Entre la nomenclatura del árbol se encuentran los nodos que son los vértices en la Teoría de Grafos. Se dice
que a es padre de b, o que b es hijo de a, donde a y b son nodos del árbol, si existe una arista que va de a
hasta b. Dos nodos se dicen hermanos si tienen el mismo padre. El nodo que no tiene padre se le llama raíz,
mientras que los nodos que no tienen hijos se llaman hojas.
El nivel de un nodo se calcula por 1+ el número de conexiones entre este y la raíz.
2.1. Proceso de selección de un computador armado
Los pasos del sistema cuando el usuario selecciona el computador ya ensamblado se dan detallan en el
Algoritmo 1.
Algoritmo 1: De Selección del Computador Ensamblado
1. Cada usuario crea o accede a una cuenta. Esto se hace accediendo a una serie de ventanas
visuales que interactúan con el Sistema Experto.
2. Aparece un asistente para determinar la selección del usuario sobre el hardware que desea
obtener.
3. El usuario selecciona el tipo de computador que desea.
De escritorio
Portátil
4. El usuario selecciona el uso que le dará al computador. Véase Figura 1.
Para el hogar o la pequeña oficina
Para juegos
Para diseño gráfico
Profesional
5. El usuario selecciona la arquitectura con la que desea que cuente su computador
AMD
Intel
6. El usuario ingresa el presupuesto en dólares con que cuenta.
7. Si el usuario está seguro de sus opciones pulsa el botón construir.
8. El sistema devuelve las opciones de hardware con sus precios, que satisfacen las características
pedidas por el usuario.
9. Si el usuario cambia de opinión en alguna de sus selecciones puede editar de nuevo su pedido.
647
Figura 1 Pantalla que permite escoger opciones de acuerdo con el uso que se le dará al computador
2.2. Proceso de selección de un computador personalizado
En el presente artículo se definirán los conjuntos siguientes:
1.
X1: es el conjunto de posibles usos que el usuario pudiera darle a la PC que desea armar. Estos usos se
representan en forma de cadena de caracteres y son los siguientes:
Para el hogar o la pequeña oficina
Para juegos
Para diseño gráfico
Profesional
2.
3.
X2: es el conjunto de precios disponibles.
X3: es el conjunto de todos los procesadores disponibles. Cada elemento del conjunto será una cadena
de caracteres con el nombre del procesador.
X4: es el conjunto de cadenas de caracteres con el nombre de los tipos de mainboard disponibles por la
empresa.
X5: es el conjunto de cadenas de caracteres con el nombre de los tipos de memoria RAM disponibles por
la empresa.
X6: es el conjunto de cadenas de caracteres con el nombre de los tipos de discos duros disponibles por
la empresa.
X7: es el conjunto de cadenas de caracteres con el nombre de los tipos de Unidades de DVD disponibles
por la empresa.
X8: es el conjunto de cadenas de caracteres con el nombre de los tipos de Sistemas Operativos
propuestos por la empresa.
4.
5.
6.
7.
8.
La relación R de la Definición 1, se considerará de compatibilidad entre las componentes, es decir, R(x1, x2,
…, x8) es verdadera si y solo si existe compatibilidad a la vez, entre el uso que el usuario puede dar a la PC,
con el precio, con el tipo de procesador, con el mainboard, con la memoria RAM, con el disco duro, las
unidades de DVD y el sistema operativo.
Los subíndices de los conjuntos Xi significan el orden de importancia de la componente. Por ejemplo, se
considera que el uso que el usuario desea darle a su PC es lo más importante para el armado de esta, por ello
se representa por el conjunto de subíndice 1, luego lo más importante es que el usuario cuente con el
presupuesto necesario para pagar el precio de una PC según su uso, por eso se representa por el subíndice 2,
luego es más importante identificar qué tipo de procesador es necesario para cumplir con tal uso y tal precio,
por lo tanto se asocia con el subíndice 3, y así sucesivamente.
Cada conjunto Xi definido anteriormente coincide con el nivel i-ésimo del árbol. Si existe alguna PC tal que
(xi, xi+1) son compatibles, entonces en el árbol se tendrá que xi es el padre de xi+1. Véase un árbol genérico en
la Figura 2 con los tres niveles superiores. El árbol está diseñado de tal manera que cada regla de la Base de
648
Conocimiento se obtiene si se desciende desde la raíz hasta el último nivel.
Se definirá la Ecuación 1 sobre compatibilidad, dado
.
(1)
i =2,…, 8.
Las Ecuaciones 2 y 3 representan la similaridad entre opciones, dadas
.
e
(2)
(3)
Para garantizar la eficiencia de los algoritmos, es importante que los elementos de cada X i tengan un orden
dentro del árbol. Por ejemplo, la Figura 2 contiene en el primer nivel un orden del uso según la complejidad,
por otro lado, en el segundo nivel el orden de los precios también está ordenado de menor a mayor. De igual
manera se pueden ordenar los procesadores, de los más simples a los más complejos etc. Esto permite que
haya una concentración entre los diferentes tipos de PC, por ejemplo, se espera que una PC de uso para el
hogar (ubicado más a la izquierda del árbol), tenga un precio más barato que los demás tipos, y así
sucesivamente.
Uso
Hogar o Pequeña
Oficina
Juegos
Diseño gráfico
Profesional
$350
$700
$450
$1000
Figura 2 Gráfico hipotético de representación del árbol para los tres niveles superiores.
Basado en lo anterior se propone el Algoritmo 2.
Algoritmo 2: De Selección del Computador Personalizado
1. Cada usuario crea o accede a una cuenta. Esto se hace accediendo a una serie de ventanas
visuales que interactúan con el Sistema Experto.
2. Aparece un asistente para determinar la selección del usuario sobre el hardware que desea
obtener.
3. El usuario debe de seleccionar la opción que desea: una PC personalizada.
4. Se inicializa i = 1.
5. El usuario propone
5.1. Si i = 1; o 1<i<8 y
, entonces hacer i = i+1. Repetir Paso 5.
5.2. Si 1< i y
, entonces el SE busca en la Base de Conocimiento algún yi tal que
, con Si((x1,x2,…,xi)) sea el más cercano a Si((x1,x2,…,yi)). Véase
Ecuaciones 1, 2 y 3.
El SE le recomienda al usuario el o los yi. Se espera a que el usuario acepte. Una vez que
acepta, esto lleva a dos pasos diferentes:
649
Si i<8, se hace i = i+1. Repetir Paso 5.
Si i = 8, Terminar.
5.3. Si i = 7 y
, Terminar.
3. RESULTADOS
Los autores de este artículo programaron adicionalmente un simulador del comportamiento de un usuario
que desearía obtener una PC personalizada, para comprobar el funcionamiento del Algoritmo 2.
Se utilizó la fórmula de desempeño
, donde N es la cantidad de veces que se realiza la
simulación y
es el número de veces que el sistema determina que hay un verdadero positivo.
Se utilizan expertos para determinar si el resultado es un verdadero positivo. Si P es la variable aleatoria que
determina si el experimento es un acierto o una fracaso, cada vez que el sistema llega a una solución que es
un verdadero positivo se asigna P = 1, de lo contrario P = 0.
Primero se evaluó P para 50 ocasiones con el simulador y se obtuvo una desviación estándar muestral sP =
0,2399 aproximadamente y una media muestral de
. Se utilizó la fórmula
para
calcular el número de simulaciones necesarias, donde
es el valor crítico para un nivel de confianza de
1-, de la distribución normal de media 0 y varianza 1. Para el nivel de confianza = 0,05 se tiene
. Mientras que e = 0,05 es el error máximo admitido. Por tanto se toma N =89.
Luego de comprobar el resultado del Algoritmo 2 con 89 casos, se obtuvo D = 95,506%, que es un número
alto de aciertos.
4.
CONCLUSIONES
En este artículo se expuso el diseño de un Sistema Experto que permite a los usuarios no necesariamente
expertos, de una manera sencilla y rápida la determinación de la compatibilidad para el ensamblaje de
computadores. Se comprobó mediante simulación que se puede esperar alrededor de un 95% de acierto del
Sistema Experto en su Algoritmo 2.
El Sistema Experto ayuda al usuario bajo dos circunstancias, cuando el usuario desea obtener un
computador ensamblado, el sistema lo ayuda a seleccionar el más adecuado a sus necesidades. Por otra
parte, si el usuario desea obtener una PC personalizada, el sistema le permite que diseñe la PC de acuerdo a
sus necesidades y corrige en caso de incompatibilidad.
Debido a que se usó un árbol, es posible incluir casos nuevos donde se diseñen nuevos computadores
compatibles. Por tanto, es posible actualizar el sistema de manera fácil. También fue posible desarrollar
algoritmos con un grado alto de exactitud debido a lo relativamente pequeño que es el número de
combinaciones para armar una PC. Si se tuviera un número mayor habría que considerar otras
representaciones del conocimiento, por ejemplo árboles de decisión que usan algoritmos como ID3 o C4.5.
RECEIVED: DECEMBER, 2018.
REVISED: JUNE, 2019
REFERENCIAS
[1] FLORES FONSECA, V. M. (2010) Generación automática de resúmenes de comportamiento de
sistemas dinámicos mediante modelos de conocimiento del dominio, Tesis Doctoral Informática.
[2] HEURTEL, O. (2009) PHP y MySQL: domine el desarrollo de un sitio web dinámico e interactivo.
Ediciones Eni.
[3] JECH, T. (2003) Set Theory. Springer-Verlag Berlin Heidelberg.
[4] LARSON, R. E., TINNEY, W. F., HAJDU, L. P. y PIERCY, D. S. (1970) State estimation in power
systems part II: Implementation and applications, IEEE Transactions on Power Apparatus and
Systems, 1970, 353-363.
[5] LÓPEZ SEPÚLVEDA, G. P. (2011) Creación de un sistema experto aplicado a estimación de
estado para centros de control de energía, Tesis Doctoral. Universidad Tecnológica de Pereira,
Pereira, Colombia.
[6] MENDOZA CÁRDENAS, S. R. y CÁCERES ZÁRATE, J. N. (2016) Sistema experto para
la selección de postulantes en puestos de una agencia bancaria usando la metodología
CommonKads. Tesina para optar el Título Profesional de Ingeniero de Sistemas, Universidad
650
Nacional Mayor De San Marcos, Lima, Perú
[7] MEX.TL. (2014). Ingeniería de Software . Obtenido de Mex.tl: Disponible en:
http://ingenieriadesoftware.mex.tl/52753_XP---Extreme-Programing.html . Consultado 20-2,
2018.
[8] PREECE, A. D. (1990) Towards a Methodology for Evaluating Expert Systems.
Expert Systems, 7, 215–293.
[9] RUIZ FLOREZ, H. A. (2009) Estimación de estado de sistemas eléctricos Usando algoritmos de
optimización combinatorial. Tesis Doctoral. Universidad Tecnológica de Pereira, Pereira, Colombia.
[10] SCHILDT, H. (2002) Fundamentos de programación en Java 2, Osborne Mc Graw Hill,
Colombia.
[11] WALLIS, W.D. (2007) A Beginner’s Guide to Graph Theory, Birkhäuser, Boston.
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SISTEM PAKAR PENDIAGNOSA PENYAKIT TUBERKULOSIS
| null | 2,017
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1. PENDAHULUAN penduduk, tetapi angka insidennya turun menjadi
185 per 100.000 penduduk di tahun 2012 [15]. Tuberkulosis
(TB)
merupakan
penyakit
menular langsung yang disebabkan oleh infeksi
bakteri
Mycobacterium
tuberculosis. TB
merupakan penyakit yang mudah menular melalui
udara dari sumber penularan yaitu pasien TB BTA
positif pada waktu batuk atau bersin, pasien
menyebarkan kuman ke udara dalam bentuk
percikan dahak. Sekali batuk dapat menghasilkan
sekitar 3000 percikan dahak [7]. Sebagian besar kuman TB menyerang paru,
tetapi dapat juga mengenai organ tubuh lainnya
yang biasa disebut dengan TB Ekstra Paru. TB Paru
merupakan bentuk yang paling sering dijumpai
yaitu sekitar 80 % dari semua penderita. TB yang
menyerang jaringan paru-paru ini merupakan satu-
satunya bentuk dari TB yang mudah menular. TB
Ekstra Paru merupakan bentuk penyakit TB yang
menyerang organ tubuh lain selain paru-paru. TB
pada dasarnya ini tidak pandang bulu karena kuman
ini dapat menyerang semua organ dari tubuh [4]. TB dapat menyerang siapa saja, terutama usia
produktif/masih aktif bekerja dan anak-anak. Sekitar 75% pasien TB adalah kelompok usia yang
paling produktif secara ekonomis (15-50 tahun). Diperkirakan seorang pasien TB dewasa akan
kehilangan rata-rata waktu kerjanya 3 sampai 4
bulan. Jika ia meninggal akibat TB, maka akan
kehilangan pendapatannya sekitar 15 tahun. Selain
merugikan secara ekonomis, TB juga memberikan
dampak buruk lainnya secara sosial bahkan
dikucilkan oleh masyarakat [3]. Masyarakat hanya mengetahui bahwa TB
menyerang bagian paru saja pada umumnya, namun
TB juga dapat menyerang organ lain selain paru
yang disebut ekstra paru. TB Ekstra Paru terjadi
ketika kuman TB menyebar ke bagian organ tubuh
lain melalui aliran darah. Diagnosis pasti untuk
penyakit TB sering sulit ditegakkan sedangkan
diagnosis kerja dapat ditegakkan berdasarkan gejala
klinis
TB
yang
kuat
(presumtif)
dengan
menyingkirkan kemungkinan penyakit lain. Indonesia berkontribusi sebesar 5,8% dari
kasus TB yang ada di dunia. TB merupakan
masalah utama kesehatan masyarakat dan menjadi
tantangan dalam masalah kesehatan masyarakat di
Indonesia dengan masih adanya sekitar 430.000
pasien
baru
per
tahun
dan
angka
insiden
189/100.000 penduduk serta angka kematian akibat
TB sebesar 61.000 per tahun atau 27/100.000
penduduk [6]. Menurut laporan WHO tahun 2013,
Indonesia menempati urutan ketiga jumlah kasus
TB setelah India dan Cina dengan jumlah sebesar
700 ribu kasus. Angka kematian masih sama
dengan tahun 2011 sebesar 27 per 100.000 Kurangnya fasilitas yang memadai, dokter ahli
yang tidak selalu ada di tempat dan kurangnya
pengetahuan
pasien
mengenai
penyakit
TB
seringkali membuat diagnosis TB terlambat yang
bisa mengancam kesehatan pasien. Nur Aini 1), Ramadiani 2), Heliza Rahmania Hatta 3) 1,2,3) Ilmu Komputer, Fakultas Ilmu Komputer dan Teknologi Informasi, Universitas Mulawarman
Jl.K.H Wahid Hasyim II, Samarinda, 75119 1,2,3) Ilmu Komputer, Fakultas Ilmu Komputer dan Teknologi Informasi, Universitas Mulawarman
Jl.K.H Wahid Hasyim II, Samarinda, 75119 E-Mail : nurshafaafi@gmail.com 1) , mmi_ugm04@yahoo.com 2), heliza.rahmania@gmail.com 3 Vol. 12, No. 1, Februari 2017 Vol. 12, No. 1, Februari 2017 Jurnal Informatika Mulawarman
ISSN 1858-4853 56 ABSTRAK Tuberkulosis merupakan penyakit infeksi yang mudah menular melalui udara yang disebabkan oleh kuman
Mycobacterium Tuberculosis. Tuberkulosis sebagian besar menyerang paru namun penyakit ini juga menyerang
organ tubuh lain yang disebut ekstra paru. Kurangnya fasilitas dan pengetahuan masyarakat dapat memperlambat
diagnosa awal Tuberkulosis Ekstra Paru sehingga dapat membahayakan keselamatan masyarakat. Maka
diperlukannya sistem pakar yang berguna untuk mendiagnosa penyakit Tuberkulosis yang dapat mempermudah
penderita dalam melakukan diagnosa awal penyakit Tuberkulosis agar segera mendapatkan penanganan yang
tepat. Tujuan dari penelitian ini adalah membangun sistem pakar pendiagnosa penyakit Tuberkulosis dengan
menggunakan metode Certainty Factor. Aplikasi ini akan mendiagnosa penyakit dengan melakukan penelusuran
gejala-gejala yang ada berdasarkan inferensi forward chaining. Penelitian ini menghasilkan sebuah sistem pakar
berbasis web dengan tingkat akurasi sebesar 85% yang dimanfaatkan untuk membantu tenaga kesehatan dan
masyarakat umum dalam mendiagnosa awal penyakit Tuberkulosis. Kata Kunci : Tuberkulosis, Sistem Pakar, Certainty Factor, Forward Chaining d. Tubekulosis Tuberkulosis (TB) adalah suatu infeksi
menular dan bisa berakibat fatal disebabkan oleh
Mycobacterium tuberculosis, Mycobacterium bovis
atau Mycobacterium africanum. TB menunjukkan
penyakit yang paling sering disebabkan oleh
Mycobacterium
tuberculosis,
tetapi
kadang
disebabkan
oleh
Mycobacterium
bovis
atau
Mycobacterium africanum. Di negara berkembang,
anak-anak terinfeksi oleh mikobakterium lainnya
yang menyebabkan TB. Organisme ini disebut
Mycobacterium bovis, yang bisa disebarkan melalui
susu yang tidak disterilkan [10]. Terdapat penelitian “Sistem Pakar untuk
Mendiagnosa Penyakit Polip Nasi (Polip Hidung)
Menggunakan metode Certainty Factor”. Hasil dari
penelitian ini adalah memberikan keputusan yang
cukup akurat mengenai diagnosa penyakit polip
nasi berdasarkan penerapan metode Certainty
Factor yang digunakan [1]. Penyakit TB merupakan penyakit kronis atau
menahun yang telah lama dikenal oleh masyarakat
luas. Penemuan Robbert Kock pada tahun 1882
secara meyakinkan telah dapat memberikan bukti
bahwa tuberkulosis adalah suatu penyakit infeksi
yang disebabkan oleh bakteri yang diberi nama
Mycobacterium tuberculosis. Orang yang pertama
kali dapat membuktikan bahwa TB adalah suatu
penyakit yang dapat ditularkan yaitu Villenim yang
hidup pada tahun 1827-1894. Penyakit TB biasa
terdapat pada paru-paru, tetapi mungkin juga pada
organ lain seperti kelenjar getah bening (nodus
lymphaticus) [11]. Berdasarkan latar belakang diatas, penulis
membangun Sistem Pakar Pendiagnosa Penyakit
Tuberkulosis Menggunakan Metode Certainty
Factor yang mampu memberikan diagnosis akan
kemungkinan seorang pasien menderita penyakit
TB beserta cara pengobatannya. 1. PENDAHULUAN Sebuah aplikasi
komputer yang sistematis sebagai alat bantu untuk
melakukan diagnosis awal penyakit TB sangat
diperlukan untuk memudahkan tenaga ahli dalam
menemukan bagian organ tubuh mana yang
terserang penyakit TB dan dapat mempercepat hasil
diagnosa sehingga tenaga ahli dapat memberikan
penanganan yang tepat. Vol. 12, No. 1, Februari 2017
57 Jurnal Informatika Mulawarman
ISSN 1858-4853 e. Gejala Penyakit Tuberkulosis Beberapa Penyakit TB yang sering diderita
oleh masyarakat adalah: Beberapa Penyakit TB yang sering diderita
oleh masyarakat adalah: 1. Tuberkulosis Paru
TB Paru adalah penyakit radang parenkim paru
yang
disebabkan
oleh
infeksi
kuman
Mycobacterium
Tuberculosis. TB
Paru
mencakup 80% dari keseluruhan kejadian
penyakit
TB
sedangkan
20%
selebihnya
merupakan TB Ekstra Paru. Jurnal Informatika Mulawarman
ISSN 1858-4853 Sistem yang dibangun dalam penelitian ini
adalah sistem yang menggunakan keahlian pakar
dalam bidang kesehatan dengan menggunakan
metode certainty factor dalam mendiagnosa sebuah
penyakit. Menurut Martin dan Oxman (1988),
sistem pakar adalah sistem berbasis komputer yang
menggunakan pengetahuan, fakta, dan teknik
penalaran dalam memecahkan masalah, yang
biasanya hanya dapat diselesaikan oleh seorang
pakar dalam bidang tertentu. Menurut Sari (2013),
metode Certainty Factor merupakan metode yang
mendefenisikan ukuran kepastian terhadap fakta
atau
aturan,
untuk
menggambarkan
tingkat
keyakinan pakar terhadap masalah yang sedang
dihadapi, dengan menggunakan Certainty Factor
dapat menggambarkan tingkat keyakinan pakar
terhadap suatu penyakit. kondisi-aksi. Dalam metode ini, data digunakan
untuk menentukan aturan mana yang dijalankan
kemudian aturan tersebut dijalankan, proses diulang
sampai ditemukan suatu hasil. Menurut Giarrattano
dan Riley, metode inferensi runut maju cocok
digunakan untuk menangani masalah pengendalian
(controlling) dan peramalan (prognosis) [8]. a. Sistem Pakar Sistem pakar (expert system) merupakan paket
perangkat lunak atau paket program komputer yang
ditujukan sebagai penyedia nasihat dan sarana
bantu dalam memecahkan masalah dalam bidang-
bidang
spesialisasi
tertentu
seperti
sains,
perekayasaan matematika, kedokteran, pendidikan
dan sebagainya [14]. b. Struktur Sistem Pakar Sistem pakar disusun oleh dua bagian utama,
yaitu lingkungan pengembangan dan lingkungan
konsultasi. Lingkungan pengembangan digunakan
untuk memasukkan pengembangan pakar ke dalam
lingkungan sistem pakar. Lingkungan konsultasi
digunakan oleh nonpakar untuk memperoleh
pengetahuan dan nasihat pakar. Kebanyakan sistem
pakar saat ini tidak berisi komponen perbaikan-
pengetahuan [14]. a. Gejala utama
Batuk terus-menerus dan berdahak selama
tiga minggu/lebih. b. Gejala tambahan yang sering dijumpai
1) Dahak bercampur darah/batuk darah. 2) Demam selama tiga minggu atau lebih
3) Sesak nafas dan nyeri dada. 4) Penurunan nafsu makan. 5) Berat badan turun. 6) Rasa kurang enak badan (malaise, lemah. 7) Berkeringat di malam hari walaupun
tidak melakukan apa-apa 1) Dahak bercampur darah/batuk darah. 2) Demam selama tiga minggu atau lebih 3) Sesak nafas dan nyeri dada. 4) Penurunan nafsu makan. 5) Berat badan turun. c. Mesin Inferensi 6) Rasa kurang enak badan (malaise, lemah. Mesin inferensi adalah program komputer
yang memberikan metodologi untuk penalaran
tentang
informasi
yang
ada
dalam
basis
pengetahuan
dan
dalam
workplace
untuk
memformulasikan
kesimpulan
[2]. Penelitian
menggunakan runut maju (Forward Chaining). Runut
maju
berarti
menggunakan
himpunan 7) Berkeringat di malam hari walaupun
tidak melakukan apa-apa 2. TB Ekstra Paru
TB Ekstra Paru merupakan penyakit yang
disebabkan oleh infeksi kuman Mycobacterium Vol. 12, No. 1, Februari 2017 58 2) Gejala Khusus c) Penurunan nafsu makan a) Rasa nyeri pada bagian punggung
atau mengalami kekakuan punggung. d) Berat badan turun e) Rasa kurang enak badan/malaise,
lemah g
p
gg
g
b) Penderita
enggan
menggerakkan
punggungnya. f) Berkeringat di malam hari walaupun
tidak melakukan apa-apa c) Penderita
menolak
untuk
membungkuk
atau
mengangkat
barang dari lantai, bila diminta
penderita akan menekuk lututunya
agar punggung tetap lurus. 2) Gejala Khusus )
j
a) Munculnya benjolan-benjolan pada
bagian yang mengalami gangguan
kelenjar seperti leher, sela paha, serta
ketiak. d) Rasa nyeri pada punggung berkurang
bila penderita beristirahat. b) Ada tanda-tanda radang di daerah
sekitar benjolan kelenjar. e) Timbulnya
benjolan
di
bagian
punggung/tulang belakang. c) Benjolan kelenjar mudah digerakkan. d) Benjolan kelenjar yang timbul terasa
kenyal. d) Benjolan kelenjar yang timbul terasa
kenyal. Jurnal Informatika Mulawarman
ISSN 1858-4853 c. TB Tulang Belakang (Spondilitis)
TB
Tulang
Belakang
atau
Spondilitis
Tuberculosis adalah penyakit radang tulang
belakang yang disebabkan oleh infeksi
kuman Mycobacterium Tuberculosis. Tuberculosis yang menyerang organ tubuh
selain paru. Penyakit ini biasanya terjadi karena
kuman menyebar dari bagian paru ke bagian
organ tubuh lain melalui aliran darah. a. Tuberkulosis Kelenjar Getah Bening
TB Kelenjar atau Limfadenitis Tuberculosis
adalah penyakit radang kelenjar getah bening
yang
disebabkan
oleh
infeksi
kuman
Mycobacterium Tuberculosis. Kelenjar getah
bening yang biasa diserang adalah bagian
leher, ketiak, dan sela paha. 1) Gejala sistemik/umum: 1) Gejala sistemik/umum: a) Batuk terus-menerus dan berdahak
selama tiga minggu/lebih. b) Demam selama tiga minggu/lebih c) Penurunan nafsu makan d) Berat badan turun 1) Gejala sistemik/umum: 1) Gejala sistemik/umum: e) Rasa kurang enak badan/malaise,
lemah a) Batuk terus-menerus dan berdahak
selama tiga minggu/lebih f) Berkeringat di malam hari walaupun
tidak melakukan apa-apa b) Demam selama tiga minggu/lebih 2) Gejala Khusus Atau Sistem pakar untuk diagnosa penyakit
Tuberkulosis ini menggunakan metode inferensi
forward chaining (penalaran maju) dan teori faktor
kepastian (certainty factor), dalam metode ini user
akan
memilih
gejala
sesuai
dengan
yang
dialaminya, kemudian sistem akan mengecek satu
demi satu gejala yang dipilih user apakah rule di
dalam database ada yang sesuai dengan inputan
user. Jika ada maka sistem akan melakukan
perhitungan nilai CF sesuai dengan rule yang
terpilih. Sistem akan memberikan output berupa
nama penyakit, besarnya nilai persentase hasil
diagnosis sistem, daftar gejala yang telah dipilih
user, serta penjelasan solusi penanganan dan
pengobatan penyakit. Di mana:
IF E1…En
: Fakta-fakta (evidence) yang ada
H
: Hipotesis atau konklusi yang
dihasilkan
CF Rule
: Tingkat keyakinan terjadinya
hipotesis H akibat adanya fakta
1. Rule evidence E tunggal dan hipotesis H tunggal
IF E THEN H (CF rule)
CF(H,E) = CF(E) × (CF rule)
2. Rule evidence E ganda dan hipotesis H tunggal
IF E1 AND E2….AND En THEN H (CF rule)
CF(H,E) = min[CF(E1), CF(E2), ….,
CF(En)] x CF(rule)
IF E1 OR E2….OR En THEN H (CF rule)
CF(H,E) = max[CF(E1), CF(E2), ….,
CF(En)] x CF(rule)
3. Kombinasi dua buah rule dengan evidence
berbeda (E1 dan E2), hipotesis sama. IF E1 THEN H Rule 1
CF(H,E1) = CF1 = (E1)
CF(Rule1)
IF E2 THEN H Rule 2
CF(H,E2) = CF2 ൌ CሺE2ሻ
CFሺRule2ሻ Di mana: IF E1…En
: Fakta-fakta (evidence) yang ada H
: Hipotesis atau konklusi yang
dihasilkan CF Rule
: Tingkat keyakinan terjadinya
hipotesis H akibat adanya fakta CF Rule
: Tingkat keyakinan terjadinya
hipotesis H akibat adanya fakta 1. Rule evidence E tunggal dan hipotesis H tunggal
IF E THEN H (CF rule)
CF(H,E) = CF(E) × (CF rule) 1. Rule evidence E tunggal dan hipotesis H tunggal
IF E THEN H (CF rule)
CF(H,E) = CF(E) × (CF rule) 2. Rule evidence E ganda dan hipotesis H tunggal
IF E1 AND E2….AND En THEN H (CF rule)
CF(H,E) = min[CF(E1), CF(E2), ….,
CF(En)] x CF(rule)
IF E1 OR E2….OR En THEN H (CF rule)
CF(H,E) = max[CF(E1), CF(E2), ….,
CF(En)] x CF(rule) User dapat mengisi kuisioner resiko sebelum
user memilih gejala sesuai dengan yang dialaminya
dengan menjawab pertanyaan umum mengenai
resiko kejadian Tuberkulosis untuk mengetahui
besarnya resiko user untuk terserang penyakit
Tuberkulosis dengan melihat faktor resiko yang ada
pada user. Jurnal Informatika Mulawarman
ISSN 1858-4853 59 b. Faktor Resiko Kejadian Tuberkulosis Berdasarkan literatur dan wawancara dengan
Pakar Tuberkulosis yaitu Bapak Romi Hendra,
S.K.M. bahwa seseorang dikatakan memiliki
kemungkinan resiko untuk terserang Tuberkulosis
apabila memenuhi faktor resiko paling kurang satu
dari faktor berikut: Berdasarkan literatur dan wawancara dengan
Pakar Tuberkulosis yaitu Bapak Romi Hendra,
S.K.M. bahwa seseorang dikatakan memiliki
kemungkinan resiko untuk terserang Tuberkulosis
apabila memenuhi faktor resiko paling kurang satu
dari faktor berikut: Atau Sistem akan mengecek jawaban user
tersebut apakah sesuai dengan aturan faktor resiko
dan memberikan hasil user tersebut beresiko untuk
terserang penyakit atau tidak. Jika user beresiko
terserang penyakit maka sistem akan menyarankan
user untuk melakukan konsultasi dengan memilih
gejala sesuai yang dialaminya, jika tidak beresiko
maka sistem akan memberikan kebebasan kepada
user untuk tetap lanjut berkonsultasi atau tidak. Sistem
juga
memberikan
informasi penyakit
mengenai jenis penyakit Tuberkulosis sehingga
sistem ini juga berfungsi layaknya sistem informasi. Data
mengenai
gejala
penyakit
dan
nilai
keyakinannya diperoleh dari seorang pakar yaitu
Bapak Eko Haryanto, A.md.Kep dalam hal ini
adalah Koordinator Tuberkulosis, Kusta dan
Malaria pada Puskesmas Lempake Samarinda
untuk mendukung pembuatan sistem pakar ini
sehingga sistem dapat bekerja layaknya seorang
pakar dan dapat menjadi alternatif pendiagnosaan
penyakit yang lebih hemat dan efisien. 3. Kombinasi dua buah rule dengan evidence
berbeda (E1 dan E2), hipotesis sama. IF E1 THEN H Rule 1
CF(H,E1) = CF1 = (E1)
CF(Rule1)
IF E2 THEN H Rule 2
CF(H,E2) = CF2 ൌ CሺE2ሻ
CFሺRule2ሻ g. Penelitian Pemenuhan konsep sistem pakar dengan
basis pengetahuan dilakukan dengan pengumpulan
data
dan
informasi
terkait
jenis
penyakit
Tuberkulosis Ekstra Paru, dengan studi pustaka dan
konsultasi dengan pakar Tuberkulosis. Basis data
dilakukan
dengan
analisis
dan
perancangan
menggunakan Unified Modelling Language (UML). Adapun konsep inference engine dilakukan dengan
penggunaan
production
rule
(if..then)
mekanismenya melalui forward chaining serta
penilaian bobot menggunakan nilai CF dan
intrepretasinya. Adapun konsep user interace dan
dialog dikembangkan dengan pembuatan antarmuka
yang user friendly bagi kemudahan dalam pengisian
data dan fakta. Keluaran yang disajikan berupa
informasi diagnosa penyakit Tuberkulosis dan nilai
kemungkinan
penyakit
yang
didiagnosis
menyerang, sedangkan uji validitas hasil diagnosis
dikomparasi dengan data pakar. IF E1 AND E2….AND En THEN H (CF rule)
Atau
IF E1 OR E2….OR En THEN H (CF rule) metode Scoring. Dengan demikian, pengguna yang
dapat berkonsultasi menggunakan sistem pakar ini
adalah pengguna yang berusia lebih dari 12 tahun. f. Metode Certainty Factor e) Membesarnya benjolan kelenjar yang
mengakibatkan
hari
demi
hari
kondisinya semakin memburuk dan
merusak tubuh. Seorang pakar sering kali menganalisis
informasi yang ada dengan ungkapan seperti
“mungkin”, “kemungkinan besar”, “hampir pasti”,
untuk mengakomodasi hal ini kita menggunakan
Certainty Factor (CF) guna menggambarkan
tingkat keyakinan pakar terhadap masalah yang
sedang terjadi [14]. Faktor Kepastian (Certainty
Factor) menyatakan kepercayaan dalam sebuah
kejadian (fakta atau hipotesis) berdasarkan bukti
atau penilaian pakar [15]. Salah satu cara dalam
mendapatkan tingkat keyakinan (CF) dari sebuah
rule adalah dengan cara mewawancarai seorang
pakar yaitu nilai CF(Rule) didapat dari interpretasi
“term” pakar yang diubah menjadi nilai CF tertentu
sesuai tabel 1 berikut [14]. f) Benjolan
kelenjar
pecah
dan
mengeluarkan cairan seperti nanah
kotor. g) Terdapat luka pada jaringan kulit atau
kulit
yang
disebabkan
pecahnya
benjolan kelenjar getah bening. b. Tuberkulosis Payudara b. Tuberkulosis Payudara
TB
Payudara
adalah
penyakit
radang
payudara yang disebabkan oleh infeksi
kuman Mycobacterium Tuberculosis. 1) Gejala sistemik/umum: 1) Gejala sistemik/umum: 1) Gejala sistemik/umum: a) Batuk terus-menerus dan berdahak
selama tiga minggu/lebih. Tabel 1. Nilai CF dan Interpretasi
Uncertain Term
CF
Definitely not (pasti tidak)
- 1.0
Almost certainly not (hampir pasti tidak)
- 0.8
Probably not (kemungkinan besar tidak)
- 0.6
Maybe not (mungkin tidak)
- 0.4
Unknown (tidak tahu)
- 02 to 0.2
Maybe (mungkin)
0.4
Probably (kemungkinan besar)
0.6
Almost certainly (hampir pasti)
0.8
Definitely (pasti)
1.0
(Sumber: T.Sutejo dkk, 2011) Tabel 1. Nilai CF dan Interpretasi
Uncertain Term
CF
Definitely not (pasti tidak)
- 1.0
Almost certainly not (hampir pasti tidak)
- 0.8
Probably not (kemungkinan besar tidak)
- 0.6
Maybe not (mungkin tidak)
- 0.4
Unknown (tidak tahu)
- 02 to 0.2
Maybe (mungkin)
0.4
Probably (kemungkinan besar)
0.6
Almost certainly (hampir pasti)
0.8
Definitely (pasti)
1.0
(Sumber: T.Sutejo dkk, 2011) b) Demam selama tiga minggu/lebih c) Penurunan nafsu makan. c) Penurunan nafsu makan. d) Berat badan turun. d) Berat badan turun. e) Rasa kurang enak badan (malaise),
lemah. f) Berkeringat di malam hari walaupun
tidak melakukan apa-apa. 2) Gejala Khusus a) Timbulnya benjolan di payudara. b) Rasa nyeri di bagian payudara. c) Adanya tanda radang di sekitar
benjolan yang timbul di payudara. Secara umum, rule direpresentasikan dalam
bentuk sebagai berikut [14] : Vol. 12, No. 1, Februari 2017
59 Jurnal Informatika Mulawarman
ISSN 1858-4853 hari di dalam rumah
5) Tinggal di lingkungan yang kumuh
6) Minum Alkohol
7) Perokok dan mantan perokok
8) Mengalami gizi buruk atau malnutrisi
9) Menderita HIV/AIDS
10) Menderita Diabetes
11) Tinggal serumah dengan penderita TB Paru
12) Tinggal serumah dengan lebih dari satu
penderita TB Paru
13) Adanya kontak dengan penderita TB Paru di
luar rumah secara intensif seperti tempat kerja,
sekolah, dll. gejala. Masing-masing gejala direpresentasikan
dengan kode seperti digambarkan pada tabel 2. Tabel 2. Daftar Nilai CF Gejala Tuberkulosis
Kode
Gejala
Nama Gejala
Nilai
CF
G01
Batuk terus-menerus dan berdahak
selama tiga minggu/lebih
0.8
G02
Dahak bercampur darah/batuk darah
0.6
G03
Demam yang berlangsung lama
0.6
G04
Sesak nafas dan nyeri dada
0.6
G05
Penurunan nafsu makan
0.8
G06
Penurunan berat badan
0.8
G07
Rasa kurang enak badan/malaise, lemah
0.8
G08
Berkeringat di malam hari walaupun
tidak melakukan apa-apa
0.6
G01a
Batuk terus-menerus dan berdahak
selama tiga minggu/lebih
0.4
G01b
Batuk terus-menerus dan berdahak
selama tiga minggu/lebih
0.4
G01c
Batuk terus-menerus dan berdahak
selama tiga minggu/lebih
-0.6
G03a
Demam yang berlangsung lama
0.4
G03b
Demam yang berlangsung lama
0.6
G03c
Demam yang berlangsung lama
-0.4
G05a
Penurunan nafsu makan
0.6
G05b
Penurunan nafsu makan
0.4
G05c
Penurunan nafsu makan
0.6
G06a
Penurunan berat badan
0.6
G06b
Penurunan berat badan
0.4
G06c
Penurunan berat badan
0.8
G07a
Rasa kurang enak badan/malaise, lemah
1.0
G07b
Rasa kurang enak badan/malaise, lemah
1.0
G07c
Rasa kurang enak badan/malaise, lemah
1.0
G08a
Berkeringat di malam hari walaupun
tidak melakukan apa-apa
-0.4
G08b
Berkeringat di malam hari walaupun
tidak melakukan apa-apa
-0.4
G08c
Berkeringat di malam hari walaupun
tidak melakukan apa-apa
0.4
G09
Munculnya benjolan-benjolan yang
terjadi pada bagian yang mengalami
gangguan kelenjar seperti leher, sela
paha, serta ketiak. 1.0
G10
Ada tanda-tanda radang di daerah
sekitar benjolan kelenjar
0.8
G11
Benjolan kelenjar mudah digerakkan
0.8
G12
Benjolan kelenjar timbul terasa kenyal
0.8
G13
Membesarnya benjolan kelenjar yang
mengakibatkan hari demi hari
kondisinya semakin memburuk dan
merusak tubuh. 1.0
G14
Benjolan kelenjar pecah, mengeluarkan
cairan seperti nanah yang kotor. 0.8
G15
Terdapat luka pada jaringan kulit atau
kulit yang disebabkan pecahnya
benjolan kelenjar getah bening
1.0
G16
Timbulnya benjolan di payudara. 1.0
G17
Rasa nyeri di bagian payudara. 0.8
G18
Adanya tanda radang di sekitar benjolan
yang timbul di payudara. 0.8
G19
Rasa nyeri/sakit pada bagian punggung
atau mengalami kekakuan punggung. 1.0
G20
Penderita enggan menggerakkan
punggungnya. Jurnal Informatika Mulawarman
ISSN 1858-4853 1.0
G21
Penderita menolak untuk membungkuk
atau mengangkat barang dari lantai, bila
diminta penderita akan menekuk
lututunya agar punggung tetap lurus. 1.0
G22
Rasa nyeri/sakit pada punggung
berkurang bila penderita beristirahat. 1.0
G23
Timbulnya benjolan di bagian
punggung/tulang belakang
0.6 6) Minum Alkohol 7) Perokok dan mantan perokok 8) Mengalami gizi buruk atau malnutrisi 11) Tinggal serumah dengan penderita TB Paru 13) Adanya kontak dengan penderita TB Paru di
luar rumah secara intensif seperti tempat kerja,
sekolah, dll. c. Perancangan Sistem Perancangan sistem pakar diagnosa awal
penyakit
Tuberkulosis
yang
dikembangkan
menggunakan
menggunakan
diagram
unified
modeling
language
(UML),
diagram
yang
digunakan yaitu use case diagram dan activity
diagram. Usecase diagram merupakan suatu
langkah penting yang harus dilakukan dalam
menyelesaikan suatu masalah. Berisi alur untuk
membantu bagaimana suatu sistem dapat berjalan. Gambar 1 merupakan usecase diagram sistem pakar
diagnosa awal penyakit Tuberkulosis. Gambar 1 merupakan usecase diagram sistem pakar
diagnosa awal penyakit Tuberkulosis. Gambar 1. Usecase Diagram Sistem Gambar 1. Usecase Diagram Sistem a. Deskripsi Sistem 1) Rumah tempat tinggal tidak tersedia atau tidak
memiliki ventilasi Sistem
pakar
pendiagnosa
penyakit
Tuberkulosis Paru dan Ekstra Paru ini tidak
digunakan
untuk
mendiagnosa
penyakit
Tuberkulosis pada anak-anak yaitu anak berusia 0-
12 tahun. Pendiagnosaan penyakit Tuberkulosis
Paru Dan Ekstra Paru pada anak-anak memiliki
metode tersendiri yaitu dengan menggunakan 2) Jendela rumah tidak dibuka setiap hari atau
tidak memiliki jendela rumah j
3) Keadaan rumah pengap dan lembab 4) Sinar matahari yang masuk ke dalam rumah
tidak cukup yang ditandai dengan tidak adanya
cahaya terang dari sinar matahari pada siang Vol. 12, No. 1, Februari 2017 60 d. Perancangan Basisdata Fakta dan pengetahuan yang berhubungan
dengan gejala-gejala Tuberkulosis Paru dan Ekstra
Paru akan digunakan dalam mengambil suatu
kesimpulan dalam pembuatan sistem pakar. Fakta
dan pengetahuan tersebut didapatkan dari hasil
studi literatur dan wawancara. Setiap jenis penyakit
Tuberkulosis pasti memiliki gejala-gejala yang
dapat diindentifikasi berdasarkan pengetahuan dari
pakar. Fakta
dan
pengetahuan
yang
telah
didapatkan akan diterjemahkan knowledge engineer
menjadi basis pengetahuan yang tersimpan dalam
sistem pakar yang telah dibuat. Fakta tersebut
ditampilkan dalam bentuk tabel gejala dan bobot Vol. 12, No. 1, Februari 2017
61 Jurnal Informatika Mulawarman
ISSN 1858-4853 2. Menu Login User Menu
Login
User
digunakan
untuk
menampilkan halaman login user. Halaman login
user berfungsi sebagai pintu masuk yang digunakan
user untuk dapat masuk ke dalam akun user. Setiap
user yang ingin melakukan konsultasi pada sistem
ini diharuskan untuk memiliki akun terlebih dahulu
agar data dan privasi user dapat terjaga. Sistem
Pak Sistem
Pakar
diagnosa
penyakit
Tuberkulosis memiliki beberapa bagian yang selalu
tampil pada setiap halaman web, yaitu bagian
header, menu samping, dan footer. Gambar 3. Halaman Dashbord User User dapat menggunakan widget Kuisioner
Resiko yang terdapat pada halaman beranda user
untuk
mengisi
kuisioner. Kuisioner
Resiko
berfungsi sebagai pemberi informasi seberapa besar
resiko user untuk terserang penyakit Tuberkulosis. Kuisioner Resiko merupakan kuisioner yang berisi
pertanyaan mengenai beberapa faktor
resiko
kejadian Tuberkulosis. Pertanyaan yang diajukan
kepada user terdiri dari 13 pertanyaan faktor resiko
Tuberkulosis dan disediakan dua pilihan jawaban
“ya”, “mungkin”, dan “tidak”. Tampilan Halaman
Kuisioner Resiko dapat dilihat pada gambar 4. 1. Halaman Utama Website Halaman utama website memiliki menu utama
yang terletak di bagian kiri yang terdiri dari menu
Login, menu Bantuan, dan menu About Me. Tampilan Halaman Utama Website dapat dilihat
pada gambar 2. Gambar 2. Halaman Utama atau Home Gambar 4. Halaman Kuisioner Resiko
User harus menjawab semua pertanyaan
Kuisioner Resiko untuk mendapatkan hasil resiko
Tuberkulosis yang dimiliki. Setelah user mengisi Gambar 2. Halaman Utama atau Home Jurnal Informatika Mulawarman
ISSN 1858-4853 61 Gambar 3. Halaman Dashbord User Setiap
jenis
penyakit
Tuberkulosis
direpresentasikan
dengan
kode
seperti
yang
tercantum pada tabel 3 berikut: Tabel 3. Daftar Jenis Penyakit Tuberkulosis. Kode
Penyakit
Nama Penyakit
P01
Tuberkulosis Paru
P02
Tuberkulosis Kelenjar Getah Bening
P03
Tuberkulosis Payudara
P04
Tuberkulosis Tulang Belakang 3. Halaman Dashboard User Halaman utama user atau Dashboard adalah
halaman utama/halaman beranda yang pertama kali
tampil ketika user berhasil login ke dalam akun user. User harus melakukan login dulu sebelum halaman
utama user tampil. Halaman utama user berisi
beberapa widget yaitu kuisioner resiko, konsultasi,
informasi penyakit & faktor resiko, dan rekam medis
yang dapat digunakan user secara langsung selain
menggunakan menu utama. Tampilan Halaman
Utama User dapat dilihat pada gambar 3. Gambar 4. Halaman Kuisioner Resiko User harus menjawab semua pertanyaan
Kuisioner Resiko untuk mendapatkan hasil resiko
Tuberkulosis yang dimiliki. Setelah user mengisi User harus menjawab semua pertanyaan
Kuisioner Resiko untuk mendapatkan hasil resiko
Tuberkulosis yang dimiliki. Setelah user mengisi Jurnal Informatika Mulawarman
ISSN 1858-4853
Gambar 6. Form Konsultasi
2) User menekan tombol submit setelah memilih
gejala yang dialami kemudian sistem akan
menampilkan
hasil
diagnosa kemungkinan
penyakit
pada
halaman
detail
diagnosa. Tampilan Detail Diagnosa dapat dilihat pada
gambar 7. Gambar 7. Halaman Detail Diagnosa
User dapat melihat gejala yang telah diinputkan Jurnal Informatika Mulawarman
ISSN 1858-4853
Gambar 6. Form Konsultasi Vol. 12, No. 1, Februari 2017 62 kuisioner resiko kejadian Tuberkulosis maka
halaman hasil Kuisioner Resiko akan tampil. Halaman hasil Kuisioner Resiko berisi hasil apakah
user memiliki kemungkinan resiko untuk terserang
penyakit Tuberkulosis atau tidak, daftar faktor
resiko yang dimiliki user berdasarkan jawaban yang
telah dipilih user untuk setiap pertanyaan resiko. Tampilan Halaman Hasil Kuisioner Resiko dapat
dilihat pada gambar 5. Gambar 5. Halaman Hasil Kuisioner Resiko Gambar 5. Halaman Hasil Kuisioner Resiko Gambar 6. Form Konsultasi Gambar 6. Form Konsultasi 4. KESIMPULAN [11] Misnadiarly. 2006. Diabetes
Melitus
Gangren, Ulcer, Infeksi, Mengenali gejala,
Menanggulangi,
dan
Mencegah
komplikasi. Jakarta: Pustaka Obor Populer. Berdasarkan
hasil
penelitian
mengenai
sistem pakar pendiagnosa penyakit Tuberkulosis
Paru dan Ekstra Paru dengan menggunakan metode Berdasarkan
hasil
penelitian
mengenai
sistem pakar pendiagnosa penyakit Tuberkulosis
Paru dan Ekstra Paru dengan menggunakan metode
Certainty Factor, dapat diambil kesimpulan: Certainty Factor, dapat diambil kesimpulan: [12] Sari, W.W. 2013. “Perancangan Sistem
Pakar untuk Mengetahui Infertilitas Pada
Perempuan
Menggunakan
Metode
Certainty Factor”. Pelita Informatika Budi
Darma, Vol. V Nomor 1. November 2013. 1. Telah dihasilkan sebuah sistem pakar untuk
diagnosa awal penyakit Tuberkulosis Paru dan
Ekstra Paru pada orang dewasa (usia lebih dari
12 tahun) berbasis web yang dibangun dengan
menggunakan
metode
inferensi
forward
chaining dan teori Certainty Factor (CF) serta
menggunakan bahasa pemrograman PHP dan
database MySQL. [13] Sutejo, T., Mulyanto, E., dan Suhartono,
V. 2011. Kecerdasan Buatan. Yogyakarta:
Andi. [14] Turban, 2005. Decision Support System,
Intelligent System. Jilid II. Edisi 7. Yogyakarta. Percetakan
Andi
Offset. Penerbit Andi. 2. Sistem pakar ini bekerja berdasarkan gejala
yang dipilih user kemudian diproses oleh
sistem sehingga menghasilkan output yaitu
nama penyakit, presentase keyakinan diagnosa
dan informasi penanganan penyakit. [15] WHO. 2013. WHO Report 2013-Global
Tuberculosis
Control. www.who.int/tb/data. diunduh
pada
tanggal 05 April 2016. 3. Sistem
Pakar
Pendiagnosa
Penyakit
Tuberkulosis
mempunyai
tingkat
akurasi
diagnosa penyakit sebesar 85%. Jurnal Informatika Mulawarman
ISSN 1858-4853 [2] Depkes RI, Ditjen PP dan PL. 2008. Pedoman
Nasional
Penanggulangan
Tuberkulosis dan Standar Internasional
Untuk
Pelayanan
Tuberkulosis. Departemen
Kesehatan
Republik
Indonesia. nilai kemungkinan dan info penanganannya pada
halaman detail diagnosa. User dapat melihat info
penanganan penyakit dengan menekan tombol “info
penanganan” pada halaman detail diagnosa. Di
bawah ini gambar info penanganan salah satu
diagnosa penyakit yaitu Tuberkulosis TB Kelenjar
Getah Bening. Tampilan Informasi Penanganan
dapat dilihat pada gambar 8. [3] Hiswani 2008. “Tuberkulosis Merupakan
Penyakit Infeksi yang Masih Menjadi
Masalah Kesehatan Masyarakat”. Skripsi. Universitas Sumatera Utara. Gambar 8. Halaman Informasi Penanganan TB
Kelenjar Getah Bening [4] Kadir, Abdul. 2008. Dasar Pemrograman
Web Dinamis Menggunakan PHP – Edisi
Revisi, ANDI, Yogyakarta. [5] Kementrian Kesehatan RI. 2011. Pedoman
Nasional Penanggulangan Tuberkulosis. Jakarta: Kementrian Kesehatan RI. [6] Kementrian Kesehatan RI. 2014. Pedoman
Nasional
Pengendalian
Tuberkulosis. Jakarta: Kementrian Kesehatan RI. 2014. [7] Kusrini. 2008. Aplikasi Sistem Pakar. Yogyakarta: Andi Offset. [8] Madcoms. 2011. Adobe Dreamweaver CS5
dengan Pemrograman PHP & MySQL. Yogyakarta : Penerbit Andi. [9] Mahdiana, R. 2010. Panduan Lengkap
Kesehatan
Mengenal,
Mencegah
dan
Mengobati
Penularan
Penyakit
dari
Infeksi. Yogyakarta: Citra Pustaka. Gambar 8. Halaman Informasi Penanganan TB
Kelenjar Getah Bening [10] Martin, J. Dan Oxman, S. 1988. Building
Expert Systems a Tutorial. New Jersey:
Prentice Hall. a.
Menu Konsultasi Menu
konsultasi
adalah
menu
yang
menampilkan halaman Konsultasi. User dapat
memulai konsultasi pada halaman ini dengan
memilih gejala-gejala yang ada sesuai yang dialami
oleh user. Inputan gejala user akan menjadi
masukan sistem pakar untuk diproses. Sistem akan
mengecek apakah inputan gejala-gejala dari user
terdapat rule yang sesuai atau tidak. Jika inputan
gejala user sesuai dengan rule penyakit yang ada
maka akan dilakukan proses perhitungan dengan
menggunakan
metode
Certainty
Factor
dan
menghasilkan hasil diagnosa kemungkinan penyakit
TB Paru dan Ekstra Paru beserta persentasi nilai
kemungkinannya. Jika tidak sesuai dengan rule
penyakit yang ada maka sistem akan memberikan
hasil output bahwa user tidak terdiagnosa terkena
penyakit TB. Di bawah ini langkah-langkah yang
dapat dilakukan user untuk melakukan konsultasi: Menu
konsultasi
adalah
menu
yang
menampilkan halaman Konsultasi. User dapat
memulai konsultasi pada halaman ini dengan
memilih gejala-gejala yang ada sesuai yang dialami
oleh user. Inputan gejala user akan menjadi
masukan sistem pakar untuk diproses. Sistem akan
mengecek apakah inputan gejala-gejala dari user
terdapat rule yang sesuai atau tidak. Jika inputan
gejala user sesuai dengan rule penyakit yang ada
maka akan dilakukan proses perhitungan dengan
menggunakan
metode
Certainty
Factor
dan
menghasilkan hasil diagnosa kemungkinan penyakit
TB Paru dan Ekstra Paru beserta persentasi nilai
kemungkinannya. Jika tidak sesuai dengan rule
penyakit yang ada maka sistem akan memberikan
hasil output bahwa user tidak terdiagnosa terkena
penyakit TB. Di bawah ini langkah-langkah yang
dapat dilakukan user untuk melakukan konsultasi: 2) User menekan tombol submit setelah memilih
gejala yang dialami kemudian sistem akan
menampilkan
hasil
diagnosa kemungkinan
penyakit
pada
halaman
detail
diagnosa. Tampilan Detail Diagnosa dapat dilihat pada
gambar 7. g
Gambar 7. Halaman Detail Diagnosa
User dapat melihat gejala yang telah diinputkan
dan kemungkinan penyakit TB yang terkena beserta 1) User memilih gejala penyakit Tuberkulosis
yang dialami. Tampilan Form Konsultasi dapat
dilihat pada gambar 6. Gambar 7. Halaman Detail Diagnosa User dapat melihat gejala yang telah diinputkan
dan kemungkinan penyakit TB yang terkena beserta User dapat melihat gejala yang telah diinputkan
dan kemungkinan penyakit TB yang terkena beserta Vol. 12, No. 1, Februari 2017
63 5. DAFTAR PUSTAKA [1]. Alfaris, S. 2014. “Sistem Pakar untuk
Mendiagnosa Penyakit Polip Nasi (Polip
Hidung) Menggunakan Metode Certainty
Factor”. Pelita Informatika Budi Darma, Vol. VII Nomor 2. Agustus 2014. [2]. Arhami, M. 2005. Konsep Dasar Sistem
Pakar. Yogyakarta: Andi.
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The Values of Inclusive Education as a Basis for Building Students Character of Mambaus Sholihin Islamic Boarding School Gresik Indonesia
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I. INTRODUCTION The issue of inclusive religion recently becomes a central issue in developing theology. This issue appears because of the
more the escape awareness about plurality that requires multi-ethnic and multi-religious growth in a diverse society. At the moment,
tolerance of ethnicity and religion in Indonesia is an important agenda since rampant violence in ethnicity, religion, and incessant
cases of terror are spread in the name of religion. The presence of Islamic boarding schools in the middle Public is not only as an
institution of education, but also as an institution of religious and social broadcasting religious. A survey from the Wahid Institute in 2020 showed that intolerance in Indonesia increased from 46 % to 54 %. The Setara
Institute report also found that throughout 2020 there had been 180 incidents of violations of freedom of religion and belief (KBB)
in Indonesia. Husni Mubarok (Jakarta: wahidfoundation.org, 2021), 02, http://wahidfoundation.org//source/eBook - Guide to
Writing a Narrative.pdf. Incidents of violence on the basis of intolerance or extremism are not impossible if they are not addressed
from the level of forming a counter-extremism narrative. In the life of the state, the narrative of intolerance and violent extremism
is contrary to a number of laws and regulations. Such as the 1945 Constitution Article 28B paragraph 2 and Article 31, the Criminal
Code Articles 156, 157, Law Number 40 of 2008 concerning the Elimination of Racial and Ethnic Discrimination Article 4 (Ahmad
Gaddafi and Kalis Mardiasih, 2021). The emergence of the practice of intolerance among young Indonesians is influenced by
psychological factors, social and environmental factors, domestic and international political conditions, understanding of religious
texts as limited to textually, the loss of role models so that they look for new charismatic figures Faiz Yunus, "Radicalism,
Liberalism and Terrorism: Their Effects" Against Islam,” Journal of the Study of the Qur'an 13, no. I (2017): 76–94,
http://journal.unj.ac.id/unj/index.php/jsq/article/view/3217/2818. Educational institutions as a place to study are actually a safe
space in rejecting and recruiting young people into extremist organizations. Educational institutions such as Islamic boarding schools
must teach tolerant character education to students and introduce them to the Islamic values of rahmatan lil alamin. In Indonesia, an Islamic boarding school (Pondok Pesantren) is one of the oldest Islamic education systems which has a
special characteristic, unique, and have a root charismatic tradition. 4176 *Corresponding Author: M. Athoiful Fanan The Values of Inclusive Education as a Basis for Building Students
Character of Mambaus Sholihin Islamic Boarding School Gresik Indonesia M. Athoiful Fanan1, Maskuri2, M. Mas’ud Said3
1 STAI An Najah Indonesia Mandiri Sidoarjo, East Java, Indonesia
2,3 University of Islam Malang, East Java, Indonesia M. Athoiful Fanan1, Maskuri2, M. Mas’ud Said3
1 STAI An Najah Indonesia Mandiri Sidoarjo, East Java, Indonesia
2,3 University of Islam Malang, East Java, Indonesia ABSTRACT: This study discusses the values of inclusive Education as a base for building students' characters at the Islamic
boarding school of Mamba'us Sholikhin (PPMBS) Gresik Indonesia. The research paradigm of this study use phenomenology and
qualitative approach, also using a case study. The data collection techniques use three types of strategies, namely interviews,
observation, and documentation. This study found the conclusion that building the values of inclusive education as the basis for
building the character of students was carried out by applying the value of tasamuh (tolerance), Al-Infitah (openness), tanawue
(diversity) and the value of Islam Rahmatan lil Alamin (peaceful Islam). KEYWORDS: Inclusive Education, Islamic Boarding School, Students Characters. KEYWORDS: Inclusive Education, Islamic Boarding School, Students Characters. International Journal of Current Science Research and Review
ISSN: 2581-8341
Volume 05 Issue 11 November 2022
DOI: 10.47191/ijcsrr/V5-i11-13, Impact Factor: 5.995
IJCSRR @ 2022
www.ijcsrr.o www.ijcsrr.org International Journal of Current Science Research and Review It can be seen that the interaction of learning in PPMBS still uses their vernacular language like
Javanese to keep the existing local language. The presence of PPMBS in the middle of society is not only as an institution of
education, but it is also a religious institution and become social broadcasting religion. The existence of PPMBS as a public institution since the beginning had been capable to accommodate various types of change, both
on the side of education structural and systematic learning. This matter is going to become very important whether the existence of
Islamic boarding schools provides any contribution of inclusiveness and multicultural in the world of Islam, this can be measured
by a lot of students who learn at PPMBS. In addition to the interesting inclusive value (openness ) of boarding schools is an education
Vision that does not recognize dichotomy and management model the institution use that combines Among diniyah with formal
education: include Madrasah Aliyah (Islamic Senior High Level), Madrasah Tsanawiyah (Islamic Junior High Level), Madrasah
Ibtidaiyah (Islamic Elementary School), University. The existence of PPMBS as a public institution since the beginning had been capable to accommodate various types of change, both
on the side of education structural and systematic learning. This matter is going to become very important whether the existence of
Islamic boarding schools provides any contribution of inclusiveness and multicultural in the world of Islam, this can be measured
by a lot of students who learn at PPMBS. In addition to the interesting inclusive value (openness ) of boarding schools is an education
Vision that does not recognize dichotomy and management model the institution use that combines Among diniyah with formal
education: include Madrasah Aliyah (Islamic Senior High Level), Madrasah Tsanawiyah (Islamic Junior High Level), Madrasah
Ibtidaiyah (Islamic Elementary School), University. The reason why PPMBS as this research site is because it has its own uniqueness. One of the uniqueness is PPMBS has
inclusive education characteristics. That is, open to all groups, with no discrimination from various types of ethnic groups. Even
almost students from many provinces in Indonesia exist at PPMBS. The inclusive characteristics of PPMBS also invite several
students who come from a foreign countries. That was not easy to blend between local culture and foreign culture. 4177 *Corresponding Author: M. Athoiful Fanan International Journal of Current Science Research and Review This learning
model must get special attention in this research on how PPMBS has a curriculum for preparation to be accepted by all students. In addition to this uniqueness, PPMBS has its own uniqueness and characteristics. The other unique characteristic of
PPMBS is PPMBS has nine branches spread across Indonesia. Every branch of PPMBS is led by alumni. Some of these focus on
the spread of Islam in their respective regions. Such as a branch of PPMBS in Masohi area (Maluku) which is located in Muslim
and Christian settlements which has a potential conflict. Meanwhile, the other branch of PPMBS in Benjeng Gresik area east java
was established with its main function to counteract a peaceful Christianity in the area which has been intensively carried out by
missionaries since 2004. In addition to this uniqueness, in terms of learning uniforms, PPMBS has also unique characteristics in
terms of wearing robes and veils that are not like Indonesian culture students. International Journal of Current Science Research and Review w
www.ijcsrr.org DOI: 10.47191/ijcsrr/V5-i11-13, Impact Factor: 5.995
IJCSRR @ 2022 DOI: 10.47191/ijcsrr/V5-i11-13, Impact Factor: 5.995 Roudlatul Muataalimin Islamic boarding school. Gontor Islamic boarding school is the model of PPMBS in terms of Mastery of
Arabic and English as everyday languages. PPMBS also adopted a social organization system as applied at Gontor Islamic boarding
school. Langitan Islamic Boarding School as the centre of PPMBS in terms of its Salafiyah curriculum. In terms of worship, PPMBS
is oriented to the Roudhotul Muta'allimin Islamic Boarding School Sawahpolo Surabaya Indonesia. The levels of formal education of PPMBS include Madrasah Aliyah (Islamic Senior High Level), Madrasah Tsanawiyah
(Islamic Junior High Level), Madrasah Ibtidaiyah (Islamic Elementary School), Roudlotul Athfal (Playgroup). While non-formal
education is Madrasah Diniyah (Religious school) which consists of the Ula level (secondary level) and Wustha (higher level). Based on information from one of the administrators of PPMBS, alumni, he said that the students at this Islamic boarding school
almost reached 4 000-5000 students who all came from almost all regions in Indonesia, even many alumni who continued their
studies in Sudan, Yemen, and Egypt. All of the alumni of PPMBS Suci Gresik are quite friendly and compact. This can be seen
from the alumni association which they call HIMAM (Students Association of PPMBS) which is active in carrying out routine
activities. The management of this organization is spread out in every region with a routine agenda that has been scheduled. Even
once a boarding school caregiver was invited to give tausiyah (preach) to the surrounding community. As background reason for this research is the diversity of PPMBS students. The students' diverse background behind
traditions, language and culture does not decrease the integration process between students with local cultures in an education
environment boarding school. It can be seen that the interaction of learning in PPMBS still uses their vernacular language like
Javanese to keep the existing local language. The presence of PPMBS in the middle of society is not only as an institution of
education, but it is also a religious institution and become social broadcasting religion. As background reason for this research is the diversity of PPMBS students. The students diverse background behind
traditions, language and culture does not decrease the integration process between students with local cultures in an education
environment boarding school. I. INTRODUCTION His ability to be a guard of primordial values and self-subsistent
makes this institution become the only institution that can exist as an essential actor in deploying Islamic values in society. Here,
An example of Islamic boarding school with various inclusiveness ideas is the Islamic Boarding school of Mambaus Sholihin
(PPMBS) Suci Gresik Indonesia. The privileges curriculum model of PPMBS is the combination of the national curriculum and the Pesantren curriculum. PPMBS uses the national curriculum as an additional instrument to elevate the excellence of Islamic boarding schools which are
conceptualized in a modern salafiyah way. The curriculum developed at PPMBS is a combination of three Islamic Boarding Schools
for daily education. The three Islamic boarding school includes Gontor Islamic boarding school, Langitan Islamic boarding school, 4176 *Corresponding Author: M. Athoiful Fanan Volume 05 Issue 11 November 2022 Available at: ijcsrr.org
Page No.-4176-4183 International Journal of Current Science Research and Review
ISSN: 2581-8341
Volume 05 Issue 11 November 2022
DOI: 10.47191/ijcsrr/V5-i11-13, Impact Factor: 5.995
IJCSRR @ 2022
www.ijcsrr.org III. RESULT AND DISCUSSION Researchers take the meaning of the term value development of inclusive education according to the perspective of
habituation of the mind that is repeated continuously. As stated by Ibrahim Alfikiy. Habits are thoughts that a person creates in his
mind and then associates with feelings continually until those thoughts believe that they are part of his behavior and deserve the
truth. (Elfiky, n.d.) According to Bakrie and Werdiningsih, the method of cultivating values can be done in various ways including
through reading the classical yellow books, good teachers' examples as role models, spiritual practice, habituation lifestyle,
discipline, organization, ibrah (getting meaning of life from ancient people), mauidlah (good advice), scouting, sports, and
kanuragan (metaphysics self-defence). (Bakrie, Maskuri and Werdiningsih, 2017). The meaning of value comes from the latin vale're
which means useful, capable, empowered, and efficient, so that value is defined as something that is considered good, useful and
correct according to the beliefs of a person or group of people (Sutarjo Adisusilo, 2012). Regarding the definition of this value,
various experts have said, namely: W.J.S. Purwadarminta in the General Indonesian Dictionary defines values with the nature of
essentials things for humanity; Muhaimin and Abdul Mujib have a definition of the value of being practical and effective in human
souls and actions as well as institutionalizing objectives in society; Meanwhile, in the view of Sidi Gazalba, as quoted by Chabib
Thoha, defining value as something abstract, ideal, value is not a concrete object, not fact, not just truth. Building the value of
inclusive education can be based on various principles, such as openness, tolerance, Bhinneka Tunggal Ika (unity in diversity) and
Islam as a leader (Ulum, 2017). When applied to a multicultural environment, "inclusion" refers to a mindset that welcomes and
celebrates diversity. (Rohmadi, 2017) Hildegun Olsen stated “inclusive education means that schools should accommodate all
children regardless of physical, intellectual, social-emotional, linguistic or other conditions.. (The Salamanca Statement and
Framework for Action on Special Need Education, para 3) "According to Hildegun Olsen, schools should make reasonable
accommodations for all students regardless of physical, mental, emotional, language or other types of disabilities. (Tarmansyah,
2009) Johnsen and Skjorten in Mudjito claim that inclusive education is a service that ensures all students with special needs are
enrolled and participate fully in educational programs offered at their neighborhood schools. 4178 *Corresponding Author: M. Athoiful Fanan International Journal of Current Science Research and Review International Journal of Current Science Researc
ISSN: 2581-8341
Volume 05 Issue 11 November 2022
DOI: 10.47191/ijcsrr/V5-i11-13, Impact Factor: 5.995
IJCSRR @ 2022 III. RESULT AND DISCUSSION The goal of inclusive education is to
give all children, including those with special needs, access to the same high-quality education available to developing children. An
open and friendly classroom has several features (Mudjito, 2012). Florian agrees, saying the following philosophical concepts support inclusive education: a. Learning and playing with other
children is the right of every child. b. When a child has learning difficulties that is no reason to make fun of them or treat them
differently. c. When kids are at school, there's no good reason to keep them apart. Each child is part of a larger whole, and they must
remain intertwined. (Mudjito, 2012). Florian agrees, saying the following philosophical concepts support inclusive education: a. Learning and playing with other
children is the right of every child. b. When a child has learning difficulties that is no reason to make fun of them or treat them
differently. c. When kids are at school, there's no good reason to keep them apart. Each child is part of a larger whole, and they must
remain intertwined. (Mudjito, 2012). In addition, Mulyono explained the principles of inclusive education as nine important factors that enable its implementation: a. Positive teachers and acceptance of differences, b. Promotive interaction, c. Success in school and in social situations, d. adapt
quickly to new situations, e. having Consultation with groups, f. Learn from the people around the environment, g. a Collaborative
efforts both home and class, h. Develop insight and learn to think independently, i. long life learning. Some definitions of “inclusive
education” boil down to the idea that all students should be able to work and socialize in the same classroom while still being
respected for their unique perspective on how best to achieve their educational goals. (Budiyanto, 2005). In addition, Mulyono explained the principles of inclusive education as nine important factors that enable its implementation: a. Positive teachers and acceptance of differences, b. Promotive interaction, c. Success in school and in social situations, d. adapt
quickly to new situations, e. having Consultation with groups, f. Learn from the people around the environment, g. a Collaborative
efforts both home and class, h. Develop insight and learn to think independently, i. long life learning. III. RESULT AND DISCUSSION Some definitions of “inclusive
education” boil down to the idea that all students should be able to work and socialize in the same classroom while still being
respected for their unique perspective on how best to achieve their educational goals. (Budiyanto, 2005). In addition, Mulyono explained the principles of inclusive education as nine important factors that enable its implementation: a. Positive teachers and acceptance of differences, b. Promotive interaction, c. Success in school and in social situations, d. adapt
quickly to new situations, e. having Consultation with groups, f. Learn from the people around the environment, g. a Collaborative
efforts both home and class, h. Develop insight and learn to think independently, i. long life learning. Some definitions of “inclusive
education” boil down to the idea that all students should be able to work and socialize in the same classroom while still being
respected for their unique perspective on how best to achieve their educational goals. (Budiyanto, 2005). The values contained in inclusive education at the PPMBS include Tasamuh (tolerance), Dynamic, diversity and Islam
Rahmatan lil alamin values. When people adopt an inclusive view (that values of diversity), they are more likely to be inspired to
learn more about those differences and pursue universal aspects of them that can help them in achieving their life goals. Knowing
that diversities are natural to help people in developing a more accepting view of them, so they are less likely to reject them and
more likely to see the potential for universal similarity The following are examples of an inclusive Islamic perspective: The values contained in inclusive education at the PPMBS include Tasamuh (tolerance), Dynamic, diversity and Islam
Rahmatan lil alamin values. When people adopt an inclusive view (that values of diversity), they are more likely to be inspired to
learn more about those differences and pursue universal aspects of them that can help them in achieving their life goals. Knowing
that diversities are natural to help people in developing a more accepting view of them, so they are less likely to reject them and e likely to see the potential for universal similarity. The following are examples of an inclusive Islamic pe 1. The potential for truth exists in every member of society. International Journal of Current Science Research and Review
ISSN: 2581-8341
Volume 05 Issue 11 November 2022
DOI: 10.47191/ijcsrr/V5-i11-13, Impact Factor: 5.995
IJCSRR @ 2022
www.ijcsrr.org International Journal of Current Science Research and Review
ISSN: 2581-8341
Volume 05 Issue 11 November 2022
DOI: 10.47191/ijcsrr/V5-i11-13, Impact Factor: 5.995
IJCSRR @ 2022
www.ijcsrr.org II. METHOD This study uses a paradigm phenomenology with a qualitative approach. The type of this research is a case study. This
approach is used to get a complete data, and a deep and thorough understanding to have more focus on this study. This study is
xpected to uncover phenomena in deep, discover by comprehensive and complete, describe, analyze, and provide interpretation
about inclusive education as a base in building student's character with research focus which is appropriate with findings or thesis
statements. To describe inclusiveness in PPMBS, this research was conducted by relying on case study research. Furthermore, this
type of case study research is carried out to uncover and examine phenomena that are specifically born and developed in a certain
place or region that are sustainable from the past, present and future. 4177 *Corresponding Author: M. Athoiful Fanan Volume 05 Issue 11 November 2022
Available at: ijcsrr.org
Page No.-4176-4183 Volume 05 Issue 11 November 2022
Available at: ijcsrr.org
Page No.-4176-4183 Issue 11 November 2022
Available at: ijcsrr.org
Page No.-4176-4183 A. Tasamuh (Tolerance) The word tolerance comes from the English language "Tolerance" which means to let. In Indonesia, it is defined as the
nature or attitude of tolerance, and silence, moreover (Depdiknas, 2008). Tolerance in Arabic is called "tasamuh" which means
generosity, ease or permit. According to Umar Hasyim, tolerance is interpreted as providing convenience and freedom to fellow
human beings to carry out the rules of life or their beliefs in determining the path of their respective fates (Hasyim, 1979), as long
as carrying out and carrying out these do not violate and do not conflict with the requirements of the norms of society. Muhammad
Yasir, “The Meaning of Tolerance in the Qur'an”, Journal of Ushuluddin Xxii, No. 2 (2014): 171. According to Rainer Forst, the topic is discussed in four different settings: a) majority-minority relations; b) the existence
of social groups with other people; c) actualization of tolerance norms and attitudes of tolerance; and d) tolerance in multicultural
society M Nur Prabowo, "Tolerance: From Historical Experience to Theoretical Construction", In Book Review, Ed. 4. when
meeting the teachers, and not fighting against senior students, as well as respecting and helping each other towards fellow students
at the boarding school. The value of tolerance that exists in PPMBS is used as the basis for religious learning at PPMBS which uses classical
yellow book studies like most modern Salaf (ancient) boarding schools. According to Dhofier's explanation in Anam, this type of
Islamic school is distinguished by its strict adherence to a traditional curriculum based on the study of classical books. While using
the sorogan (students read the classical book by their own self) and bandongan (learning in a group class) or wetonan (students
listen to the teacher read classical yellow books) approach (Anam, 2017) for education. The classics of this period, also known as
the classical yellow book, are simply restatements of repeated reading (qira'ah al-tikrar) of the achievements of earlier scholars. According to Kenneth E. Nollin, this has resulted in a “corpus of conservative traditionalism” in academic writing since then. Later,
it was incorporated into the pesantren curriculum and is still known as the classical yellow book "Kitab Kuning" in Islamic boarding
schools because of the yellow paper that use to write on it. Indonesian Muslim scholars carried out a rigorous selection process based on a Sunni ideological framework to produce
the classical Yellow Book which was accepted by Islamic boarding schools. 4179 *Corresponding Author: M. Athoiful Fanan International Journal of Current Science Research and Review 4. Inclusive attitude in communication. Understanding the differences that exist and developing the habit of communicating
with them can help avoid the harmful effects of one's biased point of view (Hanafi, 2016). 4. Inclusive attitude in communication. Understanding the differences that exist and developing the habit of communicating
with them can help avoid the harmful effects of one's biased point of view (Hanafi, 2016). A. Tasamuh (Tolerance) The only Sunni books or literatures are included in the
classical yellow book, and even then only includes the four sekt (mahdzab). The concept of fiqh followed the Ash'ariyah and
Maturidiyah beliefs, and the Sufism of al-Ghazali, Junaid al-Baghdadi, and Abd al-Qadir al-Jaelani (Hanani, 2017). The sekt
(mahdzab) commentators provided the main source material for the conservative traditionalism accepted by pesantren (Islamic
boarding schools). The fiqh of al-Akbar Abu Hanifah, and al-Umm Imam Shafi'i is among the works discussed, as well as Ibn al-
Fath Qasim's al-Qarib, al-Mahali by al-Qulyubi and Umayrah, al-Wahab Zakaria al- Fath Ansari and Fainudin bin Abdul al-Aziz
al-Malibari'. Likewise in PPMBS after listening to the theory above, researchers present empirical findings in which it turns out that
PPMBS also uses the book of Fath al-Mu'in as a scientific study of Fiqh. The planting of tolerance values contained in PPMBS is
also illustrated by the icon of the pesantren which always voices as a Islamic boarding school (Pesantren) that stands above all
groups. PPMBS teaches its students to think broadly, be patient, and be tolerant when faced with issues that come from western
culture. This PPMBS follows the guidelines set by the Ministry of Religion and Education, which means that along with reading
books, students are taught and required to use English in all aspects of daily life and general science. The kiyai (head of Islamic
boarding school)’s tolerant outlook ensures that ambitious high school students will never be short of opportunities to put their ideas
into action. In addition, the value of tolerance is also taught in providing students with an understanding of religious differences,
because it is important for students to know that excessive religious fanaticism will also have an effect on radical views which will
have implications for religious conflicts. III. RESULT AND DISCUSSION However, to avoid excessive pragmatism towards the superiority
of himself and his group, it is necessary to realize that every member of society also has weaknesses that need the help of
others. 1. The potential for truth exists in every member of society. However, to avoid excessive pragmatism towards the superiority
of himself and his group, it is necessary to realize that every member of society also has weaknesses that need the help of
others. 2. Willingness to be inclusive of members of religious communities who are different from ours. Recognizing that there are
commonalities that can help people of different faiths (schools) work together towards a common goal is critical to
advancing the development of any community. 2. Willingness to be inclusive of members of religious communities who are different from ours. Recognizing that there are
commonalities that can help people of different faiths (schools) work together towards a common goal is critical to
advancing the development of any community. 3. Inclusive attitude in socializing. Promoting good sportsmanship in day-to-day interactions with others helps people learn
to deal with differences in a constructive way and grow as individuals despite their differences in opinion and lifestyle. 3. Inclusive attitude in socializing. Promoting good sportsmanship in day-to-day interactions with others helps people learn
to deal with differences in a constructive way and grow as individuals despite their differences in opinion and lifestyle. Volume 05 Issue 11 November 2022 Volume 05 Issue 11 November 2022 Available at: ijcsrr.org
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DOI: 10.47191/ijcsrr/V5-i11-13, Impact Factor: 5.995
IJCSRR @ 2022
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4. Inclusive attitude in communication. Understanding the differences that exist and developing the habit of communicating
with them can help avoid the harmful effects of one's biased point of view (Hanafi, 2016). International Journal of Current Science Research and Review
ISSN 2581 8341 The
openness of this pesantren is also displayed in the form of facilities available at this Islamic boarding school, such as when
researchers conducted direct observations, it turns out that the means of developing da'wah in reflecting openness is displayed in
the form of online TV and radio facilities that are owned at the PPMBS. Where the content of the online TV and radio of this
pesantren provides information on the importance of pesantren education as a forum for forming students who are tolerant and open
to anyone. To form the ideal ideas for people's lives, Pesantren (Islamic boarding schools) use brilliant minds. Furthermore, the
charisma and character of the kyai (head of Islamic boarding school) in the Islamic boarding school community and the general
public provide social and cultural legitimacy to the Islamic boarding school. This perspective shows that Islamic boarding school
as an incubator for understanding Islam can be successfully applied in Indonesia, a pluralistic, multicultural country, and based on
clear principles (Munir, 2013). Mukti Ali argues somewhat regretfully that Islamic boarding school is a reminder of the importance
of da'wah institutions, not community empowerment institutions, not community economic institutions, but Islamic boarding school,
which are community reproductive institutions with a system of cultural values, mutual cooperation, obedience to kyai (head of
Islamic boarding school), discipline, and religious understanding and deep (Yasir, 2014). Nonetheless, it persists because people
have different ideas about what Islamic boarding school is and because Islamic boarding school plays an important role in
introducing new cultural models to the public. On the one hand, it is true that Islamic boarding school function as a place of learning. The relationship between parents and Islamic boarding schools, on the other hand, can be reflected in the patron-client
model. Therefore, pesantren and the culture they help foster in Indonesia are closely intertwined. There are also competing
interpretations of Islamic teachings within Islamic boarding schools. The openness value of PPMBS is carried out by collaborating
with several Islamic boarding schools, such as the Gontor Islamic Boarding School and the Langitan Islamic Boarding School. The
openness of this pesantren is also displayed in the form of facilities available at this Islamic boarding school, such as when
researchers conducted direct observations, it turns out that the means of developing da'wah in reflecting openness is displayed in
the form of online TV and radio facilities that are owned at the PPMBS. International Journal of Current Science Research and Review
ISSN 2581 8341 Where the content of the online TV and radio of this
pesantren provides information on the importance of pesantren education as a forum for forming students who are tolerant and open
to anyone. In line with the success of several pesantren cooperation programs with external institutions, it is inseparable from
teamwork. Teamwork, as defined by Poernomo, is the coordinated effort of several individuals working towards a common goal. Poernomo argues that there are several factors at play when employees come together to form teams, including the fact that when
more than one person is invested in a task, the effort tends to be more serious. Help each other as you complete agency-mandated
projects. It is very helpful for team members to know and trust each other Imron, “The Influence of Leadership Style, Teamwork,
and Organizational Culture on Work Performance and Its Impact on Employee Performance, ”Journal of Economics and
Management 5, no. 1 (2019): 64–83, http://www.stiepertiba.ac.id/ojs/index.php/jem/article/view/66/62. In addition, the Mambaus Sholihin Islamic boarding school (PPMBS) also seeks to provide activities to support the mastery
of two languages, including holding the Markaz Al Lughoh program (language centre), a program that is engaged in the field of
language under the leadership of the son-in-law of KH. Masbuhin Faqih, namely Dr. H. Mohammad Najib, MA. This two language
course programs is conducted 1 week 5 times. In the teaching system, there are different materials every day. For the Arabic language
course, the material is speaking, reading, listening, and writing and for the English language, the following materials are used:
pronunciation, grammar, speaking and entertainment. The course activities are expected to support the language development of
students on a daily learning. 4180 *Corresponding Author: M. Athoiful Fanan International Journal of Current Science Research and Review
ISSN 2581 8341 DOI: 10.47191/ijcsrr/V5-i11-13, Impact Factor: 5.995
IJCSRR @ 2022 disclosure to understand the setting in which they operate, “Wisdom is the lost treasure of a believer. Therefore, wherever the
believer finds wisdom, he will pick it up. Take that wisdom, even from a hypocrite!” In addition, the Prophet also said, "The religious
character that I love the most is "alhanifiyah assamhah. (straight and tolerance)" disclosure to understand the setting in which they operate, “Wisdom is the lost treasure of a believer. Therefore, wherever the
believer finds wisdom, he will pick it up. Take that wisdom, even from a hypocrite!” In addition, the Prophet also said, "The religious
character that I love the most is "alhanifiyah assamhah. (straight and tolerance)" To form the ideal ideas for people's lives, Pesantren (Islamic boarding schools) use brilliant minds. Furthermore, the
charisma and character of the kyai (head of Islamic boarding school) in the Islamic boarding school community and the general
public provide social and cultural legitimacy to the Islamic boarding school. This perspective shows that Islamic boarding school
as an incubator for understanding Islam can be successfully applied in Indonesia, a pluralistic, multicultural country, and based on
clear principles (Munir, 2013). Mukti Ali argues somewhat regretfully that Islamic boarding school is a reminder of the importance
of da'wah institutions, not community empowerment institutions, not community economic institutions, but Islamic boarding school,
which are community reproductive institutions with a system of cultural values, mutual cooperation, obedience to kyai (head of
Islamic boarding school), discipline, and religious understanding and deep (Yasir, 2014). Nonetheless, it persists because people
have different ideas about what Islamic boarding school is and because Islamic boarding school plays an important role in
introducing new cultural models to the public. On the one hand, it is true that Islamic boarding school function as a place of learning. The relationship between parents and Islamic boarding schools, on the other hand, can be reflected in the patron-client
model. Therefore, pesantren and the culture they help foster in Indonesia are closely intertwined. There are also competing
interpretations of Islamic teachings within Islamic boarding schools. The openness value of PPMBS is carried out by collaborating
with several Islamic boarding schools, such as the Gontor Islamic Boarding School and the Langitan Islamic Boarding School. B. Al-Infitah (Openness) Islamic teachings about openness are universal. Muslims would benefit greatly from adopting such an approachable
mentality. The application of fair openness requires that the person performing the application is aware of the context in which they
are working. The Muslim community should adopt this kind of openness character. It is important for people who practice self- Volume 05 Issue 11 November 2022
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Page No.-4176-4183 International Journal of Current Science Research and Review DOI: 10.47191/ijcsrr/V5-i11-13, Impact Factor: 5.995 Maghribi (Gresik), two of the nine prominent figures in the introduction of Islam to Java; however, Sunan Ampel (Raden Rahmat)
is considered to have made the greatest strides in this area) (Anam, 2017) PPMBS is a mirror of multicultural Islamic boarding schools because of the diversity of students and cultures to produce
values of tolerance and mutual respect. As previously mentioned, PPMBS, a boarding school in Gresik, focuses on inculcating the
values of diversity among its students. One of these values is the importance of community (Akhmadi, 2019) On the other hand, the diversity that is owned in this pesantren is contained in the rules of the pesantren, where this rule is
part of the code of ethics that must be carried out by all students here, regardless of ethnicity, regardless of the kiai's or not, all of
them are required to follow the existing rules, so the sanctions that we give are also the same, this is to teach students the importance
of the principle of justice that we frame in the diversity of rules that we set. 4181 *Corresponding Author: M. Athoiful Fanan D. Islam rahmatan lil Alamin (Peaceful Islam) Islam as rahmatan lil alamin (peaceful Islam) is best understood based on Islamic teachings on religion, ethics, and
spirituality. The Islamic concept of rahmatan lil alamin (peaceful Islam) is patterns of compassion for human interaction that are
inclusive, open-minded, dialogue-based, and tolerant Miftah Mucharomah, "Teachers in the Millennial Era in the Frame of
Rahmatan Lil Alamin (peaceful Islam)," Edukasia Islamika: Journal Islamic Education 2, No. 2 (2017): 172–190, http://E Journal. Iainpekalongan. ac. id/Index.Php/Edukasiaislamika/Article/View/1667. Pluralism is in the sense of having relationships with people
of different ethnicity, nationality, religion, race, and so on. The belief in and defence of universal human dignity and the protection
of individual human rights constitutes a humanist worldview. In a truly dialogical society, all the problems that arise as a result of
people interacting with each other are thoroughly debated and different points of view are respected. And peacefully allow others
to follow their own beliefs; this is the tolerance of Muhammad Makmun Rasyid, “Islam Rahmatan Lil Alamin (Islam of peace)
Perspective Kh. Hasyim Muzadi,” Journal of Episteme 11, No. 1 (2016): 93–116, Http://178.128.61209/ Index.Php/
Epis/Article/View/189/134. p
Despite these widespread misconceptions, most Muslims around the world continue to believe that Islam is a religion of
peace. The Islamic concept of Rahmatan Lil'alamin (Islam of peace). The solution to the problem, if there is a conflict in PPMBS
should be clarified (tabayun) first then confirm and if it is proven that there is tolerance then both parties will do mediation (tahkim)
and deliberation to find a middle way then when the situation has subsided, encourage both parties to mutually forgive or have the
nature of al-'afwu (forgiveness). This is in line with Azyumardi Azra's thinking which reveals four aspects of forgiveness, (1) Making
a moral judgment on a painful event is the first step in forgiving him; in the Islamic context, this is known as muhasabah. (2)
Determine whether the perpetrator will be punished or compensated in a certain way. Forgiveness may not negate consequences at
all, but it should prevent retaliation. Third, considering that the perpetrators are still human and should be pitied. Accept each other's
flaws and learn to coexist peacefully regardless; this is the only way to restore trust in human relations and reap the benefits of Siti
A'isyah's sincere forgiveness, “Forgiveness for the Settlement of Past Crimes: Conceptual Analysis of Islamic Perspectives 1,”
Maqashid: Journal of Islamic Law 4, No. C. Tanawue (Diversity) The form of diversity in PPMBS can be observed from the number of students who are accepted from several regions, even
some students who come from abroad and have cultural differences from Indonesia. It has become a reality that big lodges in
Indonesia, especially in East Java, are always crowded with students who want to study religion. This is inseparable from historical
records which state that the history of the development of Islamic boarding schools in Indonesia is closely related to the history of
the entry of Islam into Indonesia. As a boarding school as a medium for the spread of Islam, especially during the time of Wali
Songo (nine Muslim missionaries) who at that time spread Islam on the island of Java. Historical records show that there is a close
relationship between the entry of Islam and the establishment of Islamic boarding schools in Indonesia. During the time of the
Islamic missionary Wali Songo, pesantren played an important role in the spread of religion throughout Java. Wali Songo is credited
with establishing many pesantren in Java; the first pioneers are often called Sheikh Maulana Malik Ibrahim or Sheikh Maulana Volume 05 Issue 11 November 2022
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Page No.-4176-4183 4182 *Corresponding Author: M. Athoiful Fanan International Journal of Current Science Research and Review (tawazun), according to which we must achieve a just and equitable balance between this world and the hereafter. and the value of
tolerance (tasamuh), which we think the students at the Mamba'us Sholihin Islamic Boarding School learn to accept people who are
different from themselves, and that is a trait that we actively cultivate. If there are visitors of different religions to the pesantren, we
try our best to accommodate them. IV. CONCLUSION Building the values of inclusive education as the basis for building the student's character at the Mambaus Solikhin Suci
Islamic Boarding School (PPMBS) is done by applying the tasamuh (tolerance) concept through the studies of the classical yellow
book and the value of tolerance is illustrated by the icon of the pesantren which always voices as a pesantren that stands above all
groups and teaches about friendship and brotherhood. In addition, reciting the book and providing students with an understanding
of religious differences and implementing a system of student service to the community. In addition, there are dynamic values
(openness) that are carried out through humanitarian activities and collaboration between students and the community. as well as
the value of diversity carried out at PPMBS by inculcating mentality and an attitude of unity in diversity through the principle of
Unity in Diversity. Finally, the inclusive values that exist in the PPMBS are besed on the Islamic values of rahmatan lil alamin
which are built in the form of religious commitment and the values of, adl (fair), Al-Infitah (openness), tasamuh (tolerance) and
tawassuth (moderate) and reject the views of extremist students. REFERENCES FERENCES
1. A Faiz Yunus. (2017). Radicalism, Liberalism and Terrorism: Their Influence on Islam. Journal of Qur'anic Studies, 13(I),
76–94. 1. A Faiz Yunus. (2017). Radicalism, Liberalism and Terrorism: Their Influence on Islam. Journal of Qur'anic Studies, 13(I),
76–94. 2. Ahmad Gaddafi and Kalis Mardiasih. (2021). A Guide to Composing Counter-narratives and Alter
Tolerance and Peace. In Husni Mubarok (Ed.), Research Results (p. 02). wahidfoundation.org. 2. Ahmad Gaddafi and Kalis Mardiasih. (2021). A Guide to Composing Counter-narratives and Alternative Narratives for
Tolerance and Peace. In Husni Mubarok (Ed.), Research Results (p. 02). wahidfoundation.org. 3. Akhmadi, A. (2019). Religious Moderation in Indonesia's Diversity Religious Moderation in Ind
Journal of Religious Education and Training, 13(2), 47. 4. Anam, S. (2017). Characteristics and Islamic Education System: Knowing the History of Islamic Boarding Schools, Surau
and Meunasah in Indonesia. JALIE: Journal of Applied Linguistics and Islamic Education, I(I), 145–149
5. https://doi.org/10.33754/jalie.v1i1.52 6. Bakrie, Maskuri and Werdiningsih, D. (2017). Grounding Character Values Based on Islamic Boarding Schools. Nirma
Media. 7. Budiyanto, B. (2005). Introduction to Local Culture-Based Inclusive Education. Ministry of Nationa 7. Budiyanto, B. (2005). Introduction to Local Culture Based Inclusive Education. Ministry of National Education. 8. Ministry of National Education, D. (2008). Indonesian dictionary. Language Center. 8. Ministry of National Education, D. (2008). Indonesian dictionary. Language Center. 8. Ministry of National Education, D. (2008). Indonesian dictionary. Language Center 9. Elfiky, I. (n.d.). Positive Thinking Therapy. 10. Habibah, N. (2016). Artificial environment in learning Arabic. 3(2), 173–196. 11. Hanafi. (2016). Multiculturalism in the Qur'an, Hadith and the Madina Charter. Scientific Islamica: Journal of Islamic
Studies, 3(2), 169–190. 17). Yellow Book Learning Development Management. Journal of Reality, 15(2), 4–6. 12. Hanani, N. (2017). Yellow Book Learning Development Management. Journal of Reality, 15(2), 4–6 13. Hasyim, U. (1979). Religious Tolerance and Freedom in Islam as a Basis for Dialogue and Harmony Between Religious
People. Science Building. 14. Imron. (2019). The Influence of Leadership Style, Teamwork, And Organizational Culture On Work Performance And Its
Impact On Employee Performance. Journal of Economics and Management, 5(1), 64–83. The Influence of Leadership Style, Teamwork, And Organizational Culture On Work Performance And Its
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t 5(1) 64 83 15. Mucharomah, M. (2017). Teachers in the Millennial Era in the Frame of Lil Alamin's Grace. Edukasia Islamika: Journal
of Islamic Education, 2(2), 172–190. 16. Mudjito, M. (2012). Inclusive Education. Baduose Media. 16. D. Islam rahmatan lil Alamin (Peaceful Islam) 1 (2021): 1–17, Http://Www.Ejournal.Alqolam.Ac.Id/Index.Php/Maqashid/Article/View/614/444. Islamic faith prioritizes humans to develop a compassionate character towards one another, so it makes sense that forgiving
one another would be considered a noble act. The issue of forgiveness is so important that it is mentioned 35 times in the Qur'an of
Amirullah Bagus Purbianto, "The Concept of Forgiveness in the Perspective of the Qur'an and Its Relevance to Mental Health"
(Thesis: Iain Jember, 2016), Http://Digilib.Iain -Jember.ac.Id/Id/Eprint/234., in addition to dozens of authentic hadiths of the Prophet
Muhammad SAW, they are also dlo'if (weak). Furthermore, the Islamic values taught at PPMBS are Islam rahmatan lil 'alamin (Peaceful Islam), Islamic values rahmatan
lil alamin (Peaceful Islam), or Islam that is beneficial to everyone, being the core of inclusive education organized by Islamic
boarding school. The inclusion of rahmatan lil 'alamin (Peaceful Islam) as a quality is very important. However, in some parts of
the world today, Islam presents itself in a violent, extreme, and intolerant way. Meanwhile, the Islam that will be grown here is not
Islam that is violent, extremist, or intolerant. But Islam is merciful because it is kind, accepting, and tolerant. If we look closely, the values of rahmatan lil alamin that are practiced at PPMBS are in line with the multiethnic ethos of
the pesantren. Therefore, there are several principles, including moderate values (tawasuth), according to which this pesantren does
not stand on both sides but stands neutrally; the value of justice ('adl), namely treating students and fairly; the value of balance Volume 05 Issue 11 November 2022 Available at: ijcsrr.org
Page No.-4176-4183 International Journal of Current Science Research and Review 19. Prabowo, M.N. (2013). Tolerance: From Historical Experience To Theoretical Construction. In Ciaran Cronin (Ed.),
book review (p. 4). Cambridge University Press. 19. Prabowo, M.N. (2013). Tolerance: From Historical Experience To Theoretical Construction. In Ciaran Cronin (Ed.),
book review (p. 4). Cambridge University Press. 20. Purbianto, A. B. (2016). The Concept of Forgiveness in the Perspective of the Qur'an and Its Relevance to Mental Health. Thesis: IAIN Jember. 21. Rohmadi, S. H. (2017). Islamic Boarding School Inclusive Islamic Education (Historical-Sociological Studies in
Indonesia). Fikrotuna, 5(1). 22. Siti A'isyah. (2021). Forgiveness for Resolving Past Crimes: Conceptual Analysis of Islamic Perspectives 1. Maqashid:
Journal of Islamic Law, 4(1), 1–17. 23. Sutarjo Adisusilo, J. . (2012). Learning the Values of Constructivism and VCT Characters as Innovations in Affective
Learning Approaches. Press Eagle. 23. Sutarjo Adisusilo, J. . (2012). Learning the Values of Constructivism and VCT Characters as Innovations in Affective
Learning Approaches. Press Eagle. 24. Tarmansyah, T. (2009). Implementation of Inclusive Education in SD Negeri 03 Alai Padang Utara, Padang City (Study
on the Implementation of Education in Trial Schools of the Inclusive Education System). Pedagogy. Scientific Journal of
Education, 9(1). 24. Tarmansyah, T. (2009). Implementation of Inclusive Education in SD Negeri 03 Alai Padang Utara, Padang City (Study
on the Implementation of Education in Trial Schools of the Inclusive Education System). Pedagogy. Scientific Journal of
Education, 9(1). 25. Ulum, M. B. (2017). The Values of Multicultural Education in Al-Ishlah Islamic Boarding School Lamongan and
Qomaruddin Islamic Boarding School Gresik. UIN Sunan Ampel Surabaya. 25. Ulum, M. B. (2017). The Values of Multicultural Education in Al-Ishlah Islamic Boarding School Lamongan and
Qomaruddin Islamic Boarding School Gresik. UIN Sunan Ampel Surabaya. g
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26. Yasir, M. (2014). The Meaning of Tolerance in the Qur'an. Journal of Ushuluddin, XXII(2), 171. 4183 *Corresponding Author: M. Athoiful Fanan Volume 05 Issue 11 November 2022
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Cite this Article: M. Athoiful Fanan, Maskuri, M. Mas’ud Said (2022). The Values of Inclusive Education as a Basis for Building
Students Character of Mambaus Sholihin Islamic Boarding School Gresik Indonesia. International Journal of Current Science
Research and Review, 5(11), 4176-4183 Cite this Article: M. Athoiful Fanan, Maskuri, M. Mas’ud Said (2022). The Values of Inclusive Education as a Basis for Building
Students Character of Mambaus Sholihin Islamic Boarding School Gresik Indonesia. Cite this Article: M. Athoiful Fanan, Maskuri, M. Mas’ud Said (2022). The Values of Inclusive Education as a Basis for Building
Students Character of Mambaus Sholihin Islamic Boarding School Gresik Indonesia. International Journal of Current Science
Research and Review, 5(11), 4176-4183 4183 *Corresponding Author: M. Athoiful Fanan g
p
y
6. Yasir, M. (2014). The Meaning of Tolerance in the Qur'an. Journal of Ushuluddin, XXII(2), 171. 25. Ulum, M. B. (2017). The Values of Multicultural Education in Al-Ishlah Islamic Boarding School Lamongan and
Qomaruddin Islamic Boarding School Gresik. UIN Sunan Ampel Surabaya. REFERENCES Mudjito, M. (2012). Inclusive Education. Baduose Media. 17. Muhammad Makmun Rashid. (2016). Islam Rahmatan Lil Alamin Perspective Kh. Hashim Muzadi. Journal of Episteme,
11(1), 93–116. https://doi.org/10.21274/epis.2016.11.1.93-116 18. Munir, M. (2013). Educational Values in the Minangkabau Traditional Petitih Petatah (Alternatives for Building
Character Education). Al Hurriyah (Journal of Islamic Law), 14(1), 96–104. Volume 05 Issue 11 November 2022 International Journal of Current Science Research and Review
ISSN: 2581-8341
Volume 05 Issue 11 November 2022
DOI: 10.47191/ijcsrr/V5-i11-13, Impact Factor: 5.995
IJCSRR @ 2022
www.ijcsrr.org International Journal of Current Science Research and Review International Journal of Current Science
Research and Review, 5(11), 4176-4183 Volume 05 Issue 11 November 2022
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https://openalex.org/W2611275663
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https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0176247&type=printable
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English
| null |
A statistically compiled test battery for feasible evaluation of knee function after rupture of the Anterior Cruciate Ligament – derived from long-term follow-up data
|
PloS one
| 2,017
|
cc-by
| 10,816
|
RESEARCH ARTICLE Methods In total 48 outcome variables related to knee function, all potentially relevant for a long-term
follow-up, were included from a cross-sectional study where 70 ACL-injured (17–28 years
post injury) individuals were compared to 33 controls. Cluster analysis and logistic regres-
sion were used to group variables and identify an optimal test battery, from which a summa-
rized estimator of knee function representing various functional aspects was derived. Copyright: © 2017 Schelin 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: In accordance with
the Swedish legislation, including the Ethical
Approval Act concerning the Ethical Review of
Research Involving Humans (2003: 460) and the
Swedish Personal Data Act (SFS 1998: 204),
specific data at an individual level cannot be
deposited in a public repository. The region is also
rather small population-wise, and there is
substantial data on each individual which could
therefore make it possible to identify individuals. The data could however be made available on Purpose Citation: Schelin L, Tengman E, Ryden P, Ha¨ger C
(2017) A statistically compiled test battery for
feasible evaluation of knee function after rupture of
the Anterior Cruciate Ligament – derived from
long-term follow-up data. PLoS ONE 12(5):
e0176247. https://doi.org/10.1371/journal. pone.0176247 Clinical test batteries for evaluation of knee function after injury to the Anterior Cruciate Liga-
ment (ACL) should be valid and feasible, while reliably capturing the outcome of rehabilita-
tion. There is currently a lack of consensus as to which of the many available assessment
tools for knee function that should be included. The present aim was to use a statistical
approach to investigate the contribution of frequently used tests to avoid redundancy, and
filter them down to a proposed comprehensive and yet feasible test battery for long-term
evaluation after ACL injury. Editor: David S. Logerstedt, University of the
Sciences in Philadelphia, UNITED STATES
Received: June 17, 2015
Accepted: March 16, 2017
P bli h d M
1 2017 Editor: David S. Logerstedt, University of the
Sciences in Philadelphia, UNITED STATES A statistically compiled test battery for
feasible evaluation of knee function after
rupture of the Anterior Cruciate Ligament –
derived from long-term follow-up data Lina Schelin1,2☯*, Eva Tengman1☯, Patrik Ryden3☯, Charlotte Ha¨ger1☯ 1 Department of Community Medicine and Rehabilitation, Physiotherapy, Umeå University, Umeå, Sweden,
2 Department of Statistics, Umeå School of Business and Economics, Umeå University, Umeå, Sweden,
3 Department of Mathematics and Mathematical Statistics, Umeå University, Umeå Sweden 1 Department of Community Medicine and Rehabilitation, Physiotherapy, Umeå University, Umeå, Sweden,
2 Department of Statistics, Umeå School of Business and Economics, Umeå University, Umeå, Sweden,
3 Department of Mathematics and Mathematical Statistics, Umeå University, Umeå Sweden a1111111111
a1111111111
a1111111111
a1111111111
a1111111111 ☯These authors contributed equally to this work. * Lina.Schelin@umu.se ☯These authors contributed equally to this work. * Lina.Schelin@umu.se 1 Department of Community Medicine and Rehabilitation, Physiotherapy, Umeå University, Umeå, Sweden,
2 Department of Statistics, Umeå School of Business and Economics, Umeå University, Umeå, Sweden,
3 Department of Mathematics and Mathematical Statistics, Umeå University, Umeå Sweden Introduction Rupture of the anterior cruciate ligament (ACL) is a common injury especially in individuals
who participate in sports [1, 2]. Treatment involves either physiotherapy in combination with
reconstructive surgery, or physiotherapy alone. Regardless of treatment, individuals still often
suffer from varying extents of impaired knee function, both in the short [3, 4] and long-term
perspective despite completing rehabilitation [5–8]. Such reduced knee function may be mani-
fested by, for instance, instability, pain, swelling, decreased range of motion, joint stiffness,
reduced physical capacity or decreased activity level in everyday tasks, but particularly with
regard to sports and recreational activities. Consequently, attempts to determine knee function
often combine several assessment tools covering different aspects of knee function based
mainly on clinical examination, knee-specific scores and functional tests. The latter are aimed
at capturing indicators of physical capacity, e.g. muscular strength, balance, motor coordina-
tion etc. There is, however, still no consensus on which outcome measures to use, which
makes comparisons across studies difficult and leads to a lack of evidence for specific interven-
tions. In the clinic, self-reported questionnaires and examiner-administrated knee scores such
as the International Knee Documentation Committee 2000 subjective form (IKDC) [9], Knee
injury and Osteoarthritis Outcome Score (KOOS) [10] or Lysholm questionnaire [11] are
commonly used, and often in combination with a strength measurement and a hop task. Rupture of the anterior cruciate ligament (ACL) is a common injury especially in individuals
who participate in sports [1, 2]. Treatment involves either physiotherapy in combination with
reconstructive surgery, or physiotherapy alone. Regardless of treatment, individuals still often
suffer from varying extents of impaired knee function, both in the short [3, 4] and long-term
perspective despite completing rehabilitation [5–8]. Such reduced knee function may be mani-
fested by, for instance, instability, pain, swelling, decreased range of motion, joint stiffness,
reduced physical capacity or decreased activity level in everyday tasks, but particularly with
regard to sports and recreational activities. Consequently, attempts to determine knee function
often combine several assessment tools covering different aspects of knee function based
mainly on clinical examination, knee-specific scores and functional tests. The latter are aimed
at capturing indicators of physical capacity, e.g. muscular strength, balance, motor coordina-
tion etc. There is, however, still no consensus on which outcome measures to use, which
makes comparisons across studies difficult and leads to a lack of evidence for specific interven-
tions. Test battery for ACL Conclusions request for scientific purposes not violating these
laws, and thus guaranteeing confidentiality. Umeå
University is responsible for the personal data and
for data requests; interested researchers should
contact the Head of the Department, Department of
Community Medicine and Rehabilitation, Umeå
University, ann.sorlin@umu.se, or principal
investigator Professor Charlotte Ha¨ger, charlotte. hager@umu.se. The proposed test battery, based on a solid statistical approach, includes assessments which
are all clinically feasible, while also covering complementary aspects of knee function. Similar
test batteries could be determined for earlier phases of ACL rehabilitation or to enable longitudi-
nal monitoring. Such developments, established on a well-grounded consensus of measure-
ments, would facilitate comparisons of studies and enable evidence-based rehabilitation. Funding: We acknowledge support from the
Swedish Scientific Research Council (project no. K2011-69X-21876-01-3), Umeå University (Young
Researcher Awardee, C Ha¨ger, and the Foundation
for medical research), Va¨sterbotten county council,
Swedish National Centre for Research in Sports
(CIF, project no. P2012-0008), and Ingabritt &
Arne Lundbergs research foundation. L Schelin
was partially supported by the UMIT Research Lab
at Umeå University. The funders had no role in
study design, data collection and analysis, decision
to publish, or preparation of the manuscript. Results As expected, several variables were strongly correlated, and the variables also fell into logi-
cal clusters with higher within-correlation (max ρ = 0.61) than between clusters (max ρ =
0.19). An extracted test battery with just four variables assessing one-leg balance, isokinetic
knee extension strength and hop performance (one-leg hop, side hop) were mathematically
combined to an estimator of knee function, which acceptably classified ACL-injured individu-
als and controls. This estimator, derived from objective measures, correlated significantly
with self-reported function, e.g. Lysholm score (ρ = 0.66; p<0.001). 1 / 18 PLOS ONE | https://doi.org/10.1371/journal.pone.0176247
May 1, 2017 Introduction In the clinic, self-reported questionnaires and examiner-administrated knee scores such
as the International Knee Documentation Committee 2000 subjective form (IKDC) [9], Knee
injury and Osteoarthritis Outcome Score (KOOS) [10] or Lysholm questionnaire [11] are
commonly used, and often in combination with a strength measurement and a hop task. Regarding functional assessments, different test batteries have been suggested [12–14]. A test
battery in this context refers to a set of functional tests. A test battery consisting of three com-
monly used hop tests (vertical hop, one-leg hop for distance, and side hop), has shown a high
ability to discriminate between the injured and non-injured leg of individuals with ACL injury
[12]. Another test battery consisting of four hop tests (one-leg hop for distance, 6-m timed
hop, triple hop for distance and crossover hop for distance) has also been demonstrated to be
reliable and valid [14, 15]. Yet another test battery, consisting of knee-extension, knee-flexion
and leg-press tests, discriminates between strength of the injured and the non-injured leg [13]. The full potential of such test batteries is not always achieved, since the specific test results are
most often evaluated separately. The statistical methodology for a research question related to
a single outcome variable is often straightforward. Typically two (or more) groups are com-
pared with respect to a single variable using a statistical test [e.g. [5, 6, 8]]. Such tests are some-
times suitable to answer research questions, but single outcome variable analysis might not
reveal all of the information contained in the data. It is, for example, possible to find significant
differences between two groups when studying two variables simultaneously, while a separate
analysis for each of them would not reveal any significant group differences. Hence, it would
be desirable to analyze several variables simultaneously. Competing interests: The authors have declared
that no competing interests exist. Correlation analysis and cluster analysis can be used to understand relationships between
variables. Examples of statistical methods for dimension reduction are factor analysis, principal
component analysis (PCA) and logistic regression. Such methods may be used to combine PLOS ONE | https://doi.org/10.1371/journal.pone.0176247
May 1, 2017 2 / 18 Test battery for ACL models. The models in question should be able to discriminate between different knee func-
tion abilities. This could be quantified by considering the models misclassification rate; defined
as the proportion of incorrectly classified individuals, with and without ACL injury, when
using the model. A low misclassification rate implies in our case, that the test battery better dis-
criminates between injured individuals and healthy-knee controls. Since there may be a bilat-
eral decrease of knee function following a unilateral injury [16–18], test batteries evaluated in
both injured individuals and healthy-knee controls may provide additional information about
which functional tasks to include in the test battery. Due to the difficulties in defining optimal knee function from a rehabilitation perspective,
it would be advantageous to measure all potential aspects of knee function. However, for prac-
tical reasons the test battery often needs to be relatively small and feasible (i.e. running the test
should be relatively quick and not require extraordinary/specialized equipment). This aspect is
also considered when compiling the proposed test battery. Finally, the variables in the final test
battery are combined to an estimator of knee function using logistic regression. In addition to having a model that facilitates an estimation of the overall knee function,
it also seems highly important to understand the relationship between all outcome variables,
e.g. to identify groups of variables that are correlated to each other. This problem may be
addressed by using correlation analysis combined with cluster analysis, as we present later. Highly correlated variables might contribute with similar information, i.e. little information is
lost if a group of strongly correlated variables is represented by just one of these variables. Further, in the case of knee function, previous studies have found low or moderate correla-
tions between patient-reported outcome scores and variables from functional tests [19–22]. This suggests that single functional tests are generally not able to measure overall knee func-
tion. A compiled index derived from a representative test battery would also be more likely to
correspond with self-reported function. The aim of this paper was to investigate the possibility of applying such a statistical
approach to a large set of knee assessments, thereby detecting highly correlated variables and
filter them down to a suggestion of a comprehensive and yet feasible clinical test battery con-
sisting of only a few tests to be used in long-term evaluation after ACL injury. Participants The KACL20-study (Knee injury—Anterior Cruciate Ligament after more than 20 years [7,
23]) is a long-term follow-up with a cross-sectional design, where 70 individuals who had suf-
fered unilateral ACL injury, on average 23 (17–28) years ago, were compared to 33 healthy-
knee controls matched for age and sex. Basic individual characteristics are found in Table 1,
and detailed outcome aspects related to physical activity, hop performance, and knee strength
have been reported elsewhere [7, 23]. All participants were presented with written and oral
information about the study and gave their written informed consent according to the declara-
tion of Helsinki. The project was approved by the Regional Ethical Review Board in Umeå,
Sweden (Dnr. 07-155M and Dnr. 08-211M). If proven useful,
the suggested method could be applied to propose test batteries appropriate for acute and sub-
acute phases of ACL rehabilitation, as well as monitoring and evaluation of other disorders in
many clinical fields. Test battery for ACL information from several tests into a more valid estimator of knee function. However, inclusion
of many variables in one model may make interpretation difficult. Alternatively, building a model
based on a selected subset of variables may result in a model that is easier to interpret. This could
be achieved by applying a statistical approach that can determine which knee tests that would be
necessary and which would be redundant. In the present paper, a statistical approach was imple-
mented to define a comprehensive and feasible test battery (Fig 1) that would be more discrimina-
tive than each of the included single subtests applied separately. To the best of our knowledge,
such an approach has not been attempted with regard to knee function assessment. such an approach has not been attempted with regard to knee function assessment. We have utilized data from our long-term follow-up to implement the proposed statistical
approach and suggest a test battery to evaluate long-term knee function after ACL injury. The
statistical approach used to identify potential test batteries is based on logistic regression Fig 1. Illustration of the data structure and the statistical approach. First, correlation analysis combined with cluster analysis is applied to better
understand the relationship between all outcome variables. Potential test batteries are then investigated using logistic regression and subsequently
evaluated based on their misclassification rate and on their feasibility. The combined outcomes of the final test battery result in an estimator of knee function,
again using logistic regression. Finally, this new variable (estimator of knee function) is analyzed using traditional statistical approaches such as Spearman
rank correlation and Wilcoxon rank sum test. Fig 1. Illustration of the data structure and the statistical approach. First, correlation analysis combined with cluster analysis is applied to better
understand the relationship between all outcome variables. Potential test batteries are then investigated using logistic regression and subsequently
evaluated based on their misclassification rate and on their feasibility. The combined outcomes of the final test battery result in an estimator of knee function,
again using logistic regression. Finally, this new variable (estimator of knee function) is analyzed using traditional statistical approaches such as Spearman
rank correlation and Wilcoxon rank sum test. https://doi.org/10.1371/journal.pone.0176247.g001 https://doi.org/10.1371/journal.pone.0176247.g001 PLOS ONE | https://doi.org/10.1371/journal.pone.0176247
May 1, 2017 3 / 18 Test battery for ACL Table 1. Participant characteristics. Individuals with ACL injury
Healthy-knee controls
Number of participants
70
33
Surgery/non-surgery
33/37
N/A
Men/women
44/26
21/12
Age at follow-up test
46.9 (5.4)
46.7 (5.0)
BMI
28.1 (4.1)
24.6 (2.5)
The characteristics presented as number of individuals or mean (standard deviation). https://doi.org/10.1371/journal.pone.0176247.t001 Table 1. Participant characteristics. Table 1. Participant characteristics. for evaluation of ACL rehabilitation [12, 24]: we chose nine functional tests (including hop
tests, strength measurements and balance tests), four self-reported questionnaires, and three
examiner-administrated scores, resulting in a total of 48 outcome variables. Brief descriptions
of all 48 variables and information about their feasibility are found in Table 2. The different
hop tasks and the one-leg balance have been comprehensively described in earlier papers [7,
25]. The variables obtained from functional tests were recorded in a movement laboratory; U-
motion lab Umeå University. Participants performed the one-leg hop for distance (OLH),
one-leg vertical hop (VH), rise from chair (RC), side hop (SH) and one-leg balance (B) on
both the injured (i) and the non-injured (c) leg. For healthy-knee controls both the non-domi-
nant leg (i) and the dominant leg (c) were included. For each exercise both absolute measure-
ments (e.g. maximal hop distance on each leg) and relative measurements, such as the Limb
Symmetry Index (LSI), were considered. The strength variables were obtained from peak isoki-
netic measurements where knee flexion torque (representing hamstrings, H) and knee exten-
sion torque (representing quadriceps, Q) in concentric and eccentric contractions were
measured on both legs (for details see Tengman et al. b 2014 [23]). All strength variables were
quantified in relation to the body weight (Nm/kg) of the individual. LSI and the ratio between
hamstrings and quadriceps peak torque (H:Q ratio) were also calculated. All individuals answered several knee-specific and more general questionnaires including:
KOOS [10], Physical Activity Scale (PAS) [12, 26], International Physical Activity Question-
naire (IPAQ), 36-Item Short Form Health Survey (SF 36) [27]. For KOOS and SF-36 each sub
score was considered as one variable. Lysholm score, Tegner activity scale, [11] and Beighton
score were examiner administrated. See Table 2 for the complete list of all variables. In addi-
tion to the variables described above, some background variables were observed, including
age, sex, and clinical history (i.e. ACL-injured or healthy-knee control). PLOS ONE | https://doi.org/10.1371/journal.pone.0176247
May 1, 2017 Outcome variables The variables were obtained from a large set of knee tests, questionnaires and scores consid-
ered to have good measurement properties, which are commonly used in research and clinics PLOS ONE | https://doi.org/10.1371/journal.pone.0176247
May 1, 2017 4 / 18 Feasibility index A clinical test battery should be feasible. We therefore asked ten expert physiotherapists to
independently rank all included functional assessments and questionnaires according to time
requirement and to which extent specific equipment is needed. The ranking from the physio-
therapists is presented in Table 2 as a T:E-index, where T stands for time and E for equipment. Regarding time, a ranking of 1, 2 or 3 corresponds to less than 15 minutes, 15–30 minutes or
more than 30 minutes respectively. The estimated time demand includes the needed time for
preparation, execution and data registration. Regarding equipment, a ranking of 1 corresponds to basic equipment always being avail-
able, while a ranking of 2 implies advanced equipment or licenses. The T:E-indexes in Table 2
were obtained as the median of the answers given by the physiotherapists. The aim with the T:
E-index was to allow comparisons between test batteries regarding feasibility. 5 / 18 PLOS ONE | https://doi.org/10.1371/journal.pone.0176247
May 1, 2017 Test battery for ACL Table 2. A brief description of the 48 outcome variables included in the analysis. Variable
Short description
P-value
T:E- index
Functional tests (hops and balance)
One-leg hop
Absolute hop length in m
1:1
OLH-i
Non-dominant/injured leg
0.820
OLH-c
Dominant/non-injured leg
0.143
OLH-LSI
Limb symmetry index
0.001+
Vertical hop
Absolute hop height in cm
1:1
VH-i
Non-dominant/injured leg
0.106
VH-c
Dominant/non-injured leg
0.632
VH-LSI
Limb symmetry index
0.105
Rise from chair
Number of rises from a chair
1:1
RC-i
Non-dominant/injured leg
0.028+
RC-c
Dominant/non-injured leg
0.012+
RC-LSI
Limb symmetry index
0.690
Side hop
Number of side hops
1:1
SH-i
Non-dominant/injured leg
0.000+
SH-c
Dominant/non-injured leg
0.004+
SH-LSI
Limb symmetry index
0.004+
One-leg balance
Number of floor-supports
1:1
B-i
Non-dominant/injured leg
0.022-
B-c
Dominant/non-injured leg
0.000-
Functional tests (strength measurements)
Quadriceps
Concentric strength
3:2
Qc-i
Non-dominant/injured leg
0.000+
Qc-c
Dominant/non-injured leg
0.132
Qc-LSI
Limb symmetry index
0.005+
Eccentric strength
3:2
Qe-i
Non-dominant/injured leg
0.002+
Qe-c
Dominant/non-injured leg
0.179
Qe-LSI
Limb symmetry index
0.027+
Hamstrings
Concentric strength
3:2
Hc-i
Non-dominant/injured leg
0.076
Hc-c
Dominant/non-injured leg
0.159
HC-LSI
Limb symmetry index
0.401
Eccentric strength
3:2
He-i
Non-dominant/injured leg
0.259
He-c
Dominant/non-injured leg
0.536
He-LSI
Limb symmetry index
0.255
Strength ratios
Ratio between concentric (or eccentric) hamstrings and quadriceps strength
3:2
HQc-i
Non-dominant/injured leg
0.136
HQc-c
Dominant/non-injured leg
0.837
HQe-i
Non-dominant/injured leg
0.054
HQe-c
Dominant/non-injured leg
0.591
Examiner-administrated scores
Beighton
A score measuring hypermobility
0.020-
1:1
Tegner
Tegner activity scale measuring knee specific physical activity
0.000+
1:1
Lysholm
Knee function score
0.000+
1:1
Self-reported questionnaires Table 2. A brief description of the 48 outcome variables included in the analysis. Functional tests (strength measurements) Examiner-administrated scores PLOS ONE | https://doi.org/10.1371/journal.pone.0176247
May 1, 2017 6 / 18 T:E-index indicates how demanding the variable is to collect considering time (T, score 1–3) and equipment (E, score 1–2). Higher values
correspond to lower feasibility. The P-value column states if there is a significant difference or not between individuals with ACL injury and controls, controlling for age and sex. If a
significant difference is present, the symbol + indicates that the healthy-knee controls have a higher value than the ACL group while the symbol—indicates
the opposite. T:E-index indicates how demanding the variable is to collect considering time (T, score 1–3) and equipment (E, score 1–2). Higher values
correspond to lower feasibility. Test battery for ACL Table 2. (Continued)
Variable
Short description
P-value
T:E- index
KOOS
Knee injury and Osteoarthritis Outcome Score
2:1
KOOS-s
Symptoms
0.000+
KOOS-p
Pain
0.000+
KOOS-a
Function in activities of daily living
0.000+
KOOS-sp
Sports and recreation
0.000+
KOOS-q
Knee-related quality of life
0.000+
PAS
Physical Activity Scale
0.044+
1:1
IPAQ
International Physical Activity Questionnaire
0.247
1:1
SF-36
Patient-reported survey of patient health
2:1
SF36-pf
Physical functioning
0.000+
SF36-rp
Role physical
0.043+
SF36-bp
Bodily pain
0.000+
SF36-gh
General health
0.005+
SF36-vt
Vitality
0.151
SF36-sf
Social functioning
0.646
SF36-re
Role emotional
0.353
SF36-mh
Mental health
0.317
The P-value column states if there is a significant difference or not between individuals with ACL injury and controls, controlling for age and sex. If a
significant difference is present, the symbol + indicates that the healthy-knee controls have a higher value than the ACL group while the symbol—indicates
the opposite. T:E-index indicates how demanding the variable is to collect considering time (T, score 1–3) and equipment (E, score 1–2). Higher values
correspond to lower feasibility. Table 2. (Continued)
Variable
Short description
P-value
T:E- index
KOOS
Knee injury and Osteoarthritis Outcome Score
2:1
KOOS-s
Symptoms
0.000+
KOOS-p
Pain
0.000+
KOOS-a
Function in activities of daily living
0.000+
KOOS-sp
Sports and recreation
0.000+
KOOS-q
Knee-related quality of life
0.000+
PAS
Physical Activity Scale
0.044+
1:1
IPAQ
International Physical Activity Questionnaire
0.247
1:1
SF-36
Patient-reported survey of patient health
2:1
SF36-pf
Physical functioning
0.000+
SF36-rp
Role physical
0.043+
SF36-bp
Bodily pain
0.000+
SF36-gh
General health
0.005+
SF36-vt
Vitality
0.151
SF36-sf
Social functioning
0.646
SF36-re
Role emotional
0.353
SF36-mh
Mental health
0.317
The P-value column states if there is a significant difference or not between individuals with ACL injury and controls, controlling for age and sex. If a
significant difference is present, the symbol + indicates that the healthy-knee controls have a higher value than the ACL group while the symbol—indicates
the opposite. T:E-index indicates how demanding the variable is to collect considering time (T, score 1–3) and equipment (E, score 1–2). Higher values
correspond to lower feasibility. Table 2. (Continued) The P-value column states if there is a significant difference or not between individuals with ACL injury and controls, controlling for age and sex. If a
significant difference is present, the symbol + indicates that the healthy-knee controls have a higher value than the ACL group while the symbol—indicates
the opposite. PLOS ONE | https://doi.org/10.1371/journal.pone.0176247
May 1, 2017 Test battery for ACL [29], using the above explanatory variables, but no interaction terms, was used to model the
probability wb that the patient is healthy, by [29], using the above explanatory variables, but no interaction terms, was used to model the
probability wb that the patient is healthy, by logitðwbÞ ¼ bb0 þ bb1 xb1 þ þ bbk xbk: Note that wb may be interpreted as an estimator of the individuals’ relative knee function,
where 0 is bad and 1 is good. The coefficient βbi should be interpreted in the following way: a
one-unit increase in the variable xbi, holding all other variables at fixed values, corresponds to
a 100 exp(βbi)% increase in the odds of being a healthy control. Each battery was evaluated by considering the corresponding model’s misclassification rate
and how feasible the included variables are in the clinic (see below). The commonly used mis-
classification rate was estimated using leave-one-out cross validation, and should be inter-
preted as the probability of being classified into the wrong group. The models could include
both significant and insignificant variables. The correlation between the estimated knee function w from the final test battery and other
variables were calculated using Spearman’s rank correlation and, for two group comparisons,
Wilcoxon’s rank sum test was used. All statistical analyses were performed using the software
R, version 2.15.2. Compilation of test battery Altogether about 72 000 test batteries, representing different combinations of the included test
variables, were selected and evaluated. All test batteries with a misclassification rate lower than
0.2 were investigated further. This cut-off value was chosen arbitrarily to define a subset of rea-
sonable size for further investigation. The feasibility of those test batteries was estimated by the
sum of the variables’ feasibility indexes. For example, a battery including the variable OLH-i
from the one-leg hop for distance test and the variable Qc-i from the concentric contraction
representing quadriceps has an aggregated feasibility index of 7 according to Table 2. A battery
with a low index is regarded as highly feasible. Further, if a test battery includes the variable
OLH-i, the variable OLH-c will be available without increasing the feasibility. For the identi-
fied functional tests, we considered all possible combinations of variables available for these
tests. A condition for the final test battery, based on clinical relevance, was that it would mainly
consist of variables related to the injured leg. A relevant final test battery was compiled using
these established criteria, and in combination with existing clinical evidence. PLOS ONE | https://doi.org/10.1371/journal.pone.0176247
May 1, 2017 Statistical analysis In order to better understand the relationship between all outcome variables we use correlation
analysis combined with cluster analysis, and to statistically derive potential test batteries we
use logistic regression models. To evaluate the models, i.e. the test batteries; we consider each
models misclassification rate. The statistical analysis is summarized in Fig 1 and the methodol-
ogy details are presented below. Correlation analysis combined with hierarchical cluster analysis was used to identify highly
correlated variables. First, the Spearman’s rank correlation was used to calculate the correla-
tion between all pairs of variables, denoted ρij, where i and j are indexes for the variables. Next,
a dendrogram (i.e. tree describing the relative distance between the variables) was obtained
using hierarchical cluster analysis [28] with Ward linkage and a distance matrix D for which
the elements were: one minus the absolute correlation, i.e., dij = 1 −|ρij|. The cluster analysis
resulted in a dendrogram where highly correlated variables were grouped in clusters. Each
cluster corresponds to one branch of the dendrogram. Test batteries were obtained by selecting 1–30 variables from the functional assessments
defined in Table 2. Here, different selection strategies were considered. For small batteries
(1–4 variables) all possible combinations were investigated, and for all larger batteries (5–20
variables) 10,000 randomly sampled sets of variables were considered. In addition, we also
considered the complete test battery when including all 30 functional test variables. For each test battery logistic regression was used to model knee function as a function of
the variables in the respective test battery. This was done as follows. Let Y denote the binary
variable reflecting the clinical history of the patient, that is; 1 for healthy controls, and 0 for
ACL-injured. Let (Xb1,. . .,Xbk) denote the variables used in the bth battery. Logistic regression PLOS ONE | https://doi.org/10.1371/journal.pone.0176247
May 1, 2017 7 / 18 Results The data from the KACL20-study used in the present paper included data from both healthy-
knee controls and ACL-injured individuals. When applying correlation analysis combined
with hierarchical cluster analysis the variables fell into five major clusters that in fact repre-
sented clinically meaningful dimensions of knee functions. Generally, the pairwise correlation
within the clusters was significantly higher than between the clusters (p-value = 0.005), see Fig
2. Each cluster broadly represents different dimensions of knee function; Cluster I: the Hop
performance and knee strength included all absolute variables from the functional tests with the
exception of the variables from RC and B. Cluster II: the Perceived knee function included most
of the self-reported questionnaires and examiner-administrated scores related to perceived knee
function, including the five sub scores of KOOS, Lysholm, SF36-bp and SF36-pf. Cluster III:
Knee function reflected in activity and health was the most diverse group and included variables
related to activity (Tegner, PAS) and general health (SF36), RC (RC-c, RC-i, RC-LSI), and B
(B-c, B-i). Cluster IV: the Knee strength ratio and Cluster V: the Limb asymmetry were rather
closely related and contained all the relative functional tests variables with the exception of PLOS ONE | https://doi.org/10.1371/journal.pone.0176247
May 1, 2017 8 / 18 Test battery for ACL Fig 2. Results from Ward hierarchical cluster analysis based on Spearman correlation. The analysis resulted in five clusters: the Hop performance
and knee strength cluster is associated with absolute measurements of functional tests and knee strength measures; the Perceived knee function cluster is
linked with scores and questionnaires; the Knee function reflected in activity and health cluster contains a mixture of variables of different character; the Knee
strength ratio and the Limb asymmetry clusters were mainly associated with relative measurements between legs (LSI) in functional tests. Fig 2. Results from Ward hierarchical cluster analysis based on Spearman correlation. The analysis resulted in five clusters: the Hop performance
and knee strength cluster is associated with absolute measurements of functional tests and knee strength measures; the Perceived knee function cluster is
linked with scores and questionnaires; the Knee function reflected in activity and health cluster contains a mixture of variables of different character; the Knee
strength ratio and the Limb asymmetry clusters were mainly associated with relative measurements between legs (LSI) in functional tests. https://doi.org/10.1371/journal.pone.0176247.g002 https://doi.org/10.1371/journal.pone.0176247.g002 https://doi.org/10.1371/journal.pone.0176247.g002 RC-LSI. PLOS ONE | https://doi.org/10.1371/journal.pone.0176247
May 1, 2017 Results The average absolute correlations between variables within cluster I-V were 0.55
(SD = 0.11), 0.61 (SD = 0.15), 0.14 (SD = 0.13), 0.39 (SD = 0.08), and 0.29, (SD = 0.13) respec-
tively. The average absolute correlation between variables from different clusters was 0.12
(SD = 0.05). Potential test batteries, including only variables from functional tests, were obtained by
selecting 1–30 variables from the clusters. The misclassification rate, defined as the proportion
of incorrectly classified individuals with and without ACL injury, when using all 30 strength
and functional test variables, was 0.40. The median misclassification rates for test batteries
with 1–20 variables varied between 0.29 (15 variables) and 0.36 (3 variables). Interestingly, the
test batteries with the lowest (and also highest) misclassification rates were found among test
batteries with 3–5 variables, suggesting that a battery with few variables may more accurately
reflect knee function (Fig 3). We identified the following tests as typically connected to a low misclassification rate; one-
leg hop for distance (OLH), side hop (SH), one-leg balance (B), rise from chair (RC), and
quadriceps concentric (Qc) and eccentric (Qe) knee strength. Therefore, the misclassification
rates for all combinations of variables related to these functional tests were additionally inves-
tigated. Table 3 shows some of the most interesting test batteries and illustrates how the mis-
classification rate and the total feasibility-index (indicating time and equipment requirements)
change when variables are added to the model. The analyses resulted in several models with misclassification rates below 0.2 and feasibility
indexes below or equal to 11, some of which are shown in Table 3. Among them we selected a PLOS ONE | https://doi.org/10.1371/journal.pone.0176247
May 1, 2017 9 / 18 Test battery for ACL Fig 3. Misclassification rates for different sizes of test batteries. Misclassification rates for about 72 000 test batteries
of different sizes, representing different combinations of the included test variables. The size of the test battery is the
number of included variables. The misclassification rate should be as low as possible. The results for combinations
consisting of 5, 10, 15 and 20 variables are based on 10000 random samples. The horizontal line indicates our threshold
(0.2) for the highest acceptable misclassification rate. h
//d i
/10 1371/j
l
0176247 003 Fig 3. Misclassification rates for different sizes of test batteries. Misclassification rates for about 72 000 test batteries
of different sizes, representing different combinations of the included test variables. Results The size of the test battery is the
number of included variables. The misclassification rate should be as low as possible. The results for combinations
consisting of 5, 10, 15 and 20 variables are based on 10000 random samples. The horizontal line indicates our threshold
(0.2) for the highest acceptable misclassification rate. https://doi.org/10.1371/journal.pone.0176247.g003 https://doi.org/10.1371/journal.pone.0176247.g003 test battery with the variables OLH-i, SH-i, B-c, and Qc-i obtained from the functional assess-
ments OLH, SH, B, and Qc. The battery has only four variables and three of them are related
to the injured leg. The feasibility index for the battery was 11 and the estimated misclassifica-
tion rate was 0.17 (Table 3). Based on the KACL20-data, the suggested battery resulted in the
following model for estimating the patients overall knee function: logitðwÞ ¼ 1:1 6:3 OLH i þ 0:2 SH i 3:4 B c þ 2:7 Qc i: Interpret the new outcome variable, w, as an estimator of the individuals’ knee function
where 0 represents very low function and 1 indicates very good knee function. The distribu-
tions of the variables in the battery were similar between the ACL-injured and the healthy-
knee controls, while the corresponding distributions for the estimated knee function w were
more distinct, see Fig 4. PLOS ONE | https://doi.org/10.1371/journal.pone.0176247
May 1, 2017 10 / 18 Test battery for ACL Interestingly, the estimator w was positively correlated with perceived knee function taken
from Lysholm score (ρ = 0.66, p-value < 0.001), all subscales of KOOS (ρ = 0.36–0.60, p-
values < 0.001), Tegner activity scale (ρ = 0.26, p = 0.008), and three sub scores (pf, bp, and
gh) of SF36 (ρ = 0.23–0.47, p-values < 0.017) and negatively correlated with SF36-mh (ρ =
-0.22, p-value = 0.028). No significant difference between sexes was found (p-value = 0.6). Fur-
ther, there was no correlation between w and age (ρ = -0.12, p-value = 0.25). For each of the four examples, the first column shows the test battery of size one corresponding to the
lowest misclassification rate. The second column shows the misclassification rate and the total T:E-index
when one variable was added to the starting model. Each of the following rows indicated that one additional
variable was added. The final selected test battery is marked in bold in the shaded area. https://doi.org/10.1371/journal.pone.0176247.t003 Discussion The aim of this paper was to suggest a solid statistical selection process to derive a comprehen-
sive and yet feasible clinical test battery with different functional aspects to be used in Table 3. Misclassification rates and the T:E-index for a selected subset of test batteries. Variables
Misclassification rate
Total T:E- inde Table 3. Misclassification rates and the T:E-index for a selected subset of test batteries. Table 3. Misclassification rates and the T:E-index for a selected subset of test batteries. Variables
Misclassification rate
Total T:E- index
1. One-leg hop, Side hop, One-leg balance, Quadriceps concentric strength
1
Qc-i
0.29
5
2
Qc-i, OLH-i
0.28
7
3
Qc-i, OLH-i, B-c
0.26
9
4
Qc-i, OLH-i, B-c, SH-i
0.17
11
5
Qc-i, OLH-i, OLH-c, B-c, SH-i
0.16
11
6
Qc-i, OLH-i, OLH-c, B-c, SH-i, SH-c
0.15
11
7
Qc-i, Qc-c, OLH-i, OLH-c, B-c, SH-i, SH-c
0.16
11
2. One-leg hop, Rise from chair, One-leg balance, Quadriceps concentric strength
1
Qc-i
0.29
5
2
Qc-i, OLH-i
0.28
7
3
Qc-i, OLH-i, B-c
0.26
9
4
Qc-i, OLH-i, OLH-c, B-c
0.19
9
5
Qc-i, OLH-i, OLH-c, B-c, RC-i
0.17
11
6
Qc-i, Qc-c, OLH-i, OLH-c, B-c, RC-i,
0.18
11
3. One-leg hop, Rise from chair, One-leg balance, Quadriceps eccentric strength
1
OLH-LSI
0.30
2
2
OLH-LSI, B-c
0.27
4
3
OLH-LSI, B-c, Qe-i
0.24
9
4
OLH-i, OLH-LSI, B-c, Qe-i
0.22
9
5
OLH-i, OLH-LSI, B-c, Qe-i, RC-i
0.20
11
6
OLH-i, OLH-LSI, B-c, Qe-i, RC-i, RC-LSI
0.20
11
4. One-leg hop, Side hop, One-leg balance, Quadriceps eccentric strength
1
SH-i
0.32
2
2
SH-i, B-c
0.27
4
3
SH-i, B-c, OLH-i
0.22
6
4
SH-i, B-c, OLH-I, Qe-i
0.20
11
5
SH-i, B-c, OLH-i, OLH-c, Qe-i
0.17
11
6
SH-i, B-c, OLH-i, OLH-c, Qe-i, Qe-c
0.17
11 ,
,
,
,
,
,
2. One-leg hop, Rise from chair, One-leg balance, Quadriceps concentric strength
1
Qc-i
0.29
5
2
Qc-i, OLH-i
0.28
7
3
Qc-i, OLH-i, B-c
0.26
9
4
Qc-i, OLH-i, OLH-c, B-c
0.19
9
5
Qc-i, OLH-i, OLH-c, B-c, RC-i
0.17
11
6
Qc-i, Qc-c, OLH-i, OLH-c, B-c, RC-i,
0.18
11
3. lowest misclassification rate. The second column shows the misclassification rate and the total T:E index
when one variable was added to the starting model. Each of the following rows indicated that one additional
variable was added. The final selected test battery is marked in bold in the shaded area. PLOS ONE | https://doi.org/10.1371/journal.pone.0176247
May 1, 2017 Test battery for ACL Fig 4. Distributions of the estimator of knee function and the test battery variables. The distribution of each of the variables included in the
estimator of knee function (w) for each of the two groups, i.e. individuals with an ACL injury and healthy-knee controls. For the estimator of knee
function, values close to 1 indicate a good knee function, and values close to 0 indicate the opposite. Quadriceps concentric strength was measured
in Nm/kg; the one-leg hop for distance in meters, the one-leg balance in number of floor support, and the side hop in number of side hops. https://doi org/10 1371/journal pone 0176247 g004 Fig 4. Distributions of the estimator of knee function and the test battery variables. The distribution of each of the variables included in the
estimator of knee function (w) for each of the two groups, i.e. individuals with an ACL injury and healthy-knee controls. For the estimator of knee
function, values close to 1 indicate a good knee function, and values close to 0 indicate the opposite. Quadriceps concentric strength was measured
in Nm/kg; the one-leg hop for distance in meters, the one-leg balance in number of floor support, and the side hop in number of side hops. https://doi.org/10.1371/journal.pone.0176247.g004 rehabilitation after ACL injury. The test battery may be used to characterize knee function fol-
lowing an ACL injury. In this specific case, for the purpose of suggesting a test battery for long-
term follow-up after ACL injury, we used data from the KACL20-study to investigate which
combination of variables that optimally distinguished ACL-injured and healthy-knee controls,
while still being feasible and clinically relevant. We extracted a test battery with four variables
related to functional tests that may be used as a complement to questionnaires and scores in
the long-term perspective after injury. It is a true challenge to define “good knee function” and
many dimensions need to be considered. Previously reported test batteries have used data
without healthy-knee controls, i.e. they compared knee function between the injured and the
non-injured leg [12, 14, 15]. However, several studies show that there might be decreased bilat-
eral function after a unilateral ACL injury [16–18]. Therefore, it is essential that a test battery
can also reliably discriminate between persons with an ACL injury and healthy-knee controls,
since knee function may vary substantially across individuals, whether injured or not. Discussion One-leg hop, Rise from chair, One-leg balance, Quadriceps eccentric strength
1
OLH-LSI
0.30
2
2
OLH-LSI, B-c
0.27
4
3
OLH-LSI, B-c, Qe-i
0.24
9
4
OLH-i, OLH-LSI, B-c, Qe-i
0.22
9
5
OLH-i, OLH-LSI, B-c, Qe-i, RC-i
0.20
11
6
OLH-i, OLH-LSI, B-c, Qe-i, RC-i, RC-LSI
0.20
11
4. One-leg hop, Side hop, One-leg balance, Quadriceps eccentric strength
1
SH-i
0.32
2
2
SH-i, B-c
0.27
4
3
SH i B c OLH i
0 22
6 2. One-leg hop, Rise from chair, One-leg balance, Quadriceps concentric strength
1
Qc-i
0.29
5
2
Qc-i, OLH-i
0.28
7
3
Qc-i, OLH-i, B-c
0.26
9
4
Qc-i, OLH-i, OLH-c, B-c
0.19
9
5
Qc-i, OLH-i, OLH-c, B-c, RC-i
0.17
11
6
Qc-i, Qc-c, OLH-i, OLH-c, B-c, RC-i,
0.18
11 2. One-leg hop, Rise from chair, One-leg balance, Quadriceps concentric strength 3. One-leg hop, Rise from chair, One-leg balance, Quadriceps eccentric strength 4. One-leg hop, Side hop, One-leg balance, Quadriceps eccentric strength
1
SH-i
0.32
2
2
SH-i, B-c
0.27
4
3
SH-i, B-c, OLH-i
0.22
6
4
SH-i, B-c, OLH-I, Qe-i
0.20
11
5
SH-i, B-c, OLH-i, OLH-c, Qe-i
0.17
11
6
SH-i, B-c, OLH-i, OLH-c, Qe-i, Qe-c
0.17
11 For each of the four examples, the first column shows the test battery of size one corresponding to the For each of the four examples, the first column shows the test battery of size one corresponding to the
lowest misclassification rate. The second column shows the misclassification rate and the total T:E-index
when one variable was added to the starting model. Each of the following rows indicated that one additional
variable was added. The final selected test battery is marked in bold in the shaded area. lowest misclassification rate. The second column shows the misclassification rate and the total T:E-index
when one variable was added to the starting model. Each of the following rows indicated that one additional
variable was added. The final selected test battery is marked in bold in the shaded area. https://doi.org/10.1371/journal.pone.0176247.t003 11 / 18 PLOS ONE | https://doi.org/10.1371/journal.pone.0176247
May 1, 2017 PLOS ONE | https://doi.org/10.1371/journal.pone.0176247
May 1, 2017 Test battery for ACL In the clinical setting and in research several different functional tests are used. Many of
these test results are highly correlated, as corroborated in the present study (see Fig 2), and
thus may provide similar information. Our study investigated which of the nine functional
tests, and their related variables used in the KACL-study, that were able to optimally distin-
guish persons with bad knee function from those with good knee function. Even if adequate
tests are used it is often difficult to interpret the combined information from several tests. In
the construction of the new variable w, the statistical analysis identified two hop tasks from the
same cluster, but from different branches (c.f. Fig 2). They are thus correlated, but represent
different aspects of knee function. The OLH is an explosive maximal hop test for distance,
which is performed in a forward direction and is assumed to be the most common test used in
research and clinics after ACL injury [30, 31]. The SH on the other hand, is a multiple hop test,
reflecting endurance as represented by a maximum number of hops which are performed in a
medio-lateral direction. Thus, the OLH and the SH have different purposes and represent dif-
ferent coordinative function and performance. In addition, these tests challenge dynamic knee
joint stability differently, where most likely the SH exerts higher demands on rotational stabil-
ity (cf. [32, 33]) and may be more critical when evaluating capacity in the clinic [7]. For the
SH, we have recently demonstrated that the test challenges knee stability substantially (data
obtained from the same study population as in the present paper [32]). Both the OLH and SH
tests are considered to have high reliability [12] and are considered highly relevant for evalua-
tion of knee function after ACL injury [34]. In addition, the quadriceps concentric strength (Qc) and the one-leg balance test (B) were
identified in the selected test battery. Regarding knee muscle strength, a review article by Pal-
mieri-Smith et al. concludes that despite rehabilitation, knee muscle weakness is one of the
main dysfunctions following ACL injury [35]. Isokinetic concentric quadriceps strength test-
ing is frequently used in knee rehabilitation, is associated with self-reported knee function [36]
and has high reliability [37]. PLOS ONE | https://doi.org/10.1371/journal.pone.0176247
May 1, 2017 This is
accomplished by the suggested test battery, which distinguishes those with good knee function
from those with less good knee function. PLOS ONE | https://doi.org/10.1371/journal.pone.0176247
May 1, 2017 12 / 18 Balance further adds yet another dimension, where studies indi-
cate a reduced ability following ACL injury for the injured as well as the non-injured leg [16,
25]. The combination of the selected tests implies a range across various clinically relevant
physical dimensions of knee function and thus seems appropriate. In the present study, logistic regression is used to combine the information from the vari-
ables in the test battery, since the model, expressed in the contributing variables, is relatively
easy to interpret. The results showed that the estimator of knee function, w, to a high extent
discriminates individuals with ACL injury from healthy-knee controls. Other statistical
approaches such as factor analysis or principal component analysis could alternatively be used
to summarize information from several variables into a few factors or principal components. These factors or principal components may then be used in further analysis. However, our pri-
mary focus was to propose a test battery consisting of a few variables that are clinically observ-
able and relevant. Since the different test batteries were evaluated using logistic regression, it
was natural to use this approach to combine the available information. Once the dimension
of the problem has been reduced, traditional statistical analyses for univariate data can be
performed. Our final choice of w was based on the calculations of misclassification rates, combined
with expert reasoning regarding feasibility, where the latter is crucial for outcome measures to
be used in the clinic. Interestingly, the misclassification rate typically decreased when variables
were added to the model, up to models with five to six variables; then it increased again. The
misclassification rate depends on the choice of cut-off for classification, here set to 0.5. It
might not be the optimal cut-off, but it can still be used for comparing test batteries. The mis-
classification rate for the final test battery was 0.17, meaning that 17% of the individuals were
misclassified using the chosen model. It would be possible to include additional variables PLOS ONE | https://doi.org/10.1371/journal.pone.0176247
May 1, 2017 13 / 18 Test battery for ACL related to the non-injured leg and thereby reduce the misclassification rate to 0.15, i.e., to clas-
sify two additional individuals correctly. Even though the T:E-index does not increase, a
model with fewer variables is preferable due to interpretability. Moreover, calculation of the
total T:E-index for a test battery was based on the assumption that a functional test is always
performed on both legs, which is praxis in research and clinic, where the non-injured leg is
used as a reference leg for comparisons. When data for a control group is available for compar-
isons, the performance of the non-injured leg may not be as necessary to observe. Our final
estimator of knee function mainly included the variables discussed above that were related to
the injured leg (i). However, for the one-leg balance test, the non-injured leg (c) is used. Indeed, as discussed above both the injured and non-injured leg display balance deficits after
injury [16, 25]. The distributions for each of the variables included in w were similar within the two groups,
as seen in Fig 4. This was not the case for the estimator of knee function where a clear differ-
ence in distribution was shown, demonstrating the capacity to discriminate between injured
and healthy-knee controls. Nevertheless, some errors in misclassification could be expected
considering the difficulty in clarifying objective criteria for who actually has good knee func-
tion, as is a common experience of clinical experts. A wide range of knee functions across indi-
viduals is expected, which may particularly be the case a long time after injury with increasing
age and deconditioning; and also the case for non-injured individuals. In the KACL20 data set,
which included individuals mainly in their forties, there were individuals that had been suc-
cessfully rehabilitated and displayed knee function that was equally good as age-matched
healthy-knee controls. Moreover, some of the controls showed results similar to injured indi-
viduals, and were therefore classified as injured. Indeed, as shown in Fig 3, a variation in knee
function is present within both groups. Lysholm and KOOS scores are well established and commonly used in ACL rehabilitation. Both scores have high reliability and validity [38, 39]. Our proposed estimator of physical knee
function, w, correlated positively with both scores, indicating that it is concurrent with the
individuals self-reported knee function. We also investigated the potential influence of age and
sex, which are individual factors that have been shown to influence some of the outcome vari-
ables. For instance, Tegner activity level and KOOS differ between sexes and are negatively
influenced by age [40–42]. PLOS ONE | https://doi.org/10.1371/journal.pone.0176247
May 1, 2017 Acknowledgments The authors would like to acknowledge Lisbeth Brax Olofsson and Monica Edstro¨m for par-
taking in the data collection. Conclusions The present study shows that with a solid statistical approach, we were able to construct a com-
prehensive and yet feasible test battery for evaluation of knee function after ACL injury which
is appropriate in the long-term perspective. Our estimator of knee function combined several
aspects, and could be said to more coherently represent true knee function than a single vari-
able is able to. Consensus regarding clinical functional test batteries for various stages of reha-
bilitation, along with a general health score and a knee-specific health score, would ensure
evidence-based assessment of knee function in patients following an ACL injury and enable
reliable monitoring of knee function throughout the different phases of rehabilitation. Further,
it would make it possible to carry out powerful retrospective and prospective studies over lon-
ger timespans post-injury while facilitating comparisons across studies. Test battery for ACL movement; such indicators could be crucial factors for the prediction of outcome in knee reha-
bilitation [30]. Kinematic and kinetic variables aimed at capturing movement quality during
coordination tasks could provide such measures [45, 46] but are not so feasible in the clinic. Nevertheless, research using laboratory-based evaluation could be used to identify and validate
the most important clinical outcome measures. For instance, Di Stasi et al. used kinematics
and kinetic assessments during gait to validate a clinical test battery in relation to return-to-
sport criteria [47]. Kinematically-derived variables may also be used to characterize how chal-
lenging different functional tests are with regard to dynamic knee stability and compare func-
tions across groups of individuals [32, 48]. However, kinematic and kinetic analyses usually
generate huge/large numbers of variables, and there is a need to reduce these into the most
representative parameters. In this context the present statistical approach would be of particu-
lar value, especially in large data sets obtained from various functional tasks. Thus, the model
may be used to identify appropriate variables rather then arbitrarily selecting them. In addition to kinematic and kinetic recordings of functional tests, proprioception and lax-
ity may influence knee function [49] and it would be desirable to include such measures to
ensure that as many dimensions of physical knee function as possible were to be considered. Altogether, our proposed test battery is comprised of four different test variables, reasonably
feasible and, when combined, proven to reliably discriminate knee function at least in the long
term after injury to the knee. An advantage with this test battery compared to previously pro-
posed ones [12–15], is that it includes both functional coordination tests and more direct knee
muscle strength measurements. Further testing of the measurement properties (e.g., validity,
reliability, sensitivity) of the suggested test battery in other long-term study populations, more
than one year after ACL injury, is warranted. Author Contributions Conceived and designed the experiments: LS ET PR CH. Physical capacity, including balance, strength and hop ability is
likewise reduced with increasing age and lower for women than for men [43, 44]. Similarly,
our study did not show any correlation of estimated knee function with age, and no difference
between sexes. This may depend on the fact that our material is based on a long-term follow-
up of knee function after ACL injury and thus covers ages between 35 and 63 years. These
older age groups might be more homogenous than the younger athletes mainly tested in the
above-mentioned studies, although many of the individuals with ACL injury in our data were
athletes prior to injury. The controls were matched for age and sex but, although strived for,
there was no matching of physical activity level. The tests were performed by cohorts in a unique long-term follow-up with comparatively
extensive testing. However, for our aims this is a limitation, since a larger reduction in knee
function might be expected shortly after an injury. Thus, the suggested test battery needs to be
further validated in other cohorts. In addition, the statistical approach should be applied to
data obtained from shorter-term follow-ups after injury to verify the usefulness of the pro-
posed test battery for other stages of rehabilitation. The data used from the KACL20-study
includes nine different common functional tests as well as established scores and question-
naires adopted in rehabilitation after ACL injury, and hence seem well suited for our aims. Even so, there are many other existing tests that could have been used, e.g. triple-jump,
running-eight test. Recent research has also identified the lack of measures of quality of PLOS ONE | https://doi.org/10.1371/journal.pone.0176247
May 1, 2017 14 / 18 PLOS ONE | https://doi.org/10.1371/journal.pone.0176247
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Score (KOOS)—development of a self-administered outcome measure. J Orthop Sports Phys Ther. 1998; 28(2):88–96. https://doi.org/10.2519/jospt.1998.28.2.88 PMID: 9699158 11. Tegner Y, Lysholm J. Rating systems in the evaluation of knee ligament injuries. Clin Orthop Relat Res. 1985;(198):43–9. PMID: 4028566 12. Gustavsson A, Neeter C, Thomee P, Silbernagel KG, Augustsson J, Thomee R, et al. Conceived and designed the experiments: LS ET PR CH. Performed the experiments: LS ET PR CH. Performed the experiments: LS ET PR CH. Analyzed the data: LS ET PR CH. Analyzed the data: LS ET PR CH. Contributed reagents/materials/analysis tools: LS ET PR CH. Wrote the paper: CH ET LS PR. 15 / 18 15 / 18 PLOS ONE | https://doi.org/10.1371/journal.pone.0176247
May 1, 2017 Test battery for ACL 18.
Urbach D, Nebelung W, Weiler HT, Awiszus F. Bilateral deficit of voluntary quadriceps muscle activa-
tion after unilateral ACL tear. Med Sci Sports Exerc. 1999; 31(12):1691–6. PMID: 10613416 References A test battery for
evaluating hop performance in patients with an ACL injury and patients who have undergone ACL
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s00167-006-0045-6 PMID: 16525796 13. Neeter C, Gustavsson A, Thomee P, Augustsson J, Thomee R, Karlsson J. Development of a strength
test battery for evaluating leg muscle power after anterior cruciate ligament injury and reconstruction. Knee Surg Sports Traumatol Arthrosc. 2006; 14(6):571–80. https://doi.org/10.1007/s00167-006-0040-y
PMID: 16477472 14. Noyes FR, Barber SD, Mangine RE. Abnormal lower limb symmetry determined by function hop tests
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036354659101900518 PMID: 1962720 15. Reid A, Birmingham TB, Stratford PW, Alcock GK, Giffin JR. Hop testing provides a reliable and valid
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gle-leg stance in patients with untreated anterior cruciate ligament injury. Knee Surg Sports Traumatol
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11052492 18. Urbach D, Nebelung W, Weiler HT, Awiszus F. Bilateral deficit of voluntary quadriceps muscle activa-
tion after unilateral ACL tear. Med Sci Sports Exerc. 1999; 31(12):1691–6. PMID: 10613416 PLOS ONE | https://doi.org/10.1371/journal.pone.0176247
May 1, 2017 16 / 18 Test battery for ACL 19. Neeb TB, Aufdemkampe G, Wagener JH, Mastenbroek L. Assessing anterior cruciate ligament injuries:
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Sports Phys Ther. 1997; 26(6):324–31. https://doi.org/10.2519/jospt.1997.26.6.324 PMID: 9402569 20. Reinke EK, Spindler KP, Lorring D, Jones MH, Schmitz L, Flanigan DC, et al. Hop tests correlate with
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doi.org/10.1111/sms.12434 PMID: 25728035 34. Thomee R, Kaplan Y, Kvist J, Myklebust G, Risberg MA, Theisen D, et al. Muscle strength and hop per-
formance criteria prior to return to sports after ACL reconstruction. Knee surgery, sports traumatology,
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18503875 36. Moisala AS, Jarvela T, Kannus P, Jarvinen M. Muscle strength evaluations after ACL reconstruction. Int
J Sports Med. 2007; 28(10):868–72. https://doi.org/10.1055/s-2007-964912 PMID: 17357967 37. Pua YH, Bryant AL, Steele JR, Newton RU, Wrigley TV. Isokinetic dynamometry in anterior cruciate lig-
ament injury and reconstruction. Ann Acad Med Singapore. 2008; 37(4):330–40. PMID: 18461219 38. Salavati M, Akhbari B, Mohammadi F, Mazaheri M, Khorrami M. Knee injury and Osteoarthritis Out-
come Score (KOOS); reliability and validity in competitive athletes after anterior cruciate ligament
reconstruction. Osteoarthritis Cartilage. 2011; 19(4):406–10. https://doi.org/10.1016/j.joca.2011.01.010
PMID: 21255667 39. Briggs KK, Lysholm J, Tegner Y, Rodkey WG, Kocher MS, Steadman JR. The reliability, validity, and
responsiveness of the Lysholm score and Tegner activity scale for anterior cruciate ligament injuries of PLOS ONE | https://doi.org/10.1371/journal.pone.0176247
May 1, 2017 17 / 18 Test battery for ACL the knee: 25 years later. The American journal of sports medicine. 2009; 37(5):890–7. https://doi.org/
10.1177/0363546508330143 PMID: 19261899 the knee: 25 years later. The American journal of sports medicine. 2009; 37(5):890–7. https://doi.org/
10.1177/0363546508330143 PMID: 19261899 40. Briggs KK, Steadman JR, Hay CJ, Hines SL. Lysholm score and Tegner activity level in individuals with
normal knees. The American journal of sports medicine. 2009; 37(5):898–901. https://doi.org/10.1177/
0363546508330149 PMID: 19307332 41. Frobell RB, Svensson E, Gothrick M, Roos EM. Self-reported activity level and knee function in amateur
football players: the influence of age, gender, history of knee injury and level of competition. Knee Surg
Sports Traumatol Arthrosc. 2008; 16(7):713–9. https://doi.org/10.1007/s00167-008-0509-y PMID:
18350275 42. Paradowski PT, Bergman S, Sunden-Lundius A, Lohmander LS, Roos EM. Knee complaints vary with
age and gender in the adult population. Population-based reference data for the Knee injury and Osteo-
arthritis Outcome Score (KOOS). BMC Musculoskelet Disord. 2006; 7:38. https://doi.org/10.1186/
1471-2474-7-38 PMID: 16670005 43. Yoon TS, Park DS, Kang SW, Chun SI, Shin JS. Isometric and isokinetic torque curves at the knee
joint. Yonsei Med J. 1991; 32(1):33–43. https://doi.org/10.3349/ymj.1991.32.1.33 PMID: 1877253 44. Ageberg E, Zatterstrom R, Friden T, Moritz U. Individual factors affecting stabilometry and one-leg hop
test in 75 healthy subjects, aged 15–44 years. Scand J Med Sci Sports. 2001; 11(1):47–53. PMID:
11169235 45. Fox AS, Bonacci J, McLean SG, Spittle M, Saunders N. What is Normal? PLOS ONE | https://doi.org/10.1371/journal.pone.0176247
May 1, 2017 49.
Roberts D, Ageberg E, Andersson G, Friden T. Clinical measurements of proprioception, muscle
strength and laxity in relation to function in the ACL-injured knee. Knee Surg Sports Traumatol Arthrosc.
2007; 15(1):9–16. https://doi.org/10.1007/s00167-006-0128-4 PMID: 16791634 References Female Lower Limb Kinematic
Profiles During Athletic Tasks Used to Examine Anterior Cruciate Ligament Injury Risk: A Systematic
Review. Sports Med. 2014; 44(6):815–32. https://doi.org/10.1007/s40279-014-0168-8 PMID:
24682949 46. Hewett TE, Myer GD, Ford KR, Heidt RS Jr., Colosimo AJ, McLean SG, et al. Biomechanical measures
of neuromuscular control and valgus loading of the knee predict anterior cruciate ligament injury risk in
female athletes: a prospective study. The American journal of sports medicine. 2005; 33(4):492–501. https://doi.org/10.1177/0363546504269591 PMID: 15722287 47. Di Stasi SL, Logerstedt D, Gardinier ES, Snyder-Mackler L. Gait patterns differ between ACL-recon-
structed athletes who pass return-to-sport criteria and those who fail. Am J Sports Med. 2013; 41
(6):1310–8. https://doi.org/10.1177/0363546513482718 PMID: 23562809 48. Sole G, Tengman E, Grip H, Hager CK. Knee kinematics during stair descent 20years following anterior
cruciate ligament rupture with and without reconstruction. Clin Biomech (Bristol, Avon). 2016; 32:180–
6. 49. Roberts D, Ageberg E, Andersson G, Friden T. Clinical measurements of proprioception, muscle
strength and laxity in relation to function in the ACL-injured knee. Knee Surg Sports Traumatol Arthrosc. 2007; 15(1):9–16. https://doi.org/10.1007/s00167-006-0128-4 PMID: 16791634 18 / 18
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A CASE OF HUNTINGTONʼS CHOREA
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The journal of nervous and mental disease
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public-domain
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PHILADELPHIA NEUROLOGICAL SOCIETY 162 PHILADELPHIA NEUROLOGICAL SOCIETY Examination of the eyes shows that the pupils react promptly to
direct and indirect light stimulation and also to accommodation. The
ocular movements are unimpaired and the visual fields show no con¬
tracture. Media clear, optic discs are of good color; retinal arteries
and veins are of normal color and size. The patient remained under observation until February 27, 1908, when
Dr. H. Augustus Wilson performed a subcutaneous tenotomy of the
tendo-Achillis on both sides with the result that some two months later
the patient was . able to stand with assistance and to be about in a wheeled
chair with comfort. That the patient, whose case has here been placed on record suffered
from a diffuse form of adiposis dolorosa, there can be no doubt. That
cord changes, that is, a lateral sclerosis was also present, is a logical
inference from the symptoms presented. Whether in this case a" lateral
sclerosis merely occurred in a patient suffering from adiposis dolorosa or
whether there was an etiological relationship between the two affections,
the facts do not enable us to decide. Dr. Dercum said he wished to speak of the name Adiposis Dolorosa
which has been widely accepted. German writers substitute the word
adipositas ” for the word adiposis. Adiposis has been used by English
writers for several generations in spite of the objection that it is a hybrid
word compounded of both a Greek and .a Latin root. Properly speaking,
all parts of a word should be derived from roots from the same language. At the same time usage has confirmed the word adiposis, just as it has
confirmed other hybrid words freely used by German as well as by Eng¬
lish writers, such as “ terminology.” Further, adipositas, while derived
from roots both of which are Latin is also an artificial—a made—word. Not a single Latin writer ever used it. Indeed the proper Latin word
is “ obesitas ” and if we were to take the attitude of purists, we ought
to say obesitas dolorosa and not adipositas dolorosa. CHICAGO NEUROLOGICAL SOCIETY CHICAGO NEUROLOGICAL SOCIETY By Eugene Lindauer, M.D. J. Z., white, male, 43 years old, born in the United States, bridge
builder. His father developed a tremor after a fall, and had to be
committed to an insane asylum, where he died at the age of 64, several
years after the onset. Family history otherwise negative. Patient at the
age of 31 fell a distance of 50 feet to the ground; he struck the right
side of his forehead. He was paralyzed for some time, but recovered
full power ultimately. He developed a tremor a short time after the
accident, which has never left him since. At the present time he shows
well-marked choreic movements, affecting the entire body, but more
particularly the left side; these movements disappear when he is asleep,
but sometimes are so marked as to keep him from going to sleep. There
is a certain unsteadiness about the eyeballs, this being due probably to a
participation of the external eye muscles in the disease. Speech is almost
impossible, partly because of the disturbance in the muscles of the tongue,
and partly because of the patient’s mental condition. He comprehends
with difficulty, and if he answers at all, does so with a word or two, or a
shake of the head. He sits for hours without taking interest in his sur- PHILADELPHIA NEUROLOGICAL SOCIETY 163 roundings. He does not show the irascibility which patients with his
disease are said to have, nor does he soil himself or make a general
nuisance of himself or his attendants. Memory is very poor. Reflexes
are diminished, plantar irritation provokes dorsal flexion. He does not
seem to be able to smell. Dr; Alfred Gordon stated that the case was quite typical of Hunting¬
ton’s chorea. The only peculiarity about it was the involvement of the
upper part of the face. In the typical Huntington’s chorea only the
lower half of the face is affected. The eye globes are usually not in¬
volved. In this case they are. An interesting point in the history of the
case is the headache, which goes hand in hand with what we know of
the pathological anatomy. This is supposed to be a meningoencephalitis. CLINICAL OBSERVATIONS OF PSYCHOSES PRESENTING
THE EDDY CULT AS A COMPLICATION By Richard Dewey, M.D. This was a study of 8 cases in which the psychoses developed in con¬
nection with espousal of Eddyism. They were selected as typical from
a large number seen during the years since Christian Science, so-called,
became a method of treatment. The Eddyism was, of course, regarded as occasion rather than cause
of the mental disease, since insanity in all cases has a constitutional back¬
ground of instability. g
y
The paper was intended as a slight contribution to the question whether
there was anything characteristic, i. e., whether the psychopathic reactions
observed showed a particular form. The 8 cases consisted of—3 of paranoia, 4 of manic-depressive insanity
and 1 of psychasthenia. There was one of recovery (manic-depressive)
and 1 of virtual recovery (psychasthenia), as the patient again returned
to her accustomed work of teaching; the other cases all became chronic
with indications of an incurable state. One of the marked psychopathic
phases was panophobia for the pharmacopeia (in connection with these
phobias it is to be mentioned that 3 patients showed marked delusions of
poisoning). This was present in 6 of the 8 cases—a natural outgrowth
of the Eddy doctrine. Two of the cases showed a disposition to religious,
fasting; 3 manifested folie du doute; 3 had obsessions of evil spirits;
4 showed erotism; suicide and attempt at self injury were present in 5 cases. The group of the 3 paranoiacs all manifested continuous and un¬
wavering adherence to Eddyite ideas with an exalted frame of mind. These patients were in good physical condition. In the manic-depressive group there was I recovery with abandonment
of all Eddyite ideas. The three that became chronic cases showed intense
depression and a sense of failure and disappointment which might be
attributable to the peculiar contradictory and confusing method of thought
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The Analysis Of Marketing Mix Strategy Of Wood Processing In Increasing The Salling Margin
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Eduvest – Journal of Universal Studies
Volume 1 Number 12, December 2021
p- ISSN 2775-3735 e-ISSN 2775-3727 Eduvest – Journal of Universal Studies
Volume 1 Number 12, December 2021
p- ISSN 2775-3735 e-ISSN 2775-3727 Bunyamin, Sultan Iskandar (2021). The Analysis of Marketing Mix
Strategy of Wood Processing in Increasing the Salling Margin. Journal Eduvest. 1(12): 1428-1442
How to cite:
E-ISSN:
2775-3727
Published by:
https://greenpublisher.id/
THE ANALYSIS OF MARKETING MIX STRATEGY OF WOOD
PROCESSING IN INCREASING THE SALLING MARGIN
Bunyamin, Sultan Iskandar
STIE YPUP Makassar, Indonesia
E-mail: bunyaminypup66@gmail.com, Sultanbaru1965@gmail.com
ARTICLE INFO
ABSTRACT
Received:
The purpose of this study is to analyze whether the
November, 26th
application of the marketing mix strategy through the
2021
SWOT formulation can increase the sales volume of
Revised:
December, 17th
2021
Approved:
December, 19th
processed wood. This research was conducted at the
office of PT. Three "Rs" located at Jalan Sultan Abdullah
No. 75 Makassar, with the research time required by the
author is approximately 3 (three) months starting from
the beginning of May to July 2009. The population in this
study are all consumers who use processed wood
2021
products, for housing development purposes in South
Sulawesi, especially in the city of Makassar in the last
year, so the population in this study is unknown. While the
sample is as many as 300 respondents. Sampling was used
using a non-probability sampling approach and the
judgment method. Based on the results of the application
of SWOT analysis, it appears that the strategy used by PT. The three "Rs" of Makassar in increasing processed wood
sales are implementing the SO strategy, which is to make
strengths to take advantage of opportunities, then the
WO strategy by minimizing weaknesses to maintain
opportunities, then the ST strategy using strengths to
overcome threats and WT strategies that minimize
weaknesses and avoid threats. KEYWORDS
Mix Strategy, Marketing, Salling Margin INTRODUCTION Nowadays, the efforts made by every company, especially the timber industry,
are through improving the company's performance in order to maintain the continuity or
continuity of the company's life, so that to improve the company's performance it needs to
be supported by an increase in processed wood sales, in order to influence the
achievement of optimal profits (Berry & Cooper, 1999) (Palandeng, Kindangen, Tumbel,
& Massie, 2018) (Liu, Wang, & Li, 2018). g
One of the efforts made by every timber industry company in increasing sales of
processed wood is to carry out marketing activities, where marketing activities are
intended to facilitate sales from processed wood producers to consumers, so with the
important role of marketing carried out by the company it needs to be supported by the
implementation of marketing mix strategies, such as: product, price, distribution and
promotion (Thabit & Raewf, 2018) (Ciriković, 2014) (Išoraitė, 2016). Marketing mix strategy is a fundamental tool that is planned to achieve company
goals by developing a sustainable competitive advantage through the market entered and
marketing programs used to serve the target market in increasing competitive advantage
(Constantinides, 2006) (Pomering, 2014). With the importance of a marketing strategy,
one of the targets carried out by every timber industry company is to increase sales of
processed wood and increase competitiveness in the marketing of processed wood
(Pomering, 2017). To support the success of an accurate marketing mix strategy, a swot analysis is
needed, so that the company can find out the strengths, weaknesses, opportunities, and
threats faced by the company (Ling, Pham, & Hoang, 2009). Each business unit must be
evaluated for its strengths and weaknesses periodically, so that the company has relatively
greater strengths compared to its competitors (Fahriana & Huda, 2019). Opportunities
owned by timber industry companies are government policies in encouraging increasing
sales volume, domestic and foreign banks and at the same time being a large enough
opportunity for regional entrepreneurs to manage existing resources in the region in an
effort to increase timber industry activities in the region, while The threats faced by the
timber industry companies are the increasing level of competition among the timber
industry entrepreneurs, the cost of shipping transportation, the fluctuating exchange rate
of the rupiah against the dollar, making it quite difficult for the company to increase its
sales volume. This work is licensed under a Creative Commons
Attribution-ShareAlike 4.0 International This work is licensed under a Creative Commons
Attribution-ShareAlike 4.0 International INTRODUCTION The importance of the processed wood marketing mix strategy in the timber
industry company (Halaj, Sedliačiková, & Malá, 2018), the target to be achieved by the
company is to increase the competitive advantage in marketing processed wood in
addition to increasing the sales volume of processed wood, where it is necessary to know
that in marketing processed wood, it is marked by the large number of wood industries
that produce processed wood emerging, with the number of timber industries that have
sprung up resulting in intense competition in the marketing of processed wood both in
terms of quality or quality of processed wood products and from the selling price of
processed wood, timber industry companies are required to be able to develop an accurate
marketing mix strategy in increasing competitiveness in processed wood marketing
(Ginsberg & Bloom, 2004) (Adams, 2016) (Baker, 2014). The importance of the role of the processed wood marketing mix strategy for The importance of the role of the processed wood marketing mix strategy for THE ANALYSIS OF MARKETING MIX STRATEGY OF WOOD
PROCESSING IN INCREASING THE SALLING MARGIN Bunyamin, Sultan Iskandar
STIE YPUP Makassar, Indonesia
E-mail: bunyaminypup66@gmail.com, Sultanbaru1965@gmail.com
ARTICLE INFO
ABSTRACT Bunyamin, Sultan Iskandar
STIE YPUP Makassar, Indonesia
E-mail: bunyaminypup66@gmail.com, Sultanbaru1965@gmail.com STIE YPUP Makassar, Indonesia
E-mail: bunyaminypup66@gmail.com, Sultanbaru1965@gmail.com The purpose of this study is to analyze whether the
application of the marketing mix strategy through the
SWOT formulation can increase the sales volume of
processed wood. This research was conducted at the
office of PT. Three "Rs" located at Jalan Sultan Abdullah
No. 75 Makassar, with the research time required by the
author is approximately 3 (three) months starting from
the beginning of May to July 2009. The population in this
study are all consumers who use processed wood
products, for housing development purposes in South
Sulawesi, especially in the city of Makassar in the last
year, so the population in this study is unknown. While the
sample is as many as 300 respondents. Sampling was used
using a non-probability sampling approach and the
judgment method. Based on the results of the application
of SWOT analysis, it appears that the strategy used by PT. The three "Rs" of Makassar in increasing processed wood
sales are implementing the SO strategy, which is to make
strengths to take advantage of opportunities, then the
WO strategy by minimizing weaknesses to maintain
opportunities, then the ST strategy using strengths to
overcome threats and WT strategies that minimize
weaknesses and avoid threats. Received:
November, 26th
2021
Revised:
December, 17th
2021
Approved:
December, 19th
2021 Mix Strategy, Marketing, Salling Margin Bunyamin, Sultan Iskandar (2021). The Analysis of Marketing Mix
Strategy of Wood Processing in Increasing the Salling Margin. Journal Eduvest. 1(12): 1428-1442 Bunyamin, Sultan Iskandar Bunyamin, Sultan Iskandar RESEARCH METHOD This research was conducted at the office of PT. Three "Rs" located at Jalan Sultan
Abdullah No. 75 Makassar, with the research time required by the author is
approximately 3 (three) months starting from the beginning of May to July 2009. The
population in this study are all consumers who use processed wood products, for housing
development purposes in South Sulawesi, especially in the city of Makassar in the last
year, so the population in this study is unknown. While the sample is as many as 300
respondents. Sampling was used using a non-probability sampling approach and the
judgment method. While the data obtained are sourced from: primary data, namely data that comes
from the results of field research through interviews and direct observation. Secondary
data is data obtained from various reports and documentation at PT. Three "Rs", which
are made periodically and are closely related to this research. Data collection methods
used in this study were observation, interviews and documentation. The Analysis of Marketing Mix Strategy of Wood Processing in Increasing the Salling
Margin
1429 1429 Eduvest – Journal of Universal Studies
Volume 1 Number 12, December 2021 every timber industry company, it needs to be supported by an evaluation of the
marketing mix strategy used by timber companies in marketing processed wood. every timber industry company, it needs to be supported by an evaluation of the
marketing mix strategy used by timber companies in marketing processed wood. The goals and objectives of each timber industry company are to find out the
implementation of the marketing mix strategy carried out by the timber industry company
in the marketing of processed wood. Company PT. Tiga "R" Makassar is a company engaged in the timber industry,
which in carrying out its activities as a company that produces processed wood, employs
85 workers. Marketing of processed wood, then PT. Tiga "R" Makassar produces
competitive processed wood and high quality products. One of the factors causing the decline in sales of processed wood is due to the
emergence of many competing companies engaged in the timber industry, resulting in
intense competition in marketing which means that it can also affect sales volume in the
marketing of processed wood. The existence of a phenomenon that occurs in the company, it needs to be
supported by an analysis of marketing strategies. Where in analyzing the marketing
strategy, the swot model is used whose purpose is to see the advantages, weaknesses,
opportunities and threats in the marketing of processed wood. The purpose of this study is to analyze whether the application of the marketing
mix strategy through the SWOT formulation can increase the sales volume of processed
wood. Bunyamin, Sultan Iskandar The use of SWOT analysis, namely in order to develop a strategy to calm business
competition with the concept of cooperation or competition. In its current development, SWOT analysis is used in formulating strategic
business planning, which aims to develop long-term strategies so that the company's arch
and goals can be achieved clearly and decisions can be made immediately, in the face of
competitors. p
In connection with the application of SWOT analysis, PT. Tiga "R" Makassar is a
company engaged in the timber industry so that in managing its activities as a producer of
processed wood, one of the efforts made by PT. The three "Rs" of Makassar are
increasing competitive advantage in the sale of processed wood. Where in carrying out its business activities as a timber industry, the PT. Three
Makassar "Rs" intend to evaluate the development of processed wood sales over the last
three years ii, from 2006 to 2008. As supporting data, the following data will be presented on sales of processed
wood at PT. Three Makassar "Rs" from 2006 to 2008, which can be seen in the following
table. Table 1. Sales of Processed Timber at PT. Three "R" Makassar from 2006 to 2008 Table 1. Sales of Processed Timber at PT. Three "R" Makassar from 2006 to 2008
Year
Sales of Processed Wood (Rp)
2006
7.225.063.830
2007
7.757.965.355
2008
7.458.562.255
Total
22.441.591.410
Average
7.480.530.470
Source : PT. Tiga "R" Makassar From the sales data of drugs, drugs at the company PT. Three "R" Makassar for the
last three years (2006 to 2008) showed an average increase every year. y
g
y y
Based on the data above, it will then be presented the development of processed
wood sales at PT. Three "R" Makassar in 2004 to 2008 through the following table: g
g
Table 2. Sales of Processed Timber during 2006 to 2008 g
Year
Sales of Processed Wood
(Rp)
Sales Progress
Rp
%
2006
7.225.063.830
-
-
2007
7.757.965.335
532.901.505
7,37
2008
7.458.562.225
(299.403.110)
(3,86)
Average increase
116.749.197
1,75
Source : PT. Tiga "R" Makassar From the data regarding the development of processed wood sales for the last 3
years, where the average sales have increased by Rp. 116,749,197.5 per year. It can be
detailed that in 2007 sales of processed wood increased by Rp. 532,901,505 or 7.37% and
in 2008 decreased by Rp. 299,403,110 or 3.86%. A. Analysis of Processed Timber Sales Development A. Analysis of Processed Timber Sales Development A. Analysis of Processed Timber Sales Development
In the current era of globalization, it shows very fast changes, especially in all
fields. This is due to pressure from internal and external factors, so it is necessary for
business people to be able to adapt to the changes that occur. Pressures from internal factors in the form of innovations and strength in
competing through improving service quality, improving human resources and external
factors, namely technological advances, social, economic, political changes, government
regulations and competitors. These internal and external factors are the driving force that
requires business people to be able to anticipate and adapt to any changes in order to
continue to exist, so that changes that occur are not obstacles or threats, but can be
opportunities to develop businesses and achieve big profits. http://eduvest.greenvest.co.id 1430 B. Evaluation Based on SWOT Analysis in Increasing Processed Timber Sale B. Evaluation Based on SWOT Analysis in Increasing Processed Timber Sales SWOT analysis in the marketing of drugs, has a significant relationship on the
competitive situation in the timber industry business. One of the starting points in this Bunyamin, Sultan Iskandar The decline in sales of processed wood
was due to the intense competition with other processed wood competitors. With the decline in drug sales, one of the efforts made by the company is to
increase competitive advantage. Where with the increase in competitive advantage it will
be able to affect the sale of processed wood. 3. Opportunities (opportunities) 3. Opportunities (opportunities) The opportunities that affect the marketing of processed wood can be described as
ollows: a. Government policy in increasing export volume. b. Quite a number of customers. c. Large market share due to increased demand. d. Regional autonomy. 2. Weaknesses (weaknesses) 2. Weaknesses (weaknesses) In marketing processed wood, there are weaknesses faced by PT. Three "R"
M k
hi h
b d
ib d
f ll In marketing processed wood, there are weaknesses faced by PT. Three "R"
Makassar which can be described as follows: g p
,
y
Makassar which can be described as follows: Makassar which can be described as follows: a. Bargaining position with less/low ISA (International Saw Mill and Word a. Bargaining position with less/low ISA (International Saw Mill and Word
Working Association). a. Bargaining position with
Working Association). g
g p
(
Working Association). Working Association). b. Limited quality of human resources. c. Suboptimal product planning. d. Lack of product promotion. e. Old machinery and equipment. f. The human resource quality development system is not good because there is
no training for employees. The Analysis of Marketing Mix Strategy of Wood Processing in Increasing the Salling
Margin
1431 1431 Eduvest – Journal of Universal Studies
Volume 1 Number 12, December 2021 discussion is through an evaluation of the SWOT analysis, which is carried out based on
the strengths and weaknesses that have been carried out in increasing market
opportunities. discussion is through an evaluation of the SWOT analysis, which is carried out based on
the strengths and weaknesses that have been carried out in increasing market
opportunities. As for the purpose of doing a SWOT analysis in the marketing of Processed
Timber, especially at PT. The three Makassar "Rs" are as follows: 1. Knowing the right marketing strategy in increasing sales of processed wood. 1. Knowing the right marketing strategy in increasing sales of processed wood. 1. Knowing the right marketing strategy in increasing sales of processed wood. 2.By using SWOT analysis, it can be seen how the strengths, weaknesses,
opportunities and threats faced by PT. Three "R" Makassar in the face of this increasingly
fierce competition. 2.By using SWOT analysis, it can be seen how the strengths, weaknesses,
opportunities and threats faced by PT. Three "R" Makassar in the face of this increasingly
fierce competition. Referring to the goals and objectives of conducting an evaluation of the use of
SWOT analysis, in this description it will be easy to know what are the strengths and
weaknesses of PT. The three "Rs" of Makassar are what opportunities are able to support
developments in marketing performance and what threats will later be faced through the
use of SWOT analysis (Strengths, Weaknesses, Opportunities, Threats). For more details on the application of SWOT analysis at PT. Three "R" Makassar,
can be described one by one as follows: 1. Strengths: To see how the strength in the marketing of processed wood lies in the
following aspects: a. Has the quality of processed wood that meets the standards. b. Have HPH (Forest Use Rights). c. Has various types of processed wood. d. Has a low number of employees/HR. p y
e. Having sufficient funds but not being used for promotional activities optimally. f. Has an extensive distribution network. f. Has an extensive distribution network. g. Cheaper/affordable prices. h. Better processing. i. Ability to deliver (delivery) on time. Bunyamin, Sultan Iskandar b. The price of similar products from other companies is cheaper. b. The price of similar products from other companies is cheaper. c. Increased competition because the number of new companies in this industry is
increasing every year. g
y y
d. Competitor companies increase the quality and quantity of production, e. Rupiah exchange rate fluctuations. e. Rupiah exchange rate fluctuations. f. Increased export costs due to the weakening of the Rupiah exchange rate. f. Increased export costs due to the weakening of the Rupiah
C. Determination of Variables / Attributes in SWOT Analysis f. Increased export costs due to the weakening of the Rupiah exch
C D t
i
ti
f V
i bl
/ Att ib t
i SWOT A
l
i Determination of variables/attributes in the SWOT analysis in the marketing of
processed wood is intended to be able to formulate a marketing strategy through analysis
that will be used in marketing processed wood. With the formulation of a marketing strategy for processed wood, it can be seen
that which strategy is more adequate to be used in marketing processed wood, especially
at PT. Three "R" Makassar. In marketing processed wood, the results of the analysis of respondents' responses
to internal and external attributes are used in the application of SWOT analysis. For more details, the results of respondents' responses to the attributes of external
factors and internal factors will be presented which can be seen in table 5 below: p
Table 3. Results of Respondents' Responses to Attribute Internal Factors And
E
l F p
p
External Factors
Response
Description
Yes
No
People
%
People
%
A. STRENGTH
1. Has high quality processed wood
because it follows quality standards
39
2
36
8
2. Have HPH
43
7,33
32
2,67
3. Has various types of processed wood
39
2
36
8
4. Have a large number of employees/HR
and cheap
40
3,33
35
6,67
5. Have a large enough promotional fund
47
2,67
28
7,33
6. Has an extensive distribution network
43
7,33
32
2,67
7. Cheaper/affordable price
39
2
36
8
8. Better processing
41
4,67
34
5,33
9. Ability to deliver (delivery) on time
46
1,33
29
8,67
B. WEAKNESS
1. Bargaining position with less/low ISA
39
52
36
48
2. Limited quality of human resources
39
52
36
48
3. Suboptimal product planning
42
56
33
56
4. 4. Threats (threats) 4. Threats (threats) Factors that pose a threat to companies in marketing processed wood can be
described as follows: Factors that pose a threat to companies in marketing processed wood can be
described as follows: a. Competitors are mostly companies that have a lot of raw materials and skilled
labor. http://eduvest.greenvest.co.id http://eduvest.greenvest.co.id 1432 Bunyamin, Sultan Iskandar Less product promotion
41
54,67
34
45,33
5. Old machinery and equipment
40
53,33
35
46,67
6. The
human
resource
quality 42
56
33
44
development
system
is
not
good
because there is no training for
employees
7. The reward and punishment system is
not applied properly, resulting in a high
turnover rate
40
53,33
35
46,67 The Analysis of Marketing Mix Strategy of Wood Processing in Increasing the Salling
Margin
1433 Eduvest – Journal of Universal Studies
Volume 1 Number 12, December 2021 Eduvest – Journal of Universal Studies
Volume 1 Number 12, December 2021 C. OPPORTUNITY
1. Government policy in increasing export 42
6
33
44
volume
2. A large number of customers
43
7,33
32
42,67
3. Large market share due to
demand
increased 38
0,66
37
49,33
4. Regional autonomy
40
3,33
35
46,67
D. THREAT
1. Competitors are mostly companies that have a lot
of raw materials and skilled workers
43
57,33 32
42,67
2. The price of similar products from other
companies is cheaper
39
52
36
48
3. Increased competition because the number of
new companies in this industry is increasing
every year
41
54,67 34
45,33
4. Competing companies improve the quality and
quantity of production
43
56
32
44
5. Rupiah exchange rate fluctuations
42
52
33
46
6. Increased export costs due to the weakening of
the rupiah exchange rate
39
52
36
48
Source: Processed Data Based on table 5, namely the responses of respondents on the attributes of internal
factors and external factors on strength, the first statement is Having high quality
processed wood because it follows quality standardization, then those who give the
answer agree are 39 people or 52% and those who give the answer disagree are equal to
36 people or 48%. p
p
Then the strength in marketing processed wood has an HPH, 43 people who gave
an agree answer or 57.33% and 32 people who gave a disagree answer or 42.67%. Having varied types of processed wood, 39 people or 52% of those who gave agree
or disagreed answers and 36 people or 48% who gave disagreed answers. Having a large and cheap number of employees/HR, then those who give agree
answers are 40 people or 53.33% and those who give disagree answers are 35 people or
46.67%. Bunyamin, Sultan Iskandar the answer agreed were 42 people or 56%, and 33 people disagreed or 56%. the answer agreed were 42 people or 56%, and 33 people disagreed or 56%. Product planning that is less than optimal, the respondents who gave the answer
agreed by 41 people or 54.67% and disagreed by 34 people or 45.33%. Product promotion is lacking, so the respondents who gave the answer agreed were
41 people or 54.67% and disagreed by 34 people or 45.33%. p
p
g
y
p
p
Machines and equipment that are old, the respondents who gave the answer agreed
by 40 people or 53.33% and disagreed by 35 people or 46.67%. The human resource quality development system is not good because there is no
training for employees, so the respondents who give the answer agree are 42 people or
56% and disagree are 33 people or 44 The reward and punishment system that is not applied properly, so that the turnover
rate is high, the respondents who gave the answer agreed were 40 people or 53.33% and
disagreed by 35 people or 46.67%. Opportunity factor in the marketing strategy of processed wood, namely
government policy in increasing export volume, the respondents who gave the answer
agreed were 42 people or 56% and disagreed was 33 people or 44%. Other Opportunities The number of customers is quite a lot, so the respondents
who give the answer agree are 43 people or 57.33% and disagree are 32 people or
42.67%. A large market share due to increased demand, the respondents who gave the
answer agreed by 38 people or 50.66% and disagreed by 37 people or 49.33%. Another opportunity is regional autonomy, so the respondents who gave the answer
agreed were 40 people or 53.33% and 35 people did not agree or 46.67%. Then the threat factor for PT. Three "R" Makassar, namely competitors are mostly
companies that have a lot of raw materials and skilled labor, then the respondents who
gave the answer agree were 43 people or 57.33% and disagreed were 32 people or
42.67% The price of similar products from other companies is cheaper, so the respondents
who gave the answer agree by 39 people or 52% and disagree by 36 people or 48%. The Analysis of Marketing Mix Strategy of Wood Processing in Increasing the Salling
Margin
1435 Bunyamin, Sultan Iskandar Having a fairly large promotional fund, then those who give an agree answer are 47
people or 62.67% and those who give an answer disagree are 28 people or 37.33%. Having a smooth distribution network, 43 people or 57.33 percent of those who
give agree answers and 32 people who give disagree or 42.67% answers. The price is cheaper/affordable, 39 people or 52% of those who give the answer
agree or disagree with 36 people or 48%. The processing process is better, then those who give agree answers are 41 people
or 54.67% and those who give disagree answers are 34 people or 45.33%. The ability to deliver (delivery) on time, 39 people or 52% of those who gave
agreed answers or 36 people or 48% of those who gave disagreed answers. Then the weakness factor in the marketing strategy of processed wood is the
bargaining position with less/low ISA, then the respondents who gave the answer agreed
were 39 people or 52% and disagreed by 36 people or 48%. Another weakness in the
SWOT analysis is the limited quality of human resources, so the respondents who gave http://eduvest.greenvest.co.id 1434 Where: C = Number of treatments Ci = Total of each treatment Ri = Total of each block Ri = Total of each bloc
N = Number of data Ri Total of each bloc
N = Number of data N = Number of data Based on the formula above, it can be tested the respondent's similarity of opinion
about internal factors with the following steps: a. Hypothesis a. Hypothesis he opinion of respondents with the same internal Ho: the opinion of respondents with the same internal attributes
Ha : respondent's opinion with different internal attributes p
p
Ha : respondent's opinion with different internal attributes. Ha : respondent's opinion with different internal attributes. b. Decision making basis: Qcount < Qtable, Ho is accepted Qcount < Qtable, Ho is accepted
Ha rejected Qcount > Qtable Ho is rejected Ha accepted c. Decision: With a = 0.05 dk = 15, obtained Qtable (0.05: 15) = 24,996, so that in the test:
which shows that Qcount = 4.987 < Qtable = 24,996 which means Ho is accepted and Ha
is rejected, which means there is no difference of opinion respondents respondents about
the attributes of internal factors. 1) Test the similarity of respondents' opinions on the attributes of external factors
The Cochran Q calculation on the respondent's similarity test on the attributes of
external factors can be done with the following steps: The Cochran Q calculation on the respondent's
external factors can be done with the following steps: a. Hypothesis a. Hypothesis
Ho the opinion of respondents with the same external attributes Ho the opinion of respondents with the same external attributes Ha, the opinion of respondents with different external attributes. b. Decision making basis: Qcount < Qtable Ho is accepted
Ha rejected Qcount < Qtable Ho is accepted Qcount Qtable Ho is rejected Ha accepted c. Decision: With a = 0.05 dk 9, obtained Qtable (0.05: 9) = 16.919, so the test shows that
Qcount = 1.712. Thus the test decision is to accept Ho because Qcount (1.712) < Qtable (16.919), so
there is no difference in respondents' opinions about the attributes of external factors. Bunyamin, Sultan Iskandar Another threat is increasing competition because the number of new companies in
this industry is increasing every year, so the respondents who give the answer agree are
41 people or 54.67% and disagree are 34 people or 45.33%. Competitor companies increase the quality and quantity of production, then the
respondents who gave the answer agree by 42 people or 56% and disagree by 33 people
or 44%. Another threat is the fluctuation of the Rupiah exchange rate, so the respondents
who gave the answer agree by 42 people or 56% and disagree by 33 people or 44%. Another threat that costs exports increased due to the weakening of the rupiah
exchange rate, the respondents who gave the answer agreed by 39 people or 52% and
disagreed by 36 people or 48%. Based on Appendix 3 and Appendix 4, internal and external factors can be tested as
follows: 1) Test the similarity of respondents' opinions about the attributes of internal
factors In the test of the respondent's similarity of opinion about internal factors, it can be
determined as follows: c(c − 1)c 2 − (c − 1)N 2
Q =
cN − R 2 c(c − 1)c 2 − (c − 1)N 2
Q =
cN − R 2 The Analysis of Marketing Mix Strategy of Wood Processing in Increasing the Salling
Margin
1435 Eduvest – Journal of Universal Studies
Volume 1 Number 12, December 2021 A. STRENGTH A. SWOT Formula Analysis A. SWOT Formula Analysis A. SWOT Formula Analysis In marketing processed wood, it is necessary to analyze the strengths, weaknesses,
opportunities and threats. For more details, the following will present a SWOT analysis in
the marketing of processed wood which can be determined as follows: 1. Analysis of strengths and weaknesses in internal factors 1. Analysis of strengths and weaknesses in internal factors Based on the analysis of the strengths of PT. Three "Rs" of Makassar, then a
scale/weight assessment of strength in the marketing of processed wood can be carried
out which can be seen in table 6 below: Table 4. Analysis of Internal Strategic Factors in SWOT Analysis Table 4. Analysis of Internal Strategic Factors in SWOT Analysis Table 4. Analysis of Internal Strategic Factors in SWOT Analysis
Internal Strategic Factors
A. STRENGTH
Weigh
Rating
t
Score
(Weight
x
Rating)
1. Has high quality processed wood because it 0,07
3
0,21 Table 4. Analysis of Internal Strategic Factors in SWOT Analysis
Internal Strategic Factors
A. STRENGTH
Weigh
Rating
t
Score
(Weight
x
Rating)
1. Has high quality processed wood because it 0,07
3
0,21 Internal Strategic Factors 1436 Bunyamin, Sultan Iskandar follows quality standards
2. Have HPH
0,06
3
0,18
3. Has various types of processed wood
0,06
3
0,18
4. Have a large number of employees/HR and
0,06
3
0,18
cheap
5. Have a large enough promotional fund
0,06
3
0,18
6. Has an extensive distribution network
0,06
3
1,18
7. Cheaper/affordable price
0,06
3
0,18
8. Better processing
0,06
3
0,18
9. Ability to deliver (delivery) on time
0,06
3
0,21
TOTAL
1,68
B. WEAKNESS
1. Bargaining position with less/low ISA
0,05
4
0,20
2. Limited quality of human resources
0,07
3
0,21
3. Suboptimal product planning
0,06
3
0,18
4. Less product promotion
0,07
3
0,21
5. Old machinery and equipment
0,07
3
0,21
6. The human resource quality development
system is not good because there is no training
for employees
0,06
3
0,18
7. The reward and punishment system is not
applied properly, so it doesn't get a high
turnover
0,06
3
0,18
TOTAL
1,37
Source: Data Primer, 2007 Based on the results of the analysis of the variables rated and weighted in
accordance with the criteria for internal strategy factors that have been determined, the
results obtained are that PT. A. SWOT Formula Analysis The three Makassar "Rs" have a strength of 1.68 and a
weakness of 1.37, so it can be concluded that PT. The three Makassar "Rs" have a greater
strength value (positive = 0.31 of the existing weaknesses, with the following calculation: S = 1.68
W = 1.37
S-W = 0.31 S = 1.68
W = 1.37
S-W = 0.31 1. Analysis of Opportunities and Threats of External Factors 1. Analysis of Opportunities and Threats of External Factors The analysis of opportunities and threats of external factors can be seen through the
following Label 7: The analysis of opportunities and threats of external factors can be seen through the
following Label 7: The analysis of opportunities and threats of external factors can be seen through the
following Label 7: Table 5. Analysis of External Strategic Factors in SWOT Analysis Table 5. Analysis of External Strategic Factors in SWOT Analysis The Analysis of Marketing Mix Strategy of Wood Processing in Increasing the Salling
Margin
1437
Table 5. Analysis of External Strategic Factors in SWOT Analysis
Internal Strategic Factors
Weight
Rating
Score
(Weight
x
Rating)
A. STRENGTH
1. Government policy in increasing export volume 0,13
4
0,52
2. A large number of customers
0,11
4
0,44
3. Large market share due to increased demand
0,13
4
0,52
4. Regional autonomy
0,13
4
0,52
TOTAL
2
B. WEAKNESS
1. Competition is mostly companies that have a 0,09
4
0,36 The Analysis of Marketing Mix Strategy of Wood Processing in Increasing the Salling
Margin
1437 The Analysis of Marketing Mix Strategy of Wood Processing in Increasing the Salling
Margin
1437 lot of raw materials and skilled workers
2. The price of similar products from other
companies is cheaper
0,05
3
0,15
3. Increased competition as the number of new
companies in this industry is increasing every
year
0,08
3
0,14
4. Competing companies improve the quality and
quantity of production
0,09
3
0,27
5. Rupiah exchange rate fluctuation
0,10
3
0,30
6. Increased export costs due to the weakening of
the Rupiah exchange rate
0,09
3
0,27
TOTAL
1,44
Source: Data Primer, 2007 Based on the results of the analysis of the variables rated and weighted in
accordance with the criteria for external strategy factors that have been determined, the
data obtained that PT. A. SWOT Formula Analysis Three "R" Makassar has an opportunity value of 2 and a threat
value of 1.44 so it can be concluded that PT. The three Makassar "Rs" have a greater
chance of 0.056 and the threat posed is calculated as follows: O = 2
T = 1.44
O -T = 2-1.44 = 0.56 O = 2
T = 1.44
O -T = 2-1.44 = 0.56 Based on the results of the comparative analysis of strengths (S) and weaknesses
(W) and opportunities (O) with threats (T), a SWOT analysis formulation can be used in
the marketing of processed wood which can be described by the following scheme: Figure 1. SWOT Analysis Map in Determining Processed Timber Marketing
Strategy at PT. Three “R” Makassar Figure 1. SWOT Analysis Map in Determining Processed Timber Marketing
Strategy at PT. Three “R” Makassar The position of the processed wood marketing strategy at PT. The three Makassar
"Rs" are in quadrant I, this is a very favorable situation for PT. Three "R" Makassar have
very good opportunities and strengths. The strategy that must be applied in this condition
is to support an aggressive growth policy (Growth-oriented strategy). http://eduvest.greenvest.co.id 1438 The Analysis of Marketing Mix Strategy of Wood Processing in Increasing the Salling
Margin
1439 Bunyamin, Sultan Iskandar g
STRATEGY WO
-
Determination
of
competitive
promotion
strategy. -
HR
quality
control
system through training. - Updating machines and
equipment. g
STRATEGY WO
-
Determination
of
competitive
promotion
strategy. -
HR
quality
control
system through training. - Updating machines and
equipment. Bunyamin, Sultan Iskandar Results of tows matrix analysis in SWOT analysis
IFAS
(Internal
Factor)
EFAS
(External
Factor)
STRENGTHS (S)
- Has a high quality of processed
wood because it follows quality
standards
- Have HPH
- Has a variety of processed
wood types
- Have a large number of
employees/HR and cheap
-
Have
a
large
enough
promotional fund
- Has an extensive distribution
network
- Cheaper/affordable prices
- Better processing
- Ability to deliver (delivery) on
time. WEAKNESS (W)
- Bargaining
position
with less/low ISA
- Limited
quality
of
human resources
- Sub-optimal
product
planning
- Lack
of
product
promotion
- Old
machinery
and
equipment. - The
HR
quality
development system is not
good because there is no
training for employees. - The
reward
and
punishment system is not
applied properly resulting WEAKNESS (W)
- Bargaining
position
with less/low ISA - Has a high quality of processed
wood because it follows quality
standards - Limited
quality
of
human resources - Have HPH - Sub-optimal
product
planning - Has a variety of processed
wood types - Have a large number of
employees/HR and cheap - Lack
of
product
promotion - Old
machinery
and
equipment. -
Have
a
large
enough
promotional fund - The
HR
quality
development system is not
good because there is no
training for employees. - Has an extensive distribution
network - Cheaper/affordable prices - Better processing - The
reward
and
punishment system is not
applied properly, resulting
in a high turnover rate. - Ability to deliver (delivery) on
time. OPPORTUNITIES
(O)
- Government policy
in increasing export
volume. -The
number
of
customers is quite a
lot. - Large market share
due
to
increased
demand. -Regional autonomy. THREATS (T)
- Competitors
are
mostly
companies
that have a lot of raw
materials and skilled
labor. - The price of similar
products from other
companies
is
cheaper. -
Increased
competition because
the number of new
STRATEGY SO
- Understanding the marketing
area. - Increase the quality of service. - Increase production volume. STRATEGY ST
-
Improve
/
maintain
standardization
of
processed
wood. - Increase the quality and quantity
of processed wood. in a high turnover rate. STRATEGY WO
-
Determination
of
competitive
promotion
strategy. -
HR
quality
control
system through training. - Updating machines and
equipment. STRATEGY WT
-
Renewed
bargaining
position with ISA. - Improving the service
system in order to produce
efficiency
and
effectiveness. Bunyamin, Sultan Iskandar Bunyamin, Sultan Iskandar The results of the analysis of the SWOT analysis map above can be continued with
a SWOT matrix analysis, which is a tool used to develop marketing strategy factors. For
more details can be presented matrix tows for the marketing of processed wood at PT. Three "R" Makassar which can be seen through the following table 8: Table 6. Results of tows matrix analysis in SWOT analysi Table 6. Results of tows matrix analysis in
IFAS
(Internal
Factor)
EFAS
(External
Factor)
STRENGTHS (S)
- Has a high quality of processed
wood because it follows quality
standards
- Have HPH
- Has a variety of processed
wood types
- Have a large number of
employees/HR and cheap
-
Have
a
large
enough
promotional fund
- Has an extensive distribution
network
- Cheaper/affordable prices
- Better processing
- Ability to deliver (delivery) on
time. Table 6. Results of tows matrix analysis in SWOT analysis
IFAS
(Internal
Factor)
EFAS
(External
Factor)
OPPORTUNITIES
(O)
- Government policy
in increasing export
volume. -The
number
of
customers is quite a
lot. - Large market share
STRENGTHS (S)
- Has a high quality of processed
wood because it follows quality
standards
- Have HPH
- Has a variety of processed
wood types
- Have a large number of
employees/HR and cheap
-
Have
a
large
enough
promotional fund
- Has an extensive distribution
network
- Cheaper/affordable prices
- Better processing
- Ability to deliver (delivery) on
time. STRATEGY SO
- Understanding the marketing
area. - Increase the quality of service. - Increase production volume. WEAKNESS (W)
- Bargaining
position
with less/low ISA
- Limited
quality
of
human resources
- Sub-optimal
product
planning
- Lack
of
product
promotion
- Old
machinery
and
equipment. - The
HR
quality
development system is not
good because there is no
training for employees. - The
reward
and
punishment system is not
applied properly, resulting
in a high turnover rate. STRATEGY WO
-
Determination
of
competitive
promotion
strategy. -
HR
quality
control
system through training. - Updating machines and
equipment. Table 6. OPPORTUNITIES STRATEGY WO
-
Determination
of
competitive
promotion
strategy. - Government policy
in increasing export
volume. - Increase the quality of service. -
HR
quality
control
system through training. -The
number
of
customers is quite a
lot. - Updating machines and
equipment. - Large market share
due
to
increased
demand. -Regional autonomy. THREATS (T) -Regional autonomy. THREATS (T)
- Competitors
are
mostly
companies
that have a lot of raw
materials and skilled
labor. - The price of similar
products from other
companies
is
cheaper. -
Increased
competition because
the number of new
STRATEGY ST
-
Improve
/
maintain
standardization
of
processed
wood. - Increase the quality and quantity
of processed wood. STRATEGY WT
-
Renewed
bargaining
position with ISA. - Improving the service
system in order to produce
efficiency
and
effectiveness. -Regional autonomy. THREATS (T)
- Competitors
are
mostly
companies
that have a lot of raw
materials and skilled
labor. - The price of similar
products from other
companies
is
cheaper. -
Increased
competition because
the number of new
STRATEGY ST
-
Improve
/
maintain
standardization
of
processed
wood. - Increase the quality and quantity
of processed wood. STRATEGY WT
-
Renewed
bargaining
position with ISA. - Improving the service
system in order to produce
efficiency
and
effectiveness. STRATEGY WT
-
Renewed
bargaining
position with ISA. - Improving the service
system in order to produce
efficiency
and
effectiveness. - Competitors
are
mostly
companies
that have a lot of raw
materials and skilled
labor. The Analysis of Marketing Mix Strategy of Wood Processing in Increasing the Salling
Margin
1439 Eduvest – Journal of Universal Studies
Volume 1 Number 12, December 2021 companies
in
this
industry is increasing
every year. -
Competitor
companies improve
the
quality
and
quantity of
production. - Fluctuations in the
rupiah exchange rate. Increased
export
costs
due
to
the
weakening of the
rupiah exchange rate companies
in
this
industry is increasing
every year. -
Competitor
companies improve
the
quality
and
quantity of
production. - Fluctuations in the
rupiah exchange rate. Increased
export
costs
due
to
the
weakening of the rupiah exchange rate rupiah exchange rate Based on table 8, the tows matrix in the SWOT analysis, several marketing
strategies for processed wood can be presented through the results of the SWOT analysis,
namely as follows: 1. OPPORTUNITIES SO Strategy (Strengths - Opportunities) SO strategy is a strategy that uses strength to take advantage of opportunities, so
the efforts that need to be made by PT. Makassar's three "Rs" are a. Add marketing area a. Add marketing area One of the efforts made in selling processed wood is to increase the marketing
area. Efforts are being made to take a personal approach and an institutional approach to
potential business partners so that they can become new customers for the company. b. Increase service quality q
y
Other efforts that can be done by PT. The three "Rs" of Makassar are to increase
the quality of service to customers, this is done so that customers feel satisfied which in
turn can increase sales of processed wood and can increase optimal profits. Other efforts that can be done by PT. The three "Rs" of Makassar are to increase
the quality of service to customers, this is done so that customers feel satisfied which in
turn can increase sales of processed wood and can increase optimal profits. c. Increase production volume To increase sales of processed wood, the strategy taken is to increase the volume of
production so that the company can meet the demands of more and more customers. 2. WO Strategy (Weaknesses - Opportunities) WO strategy is a strategy that minimizes weaknesses to take advantage of
opportunities, so that efforts are made by PT. The three "Rs" of Makassar are: a. Determination of competitive promotion strategy In the marketing of processed wood, the strategy used is to determine a competitive
promotion strategy that can be carried out with a personal approach and an institutional
approach so that this promotional effort can increase the volume of processed wood sales. b. HR quality control system through training q
y
y
g
g
To reduce the reward and punishment system that is not applied properly so that
the turnover rate is high, the strategy that needs to be done is to implement the
development of human resource quality, such as HR training on matters related to the
production and marketing of processed wood. Eduvest – Journal of Universal Studies
Volume 1 Number 12, December 2021 Bunyamin, Sultan Iskandar a. Improving/maintaining standardization of processed wood Efforts made through ST strategy analysis are to maintain and improve the
standardization of processed wood so that the bargaining position with ISA increases and
can increase customer satisfaction. b. Increase the quality and quantity of processed wood One of the efforts made by PT. The three "Rs" of Makassar are to increase the
quality and quantity of processed wood so that old customers can be satisfied and new
customers can fulfill their demands which in the end is expected to increase the sales
volume of processed wood. One of the efforts made by PT. The three "Rs" of Makassar are to increase the
quality and quantity of processed wood so that old customers can be satisfied and new
customers can fulfill their demands which in the end is expected to increase the sales
volume of processed wood. 4. WT Strategy (Weaknesses - Threats) WT strategy is a strategy that avoids threats, so what needs to be done by PT. The
three "Rs" of Makassar are: a. Renewal of bargaining position with ISA In overcoming the weak bargaining position with ISA, PT. Three "R" Makassar try
to meet the existing requirements by improving the quality of the processed wood they
produce. In overcoming the weak bargaining position with ISA, PT. Three "R" Makassar try
to meet the existing requirements by improving the quality of the processed wood they
produce. p
b. Improving the service system to produce efficiency and effectiveness b. Improving the service system to produce efficiency and effectivenes In an effort to increase sales of processed wood, it is necessary to improve the
service system so as to produce efficiency and effectiveness that can ultimately meet and
exceed customer expectations so that they can be loyal and increase their purchases. In an effort to increase sales of processed wood, it is necessary to improve the
service system so as to produce efficiency and effectiveness that can ultimately meet and
exceed customer expectations so that they can be loyal and increase their purchases. CONCLUSION From the description of the results of the analysis and discussion regarding the
application of the processed wood marketing strategy at PT. Three "R" Makassar, it can
be drawn some conclusions from the overall results of the analysis that has been carried
out, namely as follows: Based on the results of the application of SWOT analysis, it appears that the
strategy used by PT. The three "Rs" of Makassar in increasing processed wood sales are
implementing the SO strategy, which is to make strengths to take advantage of
opportunities, then the WO strategy by minimizing weaknesses to maintain opportunities,
then the ST strategy using strengths to overcome threats and WT strategies that minimize
weaknesses and avoid threats. Based on the results of the analysis of the variables rated and weighted in
accordance with the criteria of external strategy factors that have been determined, the
position of the marketing strategy of PT. The three Makassar "Rs" are in a very
advantageous position, namely an aggressive strategy, where PT. The three "Rs" of
Makassar have excellent opportunities and strengths in creating adequate advantages
when compared to other competing companies. OPPORTUNITIES To reduce the reward and punishment system that is not applied properly so that
the turnover rate is high, the strategy that needs to be done is to implement the
development of human resource quality, such as HR training on matters related to the
production and marketing of processed wood. c. Machinery and equipment upgrades This is done by replacing old machines and equipment with new machines and
equipment so that the production volume and quality of processed wood can increase. This is done by replacing old machines and equipment with new machines and
equipment so that the production volume and quality of processed wood can increase. 3. ST Strategy (Strengths - Treat) ST strategy is a strategy that uses strength to overcome opportunities, so what
needs to be done by PT. The three "Rs" of Makassar are: ST strategy is a strategy that uses strength to overcome opportunities, so what
needs to be done by PT. The three "Rs" of Makassar are: http://eduvest.greenvest.co.id 1440 REFERENCES Adams, Meredith Elaine. (2016). Beyond the Glass: Examining Wine Tasting Room
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Margin
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Margin
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Thabit, Thabit, & Raewf, Manaf. (2018). The evaluation of marketing mix elements: A
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https://openalex.org/W3185489055
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https://periodicos.udesc.br/index.php/dapesquisa/article/download/20172/13150
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Portuguese
| null |
Pintura, fotografia e memória na produção artística de Paulo Gaiad
|
DAPesquisa
| 2,021
|
cc-by
| 8,894
|
Resumo O texto aborda a relação estabelecida entre pintura e fotografia na produção artística de Paulo Gaiad,
procurando analisar como a associação entre esses diferentes meios de expressão, ou paradigmas da
imagem, contribuiu para a constituição do projeto poético do artista, projeto esse baseado, conforme suas
declarações, no fenômeno da “memória”. Para tanto, o artigo concentra-se na fatura de determinados
trabalhos de Gaiad, especialmente aqueles pertencentes às séries As Paredes que me Cercam, A divina
comédia, Memórias da cozinha e Estudos de luz e sombra. Nesse conjunto de quadros, pode-se sugerir que o
artista procurou materializar a operação da memória, vista como processo de recriação do ocorrido, por
meio da justaposição entre o pictórico e o fotográfico. Para esse fim, cabe complementar, também convocou
e atualizou gêneros tradicionais, como a paisagem e a natureza-morta. Nessa lógica, os modos operativos de
Gaiad integram um cenário, o da arte contemporânea, no qual as relações entre os diferentes meios são de
comunhão e não de exclusão. Palavras-chave: Memória na arte; Gaiad, Paulo Renato, 1953-; Fotografia artística; Natureza-m Palavras-chave: Memória na arte; Gaiad, Paulo Renato, 1953-; Fotografia artística; Natureza-morta na arte. Painting, photography and memory in the artistic production of Paulo Gaiad Wagner Jonasson da Costa Lima
Doutor em Artes Visuais pela Universidade do Estado de Santa Catarina (UDESC) –
wagnerjonasson@hotmail.com– orcid.org/0000-0001-6983-8651 Pintura, fotografia e memória na produção artística de Paulo Gaiad ntura, fotografia e memória na produção artística de Paulo Gaiad Abstract The text addresses the relationship established between painting and photography in the artistic production
of Paulo Gaiad, seeking to analyze how the association between these different means of expression or
image paradigms contributed to the constitution of this artist's poetic project, that is, a project based,
according to his statements, in the phenomenon of “memory”. To this end, the study focuses on analyzing
certain works by Gaiad, especially those belonging to the series As Paredes que me Cercam, A divina
comédia, Memórias da cozinha e Estudos de luz e sombra. In this set of pictures, it may be suggested that
the artist sought to materialize the operation of memory, seen as a process of recreating what had
happened, through the juxtaposition between the pictorial and the photographic. To this end, it is worth
complementing that he also used and updated traditional genres, such as landscape and still life. In this
logic, the Gaiad's operating modes integrate a scenario, the contemporary art, in which the relations
between the different means are of communion and not of exclusion. Keywords: Memory in art; Gaiad, Paulo Renato, 1953-; Photography, Artistic; Still-life painting. Recebido em: 04/05/2021
Aceito em: 19/07/2021 DAPesquisa, Florianópolis, v. 16, p. 01-22, jul. 2021. DOI: https://doi.org/10.5965/18083129152021e0024 1 1 Pintura, fotografia e memória na produção artística de Paulo Gaiad
Wagner Jonasson da Costa Lima Pintura, fotografia e memória na produção artística de Paulo Gaiad
Wagner Jonasson da Costa Lima q
,
p
,
, p
, j
DOI: https://doi.org/10.5965/18083129152021e0024 DAPesquisa, Florianópolis, v. 16, p. 01-22, jul. 2021.
DOI: https://doi.org/10.5965/18083129152021e0024 1 INTRODUÇÃO Logo, essas imagens dispõem de 2 2 Pintura, fotografia e memória na produção artística de Paulo Gaiad
Wagner Jonasson da Costa Lima Pintura, fotografia e memória na produção artística de Paulo Gaiad
Wagner Jonasson da Costa Lima Pintura, fotografia e memória na produção artística de Paulo Gaiad
Wagner Jonasson da Costa Lima modos próprios de produção, armazenamento, transmissão e recepção. Porém, a mistura e a
justaposição de tais paradigmas encontram-se nos fenômenos de hibridização e de mestiçagem das
artes (CATTANI, 2006). À semelhança de vários artistas contemporâneos, Gaiad considerou, na
elaboração de suas obras, a coexistência entre a pintura e a fotografia, entre o artesanal e o
automático. Entretanto, os trabalhos de Gaiad constituem um projeto poético particular, orientado, de
acordo com suas declarações, para a “memória”. Como apontou o artista, “a base do meu trabalho
é praticamente a memória. [...] Eu vou recolhendo memórias para depois transformar isso na
minha obra. [...] Então nesse processo eu trabalho também a questão do material” (GAIAD, 2015). Tendo em vista essa observação, pode-se interrogar qual é a relação estabelecida entre pintura,
fotografia e memória na produção de Paulo Gaiad. Como resposta suposta e provisória, sugere-se
que, em muitos dos seus quadros, o artista buscou corporificar, por meio do encontro entre
recursos da pintura e da fotografia, a operação da memória, operação essa compreendida não
como recuperação e sim como recriação do passado. Para esse propósito, convém acrescentar,
Gaiad utilizou não só a combinação de diferentes meios, mas também a tradição da pintura,
valendo-se de gêneros como a paisagem e a natureza-morta. Para analisar a relação estabelecida entre pintura, fotografia e memória na produção de
Paulo Gaiad, o texto concentra-se na fatura de trabalhos pertencentes às séries As Paredes que me
Cercam, A divina comédia, Memórias da cozinha e Estudos de luz e sombra. Por “fatura”, entende-
se aqui a maneira de operar de determinado artista, que pode ser percebida, em parte, a partir das
evidências da elaboração do trabalho artístico, a sua textura de superfície, resultante do
entrelaçamento entre meios e procedimentos escolhidos em certa circunstância. Sendo assim,
aborda-se a obra de arte como produto da atividade intencional, isto é, como produto de um
“projeto poético” (SALLES, 2011), embora contra um pano de fundo de tradições artísticas. 1 INTRODUÇÃO No decorrer do século XX, diferentes artistas adotaram a fotografia como motivo e material
para a pintura. Na década de 1950, Robert Rauschenberg (1925-2008), movido pelas colagens de
Kurt Schwitters (1887-1948), introduziu imagens fotomecânicas em seus quadros, como cartões-
postais e anúncios de revista, distanciando-se com isso da “doutrina formalista segundo a qual a
pintura aspira a uma síntese cada vez mais restrita dos elementos que a compõem” (STEINBERG,
2008, p. 110). A partir do uso de materiais não tradicionais, bidimensionais e tridimensionais, o
artista norte-americano conferiu, na visão de Leo Steinberg, outra orientação para a pintura, uma
guinada do plano vertical, fundado pelo Renascimento, para o plano horizontal ou “de tipo
flatbed”, que “faz sua alusão simbólica a superfícies duras, como tampos de mesa, chão de ateliê,
diagramas, quadros de aviso” (STEINBERG, 2008. p. 117). O plano de tipo flatbed, para Steinberg,
expressaria uma transformação importante no tema da arte: a mudança da natureza para a cultura. Posteriormente, o chamado “neoexpressionismo”, tendência pictórica que despontou entre
final da década de 1970 e início da seguinte, também estabeleceu diálogo próprio com o universo
de imagens oferecidas pela fotografia e veiculadas pelos meios de comunicação de massa. Naquele
momento, diversos artistas, além de reabilitar a pintura, depois das propostas de desmaterialização
da arte, dedicaram-se à “elaboração de outros sistemas visuais significativos, criados a partir da
conjugação de imagens e procedimentos linguísticos preexistentes” (CHIARELLI, 2001, p. 257). A
fotografia e as suas imagens comparecem na produção de Anselm Kiefer (1945-), artista alemão
cujo trabalho inicial aliava símbolos da cultura germânica, apossados pelo nazismo, com uma
ênfase na densidade da superfície pictórica, derivada dos expressionismos da primeira metade do
século XX (HUYSSEN, 1996). Frequentemente, a imagem fotográfica, incorporada às telas de Kiefer,
juntamente com outros objetos, sofreu a ação de ampla variedade de materiais. É possível identificar na produção de Paulo Gaiad (1953-2016) essa estreita relação entre
pintura e fotografia que assinalou os trabalhos de Rauschenberg e Kiefer. Com formação em
arquitetura e urbanismo, Gaiad iniciou carreira artística na década de 1980, depois de fixar
residência em Florianópolis – SC. Realizou a primeira exposição individual em 1987 e, a partir de
2000, associou seguidamente pintura e fotografia, meios tidos como paradigmas da imagem. Conforme Santaella e Nöth (1998, p. 163), a pintura decorre de “processos artesanais de criação”,
enquanto a fotografia de “processos automáticos de captação”. DOI: https://doi.org/10.5965/18083129152021e0024 DAPesquisa, Florianópolis, v. 16, p. 01-22, jul. 2021.
DOI: https://doi.org/10.5965/18083129152021e0024 DAPesquisa, Florianópolis, v. 16, p. 01-22, jul. 2021.
h
//
/
/ 1 INTRODUÇÃO Logo,
antes de tratar da fatura de Gaiad, busca-se brevemente apresentar a correspondência entre
pintura, fotografia e memória, assim como, em seguida, situar o artista brasileiro. Por fim, convém
salientar que o presente texto, derivado de roda de conversa ocorrida em 2016 no Museu da Escola DAPesquisa, Florianópolis, v. 16, p. 01-22, jul. 2021. DOI: https://doi.org/10.5965/18083129152021e0024 DAPesquisa, Florianópolis, v. 16, p. 01-22, jul. 2021. DOI htt
//d i
/10 5965/18083129152021 0024 3 3 3 Pintura, fotografia e memória na produção artística de Paulo Gaiad
Wagner Jonasson da Costa Lima Pintura, fotografia e memória na produção artística de Paulo Gaiad
Wagner Jonasson da Costa Lima Catarinense (Mesc)1, não se propõe esgotar o assunto, mas lançar apontamentos para
investigações futuras. Catarinense (Mesc)1, não se propõe esgotar o assunto, mas lançar apontamentos para
investigações futuras. 1 Roda de conversa intitulada “Conversas sobre Paulo”, ocorrida em 19 de abril de 2016 no Museu da Escola Catarinense
(Mesc), com a participação dos seguintes convidados: Profa. Dra. Sandra Makowiecky, Prof. Dr. Antonio Vargas, Prof.
Dr. Edélcio Mostaço e Prof. Dr. Wagner Jonasson da Costa Lima. DOI: https://doi.org/10.5965/18083129152021e0024 DAPesquisa, Florianópolis, v. 16, p. 01-22, jul. 2021. 2 PINTURA, FOTOGRAFIA E MEMÓRIA Desde a sua invenção, a fotografia, considerada a primeira imagem técnica que prescinde da
mão do artista, diferenciou-se das artes tradicionais, notadamente da pintura. Surgida quase
simultaneamente na França e na Inglaterra, na era do maquinismo, a fotografia determinou,
segundo André Rouillé (2009), um novo protocolo de produção de imagens. Se a imagem pictórica,
resultante de operações manuais, surge durante processo de fabricação, a imagem fotográfica,
resultante de operações químicas, surge de uma só vez, em espaço adaptado para isso, o
laboratório. Em lugar de representar, a fotografia situou-se como máquina de capturar. Dessa
maneira, como máquina de produzir imagens de captura, a fotografia foi acusada de não omitir
nada, diversamente da pintura, cuja elaboração envolveria seleção. A partir de meados do século
XIX, e no decurso de mais de um século, a oposição entre pintura e fotografia pronunciou-se em
distinções entre construção e captação, entre mão do artista e máquina do operador, entre arte e
documento. Percebida como documento, ou como “testemunho de uma realidade que existiu”
(GONZÁLEZ FLORES, 2011, p. 123), a fotografia funcionaria como equivalente palpável da memória. De acordo com Laura González Flores (2011, p. 125), a memória, até o século XIX, foi explicada
“como uma seleção mecânica de dados e imagens que são concebidos como oriundos de um
arquivo/passado e levados a um exame/presente”. Por sua vez, a fotografia, no seu uso cotidiano,
recebeu a classificação de meio referencial e indicial, concepção que concorda com a interpretação
oferecida por autores como Roland Barthes (1915-1980) e Susan Sontag (1933-2004). Nessa visão, a
essência da fotografia se depreende do que sucedeu, ou seja, “sem objeto-existência banhado pela
luz não há imagem-testemunho” (GONZÁLEZ FLORES, 2011, p. 123). Diante disso, memória e
fotografia operariam de modo semelhante, deslocando imagens do passado para o presente, a
memória faria isso mentalmente, enquanto a fotografia materialmente. 4 4 Pintura, fotografia e memória na produção artística de Paulo Gaiad
Wagner Jonasson da Costa Lima Objeto de consideração desde a antiguidade clássica, a memória, já nas primeiras décadas
do século XX, integrou as reflexões de Sigmund Freud (1856-1939), Henri Bergson (1859-1941) e
Walter Benjamin (1892-1940). Mais recentemente, o assunto foi abordado em vários ensaios pelo
crítico cultural alemão Andreas Huyssen (1996). Para o autor, a memória, em si, não opera como
recuperação e sim como reapresentação do passado, reapresentação essa que atende,
especialmente, as exigências do presente. 2 PINTURA, FOTOGRAFIA E MEMÓRIA “O status temporal de qualquer ato da memória é
sempre o presente e não, como certa epistemologia ingênua pensa, o próprio passado” (HUYSSEN,
1996, p. 14-15). Além dessa compreensão, Huyssen registrou a ascensão, desde a década de 1980,
de um “discurso da memória” nas sociedades ocidentais, fenômeno relacionado, segundo ele, com
a crise da ideologia do progresso e com o esgotamento das filosofias finalistas da história. Essa
preocupação com a memória e a sua reapresentação manifestou-se, no final do século XX, nos
âmbitos político e cultural. Também na esfera da arte contemporânea notam-se numerosos trabalhos que colocam a
memória em destaque, muitos deles servindo-se da fotografia. Nesse cenário da fotografia artística,
salientou González Flores (2011, p. 137): “existe uma fotografia não fascinada com o dispositivo
mimético, que transcende a imago metonímica e assume plenamente a sua subjetividade”. Nesse
caso, a fotografia, muitas vezes apreendida como documento ou testemunho, sofreu um processo
de “artistificação”, funcionando doravante como linguagem expressiva. Dentre os recursos de
artistificação encontra-se, historicamente, a alteração da realidade fotografada, a manipulação das
técnicas fotográficas e a combinação da fotografia com outras linguagens, como a pintura. A partir
das vanguardas históricas, variados artistas investiram contra a disjunção dos meios, adotando um
método de trabalho “transgenérico” (GONZÁLEZ FLORES, 2011, p. 178). Como exemplo, pode-se
referir a produção de artistas como Paulo Gaiad. DOI: https://doi.org/10.5965/18083129152021e0024 DAPesquisa, Florianópolis, v. 16, p. 01-22, jul. 2021. DAPesquisa, Florianópolis, v. 16, p. 01-22, jul. 2021.
DOI: https://doi.org/10.5965/18083129152021e0024 3 UMA ARTE DA MEMÓRIA Nascido em Piracicaba – SP, Paulo Gaiad interessou-se desde cedo pelo desenho e pela
pintura, disposição que o levou a optar, aos 19 anos, pelas áreas de arquitetura e urbanismo
(CHEREM, 2015). Em 1972, ingressou no curso de Arquitetura e Urbanismo da Universidade de
Brasília – UnB, mas logo abandonou os estudos para atuar como arquiteto em sua cidade natal. 5 5 5 Pintura, fotografia e memória na produção artística de Paulo Gaiad
Wagner Jonasson da Costa Lima Pintura, fotografia e memória na produção artística de Paulo Gaiad
Wagner Jonasson da Costa Lima Pintura, fotografia e memória na produção artística de Paulo Gaiad
Wagner Jonasson da Costa Lima Dois anos depois, matriculou-se no curso de Desenho da Pontifícia Universidade Católica de
Campinas – PUC Campinas. Nesse período, Gaiad obteve uma bolsa de estudos para o curso de
Planejamento Urbano da Universidade de Oslo, Noruega. Em 1976, recebeu um convite para
trabalhar com Vilanova Artigas (1915-1985) em um reconhecido escritório de arquitetura de São
Paulo. Na capital paulista, frequentou o curso livre de desenho da Pinacoteca. Mais tarde, já casado
e com filho, afastou-se do escritório para exercer a atividade de projetista. Em 1981, Gaiad fixou residência em Florianópolis como funcionário do Banco do Estado do
Rio de Janeiro – Banerj, atuando paralelamente em um escritório de arquitetura para adiante, no
final da década, dedicar-se às artes visuais. Segundo depoimento do artista (CHEREM, 2015), a
experiência de um passeio de barco ao redor da Ilha de Santa Catarina, em companhia de colegas
arquitetos e fotógrafos, contribuiu para essa transição da arquitetura para as artes visuais. Realizada em 1986, a excursão promoveu a súbita produção de grande número de trabalhos que
retratavam com traços caligráficos e cores saturadas a paisagem do litoral catarinense. Posteriormente, Gaiad frequentou as Oficinas de Arte do Centro Integrado de Cultura – CIC, espaço
vinculado à Fundação Catarinense de Cultura – FCC. Nesse local, sob a orientação de outros
artistas, participou de oficinas de desenho e gravura. No final da década de 1980 e início da
seguinte, começou a exibir desenhos e pinturas em galerias e salões de arte. Durante o tempo em que Gaiad voltava-se pouco a pouco para as artes visuais, irrompiam
no cenário artístico brasileiro eventos embalados pela revalorização da pintura, depois de período
marcado pelas vertentes conceitualistas. DAPesquisa, Florianópolis, v. 16, p. 01-22, jul. 2021.
DOI: https://doi.org/10.5965/18083129152021e0024 DAPesquisa, Florianópolis, v. 16, p. 01-22, jul. 2021.
DOI: https://doi.org/10.5965/18083129152021e0024 nópolis, v. 16, p. 01-22, jul. 2021. 3 UMA ARTE DA MEMÓRIA Em 1984, por exemplo, a exposição Como vai você,
Geração 80?, realizada na Escola de Artes Visuais do Parque Lage (EAV), no Rio de Janeiro, reuniu
126 artistas brasileiros que, no entendimento dos curadores, desejavam produzir “sem os pavores
conceituais” (COSTA, 1984). Nesse ajuntamento de artistas e trabalhos encontrava-se, de acordo
com o crítico de arte Wilson Coutinho (1984, p. 6), “o neo-expressionismo, o pattern de retorcidas
formas coloridas, o rabisco rápido, a pintura agressiva, o mal pintado, o graffitismo, a recaptura da
história da arte, a gestualidade agressiva”. O evento, a despeito da presença de trabalhos que não
se configuravam por meio de recursos exclusivamente pictóricos, tornou-se emblemático da
retomada da pintura no país. Ao longo da década de 1990, Paulo Gaiad adotou expedientes como o “rabisco rápido”, “a
pintura agressiva” e a “recaptura da história da arte”, que se tornaram recorrentes em sua 6 6 Pintura, fotografia e memória na produção artística de Paulo Gaiad
Wagner Jonasson da Costa Lima Pintura, fotografia e memória na produção artística de Paulo Gaiad
Wagner Jonasson da Costa Lima Pintura, fotografia e memória na produção artística de Paulo Gaiad
Wagner Jonasson da Costa Lima produção. Em Paisagem a caminho de Dresden (Figura 1), por exemplo, a linha apresenta-se ora
como garatuja desordenada, ora como contorno que conforma árvores, vias, pontes e construções
esquemáticas. Esses traços e figuras acham-se sobre manchas de cinza, azul e verde, depositadas
com movimentos enérgicos do pincel. Ao lado da fatura ligeira, o título reforça o caráter de notação
ou recordação de viagem do trabalho. De acordo com o artista, “ao compor o retrato de uma
cidade eu vou juntando momentos e lembranças, anotações feitas na memória, e então passo a
montar a ‘minha’ Berlim, a ‘minha’ Stuttgart, a ‘minha’ Dresden” (GAIAD apud MAKOWIECKY, 2009,
p. 2722). Aqui, como em outras produções do período, Gaiad reelaborou vivências com o objetivo
de produzir nova composição gráfica ou pictórica. – PAULO GAIAD, PAISAGEM A CAMINHO DE DRESDEN, 1995, GRAFITE E GUACHE SOBRE PAPEL, 50.0 X 6
CM, COLEÇÃO MUSEU VICTOR MEIRELLES, FLORIANÓPOLIS
FONTE: MUSEU VICTOR MEIRELLES. Disponível em: https://museuvictormeirelles.museus.gov.br. Acesso em: 25 jan. 2019. DOI: https://doi.org/10.5965/18083129152021e0024 DAPesquisa, Florianópolis, v. 16, p. 01-22, jul. 2021. proximidade, preenchimento e vazio, superfície e profundidade, lembrança e apagamento”
(CHEREM, 2015, p. 1). No interior dessa produção, ainda nas palavras de Cherem, a clave do vivido serve como manancial infinitamente revisitado e estoque movente com
força de consignação poética, através do qual reconhece seu repertório, travando uma luta
contra o esquecimento e produzindo reverberações para novos trabalhos. (CHEREM, 2015,
p. 1). Essa “luta contra o esquecimento”, que mobilizou diferentes meios e procedimentos,
mostrou-se em trabalhos reunidos na exposição Impossibilias: arquivo e memória em Paulo Gaiad,
realizada na Fundação Cultural Badesc, em Florianópolis, entre os dias 26/11/2015 e 26/02/2016
(EXPOSIÇÃO..., 2015); a última do artista, que faleceu em outubro de 2016. Com curadoria de
Cherem, a exposição, que revisitou a trajetória artística de Gaiad e ocupou todas as salas da
Fundação, abarcou colagens, desenhos, pinturas, objetos e vídeos. A curadoria agrupou os
trabalhos exibidos atentando para três temas que considerou recorrentes na extensa e
heterogênea produção do artista: carne (materialidade corporal), passagem (reflexão plástica sobre
espaço) e cifra (pequenos segredos biográficos). No título da exposição, contudo, destaca-se o
assunto norteador da poética do artista, o da memória, que atravessa diversas séries, como As
paredes que me cercam, A divina comédia, Memórias da cozinha e Estudos de luz e sombra,
abordadas nas próximas seções. 3 UMA ARTE DA MEMÓRIA FIGURA 1 – PAULO GAIAD, PAISAGEM A CAMINHO DE DRESDEN, 1995, GRAFITE E GUACHE SOBRE PAPEL, 50.0 X 69.5
CM, COLEÇÃO MUSEU VICTOR MEIRELLES, FLORIANÓPOLIS FIGURA 1 – PAULO GAIAD, PAISAGEM A CAMINHO DE DRESDEN, 1995, GRAFITE E GUACHE SOBRE PAPEL, 50.0 X 69.5
CM, COLEÇÃO MUSEU VICTOR MEIRELLES, FLORIANÓPOLIS FONTE: MUSEU VICTOR MEIRELLES. Disponível em: https://museuvictormeirelles.museus.gov.br. Acesso em: 25 jan. 2019. FONTE: MUSEU VICTOR MEIRELLES. FONTE: MUSEU VICTOR MEIRELLES. Disponível em: https://museuvictormeirelles.museus.gov.br. Acesso em: 25 jan. 2019. Disponível em: https://museuvictormeirelles.museus.gov.br. Acesso em: 25 jan. 2019. A partir de 2000, reafirmando e estendendo tema já presente em trabalhos anteriores, a
memória e o seu processo de reorganização do sucedido, Gaiad associou com frequência pintura e
fotografia. Resultaram desse encontro entre meios de expressão séries como As paredes que me
cercam (2003-2007), A divina comédia (2003-2007), Memórias da cozinha (2005), Estudos de luz e
sombra (2007), Fragmentos de um Noturno (2008), Desenho das sombras (2009) e After Darkness
(2014). Como notou Rosângela Miranda Cherem, que no texto “Sensibilidades biográficas e
percepções temporais na obra de Paulo Gaiad” abordou a trajetória e o fazer do artista, a obra de
Gaiad distingue-se “por um fluxo onde a imagem e a linguagem se rebatem por meio de distância e 7 7 Pintura, fotografia e memória na produção artística de Paulo Gaiad
Wagner Jonasson da Costa Lima Pintura, fotografia e memória na produção artística de Paulo Gaiad
Wagner Jonasson da Costa Lima proximidade, preenchimento e vazio, superfície e profundidade, lembrança e apagamento”
(CHEREM, 2015, p. 1). No interior dessa produção, ainda nas palavras de Cherem, DOI: https://doi.org/10.5965/18083129152021e0024 DAPesquisa, Florianópolis, v. 16, p. 01-22, jul. 2021. DAPesquisa, Florianópolis, v. 16, p. 01-22, jul. 2021. 4 MEIOS, PROCEDIMENTOS E EFEITOS DAP
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Entre 2003 e 2007, Gaiad produziu um conjunto de pinturas inspiradas na Divina Comédia
de Dante Alighieri (1265-1321) e nas gravuras de Gustave Doré (1832-1883) que ilustram o poema
(CHEREM, 2015). Embora impelidos pelo épico dantesco e sua iconografia, os trabalhos de Gaiad
constituem um universo particular, baseado em registros fotográficos das tragédias e prazeres
humanos. Essas imagens, coletadas em livros, revistas e feiras de antiguidades, converteram-se em
suporte para os gestos do pintor, adquirindo assim outros sentidos, muitas vezes simbólicos. Nessa
situação, o objet trouvé – “qualquer objeto encontrado por um artista e apresentado sem qualquer
alteração ou somente com modificações mínimas, como obra de arte” (LUCIE-SMITH, 1990, p. 139)
– transformou-se em image trouvée. Intitulada A divina comédia e também dividida em Inferno, 8 8 8 Pintura, fotografia e memória na produção artística de Paulo Gaiad
Wagner Jonasson da Costa Lima Pintura, fotografia e memória na produção artística de Paulo Gaiad
Wagner Jonasson da Costa Lima Pintura, fotografia e memória na produção artística de Paulo Gaiad
Wagner Jonasson da Costa Lima Purgatório e Paraíso, a série de Gaiad parece reapresentar e reformular, mediante a reunião entre
pintura e fotografia, figuras e conteúdo do passado coletivo. Para realizar a pintura A porta do inferno (Figura 2), que abre e compõe a série Inferno,
Gaiad apropriou-se de uma imagem fotográfica da rendição de soldados alemães na Primeira
Guerra Mundial (CHEREM, 2015). Ampliada e transposta para uma tela de grandes dimensões, por
meio de processo que o artista denominou de “macrofotografia” (BOPPRÉ, 2014), a imagem em
preto e branco recebeu econômica intervenção pictórica. No quadro de Gaiad, os prisioneiros
amontoados, enquadrados de cima para baixo, têm os rostos ocultados por pequenas manchas de
tinta acrílica vermelha, com exceção de dois homens que encaram o observador no centro da
fotografia. O gesto pictórico, no caso, comparece como um tipo de “comentário” ao dramático
evento histórico. Com toques regulares do pincel, o artista suprimiu, uma a uma, a identidade
daqueles que compõem a multidão, em um movimento ascendente de desumanização. Associado
às imagens da guerra, o Inferno de Gaiad mostra-se como espaço no qual prevalecem a assimetria
de poder e a legitimação da violência. FIGURA 2 – PAULO GAIAD, A PORTA DO INFERNO, 2007, FOTOGRAFIA, ACRÍLICA E COLAGEM
140 CM, COLEÇÃO PARTICULAR FONTE: LIMA, Fifo. Paulo Gaiad. Florianópolis: Tempo Editorial, 2010. DAPesquisa, Florianópolis, v. 16, p. 01-22, jul. 2021. DOI: https://doi.org/10.5965/18083129152021e0024 DOI: https://doi.org/10.5965/18083129152021e0024 DAPesquisa, Florianópolis, v. 16, p. 01-22, jul. 2021. 4 MEIOS, PROCEDIMENTOS E EFEITOS FONTE: LIMA, Fifo. Paulo Gaiad. Florianópolis: Tempo Editorial, 2010. A série Purgatório, por sua vez, surgiu de uma coleção de fotografias de família, sem
identificação, adquirida pelo artista em uma feira de antiguidades em Curitiba – PR (CHEREM, DAPesquisa, Florianópolis, v. 16, p. 01-22, jul. 2021. //
/
/ 9 DOI: https://doi.org/10.5965/18083129152021e0024 9 Pintura, fotografia e memória na produção artística de Paulo Gaiad
Wagner Jonasson da Costa Lima Pintura, fotografia e memória na produção artística de Paulo Gaiad
Wagner Jonasson da Costa Lima 2015). Para Gaiad (2007), era significativo o anonimato das pessoas retratadas e o abandono desse
conjunto de imagens. Depois de impressas sobre papel jornal “quase podre” (GAIAD, 2007), as
fotografias foram cobertas por camada semitransparente de tinta branca, que atenuou detalhes e
contrastes das imagens, e fixadas sobre suporte de papelão pardo que estampa a legenda
carimbada “Estúdio de Reproduções Fotográficas Ninho do Lagarto – Campeche – Brasil – Lost in
Purgatory”. Na Figura 3, duas mulheres, vestidas com roupas de décadas passadas, olham e sorriem
para o observador, considerando-se a intervenção pictórica sobre a imagem, como se estivessem
distanciadas ou encobertas por nevoeiro. Assim, reelaborando aspectos do segundo reino da Divina
Comédia de Alighieri, no qual a expiação de faltas passadas constitui a condição para a beatitude, o
Purgatório de Gaiad é marcado pela atmosfera nebulosa, indistinta, resultante de sutil ação de
apagamento. 2015). Para Gaiad (2007), era significativo o anonimato das pessoas retratadas e o abandono desse
conjunto de imagens. Depois de impressas sobre papel jornal “quase podre” (GAIAD, 2007), as
fotografias foram cobertas por camada semitransparente de tinta branca, que atenuou detalhes e
contrastes das imagens, e fixadas sobre suporte de papelão pardo que estampa a legenda
carimbada “Estúdio de Reproduções Fotográficas Ninho do Lagarto – Campeche – Brasil – Lost in
Purgatory”. Na Figura 3, duas mulheres, vestidas com roupas de décadas passadas, olham e sorriem
para o observador, considerando-se a intervenção pictórica sobre a imagem, como se estivessem
distanciadas ou encobertas por nevoeiro. Assim, reelaborando aspectos do segundo reino da Divina
Comédia de Alighieri, no qual a expiação de faltas passadas constitui a condição para a beatitude, o
Purgatório de Gaiad é marcado pela atmosfera nebulosa, indistinta, resultante de sutil ação de
apagamento. FIGURA 3 – PAULO GAIAD, PURGATÓRIO, 2003-2007, TINTA SOBRE PAPEL JORNAL SOBRE PAPELÃO, 36 X 50 CM,
COLEÇÃO PARTICULAR
FONTE: Acervo do artista. 4 MEIOS, PROCEDIMENTOS E EFEITOS FIGURA 3 – PAULO GAIAD, PURGATÓRIO, 2003-2007, TINTA SOBRE PAPEL JORNAL SOBRE PAPELÃO, 36 X 50 CM,
COLEÇÃO PARTICULAR FIGURA 3 – PAULO GAIAD, PURGATÓRIO, 2003-2007, TINTA SOBRE PAPEL JORNAL SOBRE PAPELÃO, 36 X 50 CM,
COLEÇÃO PARTICULAR FONTE: Acervo do artista. FONTE: Acervo do artista. Já a série Paraíso teve como ponto de partida o livro Forbidden erótica, que reúne
fotografias eróticas dos séculos XIX e XX, todas pertencentes ao colecionador Mark Lee Rotenberg
(CHEREM, 2015). Desse acervo de imagens, Gaiad fotografou novamente pormenores que
considerou mais sugestivos: um olhar convidativo, uma boca aberta, um toque de mão (Figura 4). Esse movimento em direção ao detalhe eliminou referências espaciais que localizassem as figuras. O primeiríssimo plano, que no âmbito do cinema também destaca um rosto ou amplia um objeto,
“lavra um outro tento contra a ordem familiar das aparências” (EPSTEIN, 1983. p. 284). Além disso, 10 Pintura, fotografia e memória na produção artística de Paulo Gaiad
Wagner Jonasson da Costa Lima Pintura, fotografia e memória na produção artística de Paulo Gaiad
Wagner Jonasson da Costa Lima Pintura, fotografia e memória na produção artística de Paulo Gaiad
Wagner Jonasson da Costa Lima a proximidade tornou a textura granular da superfície fotográfica mais perceptível, estabelecendo
uma espécie de “tato visual”, quer dizer, o olhar como prolongamento dos dedos. Posteriormente,
as imagens foram impressas sobre papel, afixadas sobre espessas placas de gesso, quebradas e
reconstituídas, à maneira de achados arqueológicos. Nesse conjunto de trabalhos, o paraíso, uma
vez perdido, foi reconquistado. PAULO GAIAD, DIVINA COMÉDIA: PARAÍSO, 2004, PAPEL SOBRE GESSO, 78 X 78 CM,
FONTE: LIMA, Fifo. Paulo Gaiad. Florianópolis: Tempo Editorial, 2010. FONTE: LIMA, Fifo. Paulo Gaiad. Florianópolis: Tempo Editorial, 2010. Em determinadas peças da série Paraíso, além de incisões e rasgos que marcam e
desgastam a superfície das imagens, que dessa maneira parecem envelhecidas pelo uso e pelo
tempo, o artista empregou expedientes da pintura. No trabalho da Figura 5, composto de tinta,
papel e suporte de gesso, toques de magenta retocam a cor dos lábios e as sombras dos olhos de
uma figura feminina, enquanto pinceladas palpáveis de azul estabelecem um fundo para a cena. A
fotografia, a partir de tais acréscimos, assumiu feição ambígua e arbitrária, visto que não há plena
coincidência, mas certo descolamento entre imagem, matéria e cor. DAPesquisa, Florianópolis, v. 16, p. 01-22, jul. 2021.
DOI: https://doi.org/10.5965/18083129152021e0024 DAPesquisa, Florianópolis, v. 16, p. 01 22, jul. 2021.
DOI: https://doi.org/10.5965/18083129152021e0024 4 MEIOS, PROCEDIMENTOS E EFEITOS Esse efeito decorre,
principalmente, do contraste entre a superfície lisa e dessaturada da fotografia e a superfície
encorpada e intensa da intervenção pictórica. Ao constituir essa diferença de textura e coloração,
Gaiad enfatizou no mesmo suporte o processo tangível de revitalização e, por consequência, de
reconstrução da imagem. DAPesquisa, Florianópolis, v. 16, p. 01-22, jul. 2021. DOI: https://doi.org/10.5965/18083129152021e0024 11 11 Pintura, fotografia e memória na produção artística de Paulo Gaiad
Wagner Jonasson da Costa Lima FIGURA 5 – PAULO GAIAD, DIVINA COMÉDIA: PARAÍSO, 2003-2007, TINTA SOBRE PAPEL SOBRE GESSO, 40 X 40 CM,
COLEÇÃO PARTICULAR FONTE: Acervo do artista. FONTE: Acervo do artista. A pintura de Gaiad, nessa situação particular, bem como nas séries Inferno e Purgatório,
concentrou-se em fazer modificações sobre a imagem apropriada e desse jeito remodelar a sua
configuração e sentido. Todavia, cabe notar que a coloração manual de fotografias tornou-se
frequente já no século XIX. Normalmente, a sobreposição era justificada pelas “deficiências” da
imagem, como, por exemplo, a ausência de cor. Assim surgiu o retoque sobre o negativo ou a cópia
(GONZÁLEZ FLORES, 2011). Gaiad, entretanto, não buscou o aperfeiçoamento da fotografia, mas a
justaposição de diversos meios de expressão. Dessa maneira, avizinhou-se da produção de artistas
como Robert Rauschenberg e Anselm Kiefer. Em comum, o modo de operar que privilegia a
“coexistência tensa de diferentes sistemas de signos dentro de um único espaço de representação”
(CATTANI, 2006, p. 111). Nessa operação de mestiçagem, na qual concorrem a fotografia e a
pintura, o automático e o artesanal, Gaiad substancializou processos como esquecimento e
recordação. DOI: https://doi.org/10.5965/18083129152021e0024 DAPesquisa, Florianópolis, v. 16, p. 01-22, jul. 2021. DAPesquisa, Florianópolis, v. 16, p. 01-22, jul. 2021.
DOI: https://doi.org/10.5965/18083129152021e0024 5 APREENDER O MOMENTO FUGIDIO Em sua trajetória artística, Gaiad não se restringiu a trabalhos com fortes conotações
emblemáticas, como os que integram a série A divina comédia, mas também abordou a pintura de
paisagem e a sua tradição. Esse gênero pictórico, que ocupou “os níveis mais baixos da hierarquia
de gêneros que dominou a reflexão sobre arte nos séculos XVI e XVII” (MATTOS, 2008, p. 11),
ganhou relevância a partir do século XVIII, notadamente com a produção de Caspar David Friedrich 12 12 Pintura, fotografia e memória na produção artística de Paulo Gaiad
Wagner Jonasson da Costa Lima Pintura, fotografia e memória na produção artística de Paulo Gaiad
Wagner Jonasson da Costa Lima Pintura, fotografia e memória na produção artística de Paulo Gaiad
Wagner Jonasson da Costa Lima (1774-1840), William Turner (1775-1851), John Constable (1776-1837), Camille Corot (1796-1875),
Camille Pissarro (1830-1903), dentre outros. Em meados do século XIX, a pintura de paisagem, que
entre os artistas românticos esteve vinculada à projeção de sentimentos, dirigiu-se
progressivamente para a captura do contingente. Na produção de Gaiad, essas poéticas foram
agenciadas, reelaboradas e atualizadas por meio da conjunção entre pintura e fotografia. A partir
desses materiais, o artista atribuiu concretude à dinâmica entre a passagem do tempo e a atividade
da memória. Por exemplo, na pintura Noturno (Figura 6), realizada em 2003, nuvens delineadas
esquematicamente ganham densidade devido ao depósito e acúmulo de materiais como cimento,
areia e massa. Construídas sem gradientes de luz e sombra, essas nuvens, de brancura próxima da
uniformidade, destacam-se do fundo azul escuro e segmentam a parte superior da tela. Composição e cores lembram o clima dramático e amiúde espiritual de pinturas como A cruz ao
lado do Mar Báltico (1815) ou Beira-mar ao luar (1835-1836), de Caspar David Friedrich, importante
representante do romantismo alemão. Já na parte inferior da tela, Gaiad tratou de encadear
horizontalmente imagens fotográficas em instantes distintos. A partir dessa disposição, nuvens
avançam sobre o luar e tem-se quase a sensação de movimento. Desse modo, no mesmo trabalho,
o artista estabeleceu um contraste entre a unicidade da pintura e a multiplicidade da fotografia,
entre sucessão de acontecimentos e fixação desses acontecimentos, entre o ocorrido e a
rememoração. FIGURA 6 – PAULO GAIAD, NOTURNO, 2003, CIMENTO, AREIA, MASSA E FOTOGRAFIA SOBRE TELA, 200 X 140 CM,
COLEÇÃO PARTICULAR ÃO PARTICULAR
FONTE: LIMA, Fifo. Paulo Gaiad. Florianópolis: Tempo Editorial, 2010. FONTE: LIMA, Fifo. Paulo Gaiad. Florianópolis: Tempo Editorial, 2010. 5 APREENDER O MOMENTO FUGIDIO DAPesquisa, Florianópolis, v. 16, p. 01-22, jul. 2021. DOI: https://doi.org/10.5965/18083129152021e0024 13 13 Pintura, fotografia e memória na produção artística de Paulo Gaiad
Wagner Jonasson da Costa Lima Pintura, fotografia e memória na produção artística de Paulo Gaiad
Wagner Jonasson da Costa Lima A associação entre pintura de paisagem e imagem fotográfica encontra-se também em
trabalhos como Céu de Delft I e Céu de Delft II, ambos pertencentes à série Estudos de luz e sombra
e elaborados depois da visita de Gaiad à cidade holandesa (GAIAD, 2007). A viagem decorreu da
participação em 2006 no projeto Pintar um Futuro (PAINT A FUTURE, 2019), concebido pela artista
Hetty van der Linden. Após tomar parte no projeto, que visa promover o bem-estar de crianças
carentes ao redor do mundo, Gaiad foi convidado para diversos eventos na Europa, como
residências e exposições. Durante a breve excursão a Delft, de um dia, espantou-se com as
repentinas mudanças do clima e sobretudo do aspecto do céu. Como declarou em entrevista, “ele
tem uma movimentação muito rápida, tá limpo, daqui a pouco fecha, chove, limpa de novo”
(GAIAD, 2007). Impressionado pelo fenômeno, o artista realizou vários registros fotográficos que
mais tarde foram selecionados e transpostos para a superfície de duas telas. Em seguida, iniciou o
trabalho com as tintas, complementando e alterando as imagens capturadas. A pintura Céu de Delft II (Figura 7), por exemplo, mostra a imagem da silhueta da cidade –
telhados, postes, pináculos – contra um acumulado de nuvens modeladas pela luz. Manchas opacas
de azul escuro e camadas transparentes de cores terrosas, realizadas com a tinta acrílica,
completam e alteram a vista. Abaixo, pinceladas largas, juntamente com escorridos, respingos e
rasgos, ocupam ampla área do quadro. A linha do horizonte elevada, próxima à borda superior do
quadro, sublinha a passagem de uma visualidade fotográfica e sua dimensão especular para uma
gestualidade informal e seu investimento na matéria. Dessa maneira, pode-se acompanhar a
transição da figuração para a não figuração, do determinado para o indeterminado, do registro para
a reminiscência. A pintura formulou-se somente depois que o artista prolongou e reafirmou, com
base nos elementos fornecidos pela fotografia, as suas percepções e afetos. DAPesquisa, Florianópolis, v. 16, p. 01-22, jul. 2021. DOI: https://doi.org/10.5965/18083129152021e0024 DAPesquisa, Florianópolis, v. 16, p. 01-22, jul. 2021. 5 APREENDER O MOMENTO FUGIDIO DOI: https://doi.org/10.5965/18083129152021e0024 14 Pintura, fotografia e memória na produção artística de Paulo Gaiad
Wagner Jonasson da Costa Lima FIGURA 7 – PAULO GAIAD, CÉU DE DELFT II, 2007, FOTOGRAFIA, ACRÍLICA E COLAGEM SOBRE PAPEL SOBRE TELA, 200 X
140 CM, COLEÇÃO PARTICULAR FONTE: LIMA, Fifo. Paulo Gaiad. Florianópolis: Tempo Editorial, 2010. FONTE: LIMA, Fifo. Paulo Gaiad. Florianópolis: Tempo Editorial, 2010. Com as suas nuvens e o seu tratamento vigoroso, Céu de Delft II faz menção deliberada à
pintura de paisagem holandesa do século XVII e, consequentemente, à produção de William Turner
e John Constable. Como observou Kenneth Clark, nas telas de Jacob van Ruisdael (1628-1682),
principal expoente da fase clássica da pintura de paisagem holandesa, “as nuvens acumulam-se no
espaço, as sombras navegam através de planícies” (CLARK, 1961, p. 54). Esse sentido de mobilidade
dos elementos naturais tornou-se modelo para toda uma escola pictórica da Inglaterra do final do
século XVIII e início do seguinte. Entre 1821 e 1822, Constable pintou cerca de 100 estudos a óleo
de céus em diversas condições, observando formações de nuvens e seus deslocamentos. Estudioso
da tradição e atento observador da natureza, o pintor inglês alegou que sua arte visava ao “breve
momento arrancado ao tempo fugidio para uma existência permanente e sóbria” (CONSTABLE
apud GOMBRICH, 2007, p. 325). Em suas paisagens, Paulo Gaiad parece também cultivar o desejo de captura do efêmero, o
que revela afinidade com a poética romântica, caracterizada pelo culto à natureza e à imaginação,
assim como pela experiência da “caducidade do tempo”, na qual “o eterno desaparecer e morrer
parecem infinitos” (PAZ, 1992, p. 52). Ao mesmo tempo, o artista foi informado pela fotografia,
cinema e televisão da mesma maneira que uma geração de pintores que iniciaram as suas
atividades nas décadas de 1970 e 1980. A partir daí, numerosos artistas lidaram com “um universo DAPesquisa, Florianópolis, v. 16, p. 01-22, jul. 2021. DOI: https://doi.org/10.5965/18083129152021e0024 DAPesquisa, Florianópolis, v. 16, p. 01-22, jul. 2021. DOI h
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DOI: https://doi.org/10.5965/18083129152021e0024 15 Pintura, fotografia e memória na produção artística de Paulo Gaiad
Wagner Jonasson da Costa Lima Pintura, fotografia e memória na produção artística de Paulo Gaiad
Wagner Jonasson da Costa Lima de informações fragmentado, cheio de imagens das mais diversas épocas e procedências”
(CHIARELLI, 2001, p. 265), cada qual à sua maneira, dependendo dos modelos selecionados. 5 APREENDER O MOMENTO FUGIDIO Em
Noturno e Céu de Delft II, o uso de cenas fotográficas diferencialmente articuladas, ao lado de
referências à tradição da pintura de paisagem, apresenta-se como uma solução encontrada por
Gaiad para figurar as relações entre o instante que passa e a lembrança que o reelabora. DOI: https://doi.org/10.5965/18083129152021e0024 DAPesquisa, Florianópolis, v. 16, p. 01-22, jul. 2021.
DOI: https://doi.org/10.5965/18083129152021e0024 DAPesquisa, Florianópolis, v. 16, p. 01-22, jul. 2021. 6 A TRANSITORIEDADE DE TODAS AS COISAS Além da pintura de paisagem, outros gêneros pictóricos tradicionais, como a
natureza-morta, serviram ao propósito de Gaiad de materializar o processo mnemônico. A
natureza-morta, na acepção de Meyer Schapiro (2010. p. 61), “consiste de objetos que, sejam
artificiais ou naturais, estão subordinados ao homem como elementos de uso, manipulação e
prazer”. Desde a sua afirmação como gênero autônomo no século XVII, a natureza-morta engajou
pintores na representação de aspectos singulares de objetos corriqueiros. A princípio de aparência
banal, esses objetos, ampulhetas, castiçais, livros, cálices tornaram-se motivo de divagação
metafísica nas vanitas, tipo de pintura voltada para a ponderação sobre a transitoriedade de todas
as coisas que se desenvolveu plenamente nos Países Baixos a partir de 1600. Nos quadros de Gaiad,
a natureza-morta, a pintura e a fotografia aparecem como materiais que contribuem para a
concretização do entrelace entre falta e recordação. Sob esse viés, pode-se destacar a tela As Paredes que me Cercam (Figura 8), concebida em
torno da fotografia de um copo que evoca, com o seu brilho e a sua disposição, a pintura de Jean-
Baptiste-Siméon Chardin (1699-1779), artista reconhecido por naturezas-mortas como O pote de
azeitonas (1760), cuja técnica era capaz de manifestar, na visão de Denis Diderot (2006. p. 90), “a
própria substância dos objetos”. Abaixo da imagem fotográfica, Gaiad esboçou com pinceladas
rápidas uma toalha de mesa branca que se destaca do fundo composto de uma superfície marrom
e de grandes manchas verdes escuras. A partir de recursos pictóricos, o artista fez coincidir a
imagem do copo e o objeto fotografia, ambos apoiados arbitrariamente sobre um tampo
inacabado. Nessa lógica, a pintura de Gaiad contém simultaneamente uma referência ao mundo
cotidiano e um fragmento desse mundo na composição do quadro. Juntas, pintura e fotografia
instauram um jogo de ausência e presença. 16 16 Pintura, fotografia e memória na produção artística de Paulo Gaiad
Wagner Jonasson da Costa Lima Pintura, fotografia e memória na produção artística de Paulo Gaiad
Wagner Jonasson da Costa Lima FIGURA 8 – PAULO GAIAD, AS PAREDES QUE ME CERCAM, 2003, FOTOGRAFIA, ACRÍLICA E COLAGEM SOBRE TELA, 120 X
150 CM, COLEÇÃO PARTICULAR FIGURA 8 – PAULO GAIAD, AS PAREDES QUE ME CERCAM, 2003, FOTOGRAFIA, ACRÍLICA E COLAGEM SOBRE TELA, 120 X
150 CM, COLEÇÃO PARTICULAR FONTE: LIMA, Fifo. Paulo Gaiad. Florianópolis: Tempo Editorial, 2010. FONTE: LIMA, Fifo. Paulo Gaiad. Florianópolis: Tempo Editorial, 2010. 6 A TRANSITORIEDADE DE TODAS AS COISAS Em outros trabalhos de Gaiad, a natureza-morta coloca-se de modo mais explícito como
veículo para a memória autobiográfica e para a meditação sobre a finitude. É o caso de Memória da
cozinha (Figura 9), pintura em que o artista elaborou, a partir da mesa que pertenceu à sua família
e de objetos pessoais, as suas recordações da infância (LIMA, 2010). Os utensílios escolhidos e
fotografados – as cadeiras, a mesa, a comida, a bebida, a louça – remetem ao doméstico e ao
convival, sugerindo um anseio de restauração da ordem familiar. Simultaneamente, sob uma
atmosfera simbólica, congênere àquela encontrada nas vanitas do século XVII, tais objetos
comparecem como lembranças do efêmero e da morte. Em meio a esse simbolismo, rasgos e
borrões de tinta degradam e lambuzam a superfície da imagem, promovendo um contraponto
entre inclusão e exclusão do observador, entre profundidade e anteparo. Nessas condições, o
quadro enseja a sobreposição de sinais emblemáticos da passagem do tempo e de sinais concretos
da realização do trabalho. DAPesquisa, Florianópolis, v. 16, p. 01-22, jul. 2021. DOI: https://doi.org/10.5965/18083129152021e0024 17 Pintura, fotografia e memória na produção artística de Paulo Gaiad
Wagner Jonasson da Costa Lima FIGURA 9 – PAULO GAIAD, MEMÓRIA DA COZINHA, 2007, FOTOGRAFIA, ACRÍLICA E COLAGEM SOBRE TELA, 140 X 140
CM, COLEÇÃO PARTICULAR AIAD, MEMÓRIA DA COZINHA, 2007, FOTOGRAFIA, ACRÍLICA E COLAGEM
EÇÃO PARTICULAR
FONTE: LIMA, Fifo. Paulo Gaiad. Florianópolis: Tempo Editorial, 2010. FONTE: LIMA, Fifo. Paulo Gaiad. Florianópolis: Tempo Editorial, 2010. Em As paredes que me cercam e Memória da cozinha, Gaiad parece propor vias de contato,
sem desconsiderar o “anti-ilusionismo” da arte moderna, entre a tradição da natureza-morta e a
mestiçagem da pintura contemporânea. No início do século XX, Pablo Picasso (1881-1973) salientou
a concretude de suas naturezas-mortas agregando recortes de jornal à tela, entre outros materiais,
ao lado da tinta a óleo tradicional, não raro misturada com areia. Tanto a colagem quanto o efeito
de textura áspera indicam o interesse dos cubistas pelo quadro como objeto e não como janela. Posteriormente, Jasper Johns (1930-), nas décadas de 1950 e de 1960, incluiu utensílios familiares –
uma vassoura, uma xícara, uma lanterna, um prato – em Fool’s house (1962) e Souvenir (1964),
trabalhos que atenuam os limites entre o espaço cotidiano e o espaço pictórico. DOI: https://doi.org/10.5965/18083129152021e0024 DAPesquisa, Florianópolis, v. 16, p. 01-22, jul. 2021. 6 A TRANSITORIEDADE DE TODAS AS COISAS Tal como Picasso e
Johns, Paulo Gaiad, ao adotar a lógica agregadora da colagem e da assemblagem, transformou o
plano da pintura, e de suas naturezas-mortas, em um amontoamento heteróclito de materiais que
exibem as suas qualidades. No interior desse amálgama denso estabelecido por Gaiad, a fotografia, junto à pintura,
cumpriu importante papel, tanto constitutivo quanto significativo. Em muitas ocasiões, o artista
iniciou seus trabalhos a partir de imagem fotográfica fixada sobre a superfície do quadro,
modificando a sua configuração, textura e cor. Contudo, em telas como As paredes que me cercam,
ou mesmo Memória da cozinha, a fotografia apresentou-se não apenas como imagem que provoca 18 18 Pintura, fotografia e memória na produção artística de Paulo Gaiad
Wagner Jonasson da Costa Lima Pintura, fotografia e memória na produção artística de Paulo Gaiad
Wagner Jonasson da Costa Lima Pintura, fotografia e memória na produção artística de Paulo Gaiad
Wagner Jonasson da Costa Lima e dirige a ação do pintor, mas como objeto de manipulação. Nesse sentido, possui materialidade e,
tal como outros objetos, está sujeita à deterioração. Esse processo entrópico, no qual a organização
caminha para a desorganização e, em seguida, para a ruína, parece acelerar-se, no caso de
Memória da cozinha, a partir da intervenção pictórica. A tinta acrílica, que produz grumos,
escorridos e logo depois rasgões, corrompe a superfície homogênea e maquinal da imagem
fotográfica. e dirige a ação do pintor, mas como objeto de manipulação. Nesse sentido, possui materialidade e,
tal como outros objetos, está sujeita à deterioração. Esse processo entrópico, no qual a organização
caminha para a desorganização e, em seguida, para a ruína, parece acelerar-se, no caso de
Memória da cozinha, a partir da intervenção pictórica. A tinta acrílica, que produz grumos,
escorridos e logo depois rasgões, corrompe a superfície homogênea e maquinal da imagem
fotográfica. DOI: https://doi.org/10.5965/18083129152021e0024 DAPesquisa, Florianópolis, v. 16, p. 01-22, jul. 2021. DAPesquisa, Florianópolis, v. 16, p. 01-22, jul. 2021. nópolis, v. 16, p. 01-22, jul. 2021. 7 CONSIDERAÇÕES FINAIS Surgida no século XIX como máquina de produzir imagens de captura, a fotografia, no
transcorrer do século XX, foi admitida decididamente na esfera da pintura, notadamente em
produções de artistas contemporâneos como Robert Rauschenberg, Anselm Kiefer e também, a
partir de 2000, Paulo Gaiad. Tanto Rauschenberg quanto Kiefer investiram, em situações distintas,
contra a disjunção das artes, adotando um modo de operar “transgenérico”, no qual se valoriza a
conjunção de meios. À semelhança desses pintores, Gaiad levou em conta, na construção de seus
quadros, a coexistência entre o pictórico e o fotográfico. Todavia, de acordo com declarações do
artista brasileiro, essa associação serviu principalmente ao seu propósito, ou projeto poético, de
tratar da memória como recriação do ocorrido, tema que alicerça o seu trabalho artístico. Na série A divina comédia, por exemplo, imagens fotográficas preexistentes, coletadas em
livros, revistas e feiras de antiguidades transformaram-se em suporte para o trabalho do pintor. Em
tal situação, na qual a superfície da fotografia recebeu a matéria pictórica, o artista, a partir de
ocultamentos, transparências e contrastes de textura e cor, alterou a aparência e, por conseguinte,
o sentido das imagens recolhidas. Dessa maneira, a série de Gaiad, desenvolvida entre os anos de
2003 e 2007, recolocou figuras e reorganizou conteúdo do passado. Nessa operação de
reelaboração do sucedido, que envolveu a “mestiçagem” – justaposição de diferentes meios no
interior de determinado espaço de representação – Gaiad tornou sensíveis e tateáveis processos
imateriais, reconhecidamente subjetivos, como a recordação e o esquecimento. Para levar adiante esse projeto de materialização do mnemônico, Gaiad convocou ainda a
tradição da pintura, recorrendo a gêneros já repertoriados pela história da arte, como a paisagem e
a natureza-morta, e os seus conteúdos. Em suas paisagens, que remetem em alguns momentos à 19 Pintura, fotografia e memória na produção artística de Paulo Gaiad
Wagner Jonasson da Costa Lima Pintura, fotografia e memória na produção artística de Paulo Gaiad
Wagner Jonasson da Costa Lima pintura holandesa e romântica, o artista tratou das relações entre instante e lembrança. Já em suas
naturezas-mortas, que exibem atmosfera simbólica semelhante àquela encontrada nas vanitas do
século XVII, das relações entre falta e recordação. Nesses casos, gêneros reconhecidos, e as suas
iconografias características, foram utilizados como materiais, ao lado da pintura e da fotografia, na
concretização do que o artista concebe como memória. REFERÊNCIAS BOPPRÉ, F. Apontamentos sobre um olhar do crítico. In: PUNCTUM. Florianópolis : UFSC, 10. dez. 2014. Disponível em: http://www.punctum.ufsc.br/?p=2287. Acesso em: 25 jan. 2019. BOPPRÉ, F. Apontamentos sobre um olhar do crítico. In: PUNCTUM. Florianópolis : UFSC, 10. dez. 2014. Disponível em: http://www.punctum.ufsc.br/?p=2287. Acesso em: 25 jan. 2019. CATTANI, I. B. Cruzamentos e tensões: mestiçagens na arte contemporânea no Brasil e no Canadá. Interfaces Brasil/Canadá, Pelotas, v. 6, n. 1, p. 109-130, 2006. Disponível em:
https://periodicos.ufpel.edu.br/ojs2/index.php/interfaces/article/view/6920. Acesso em: 19 fev. 2019. CATTANI, I. B. Cruzamentos e tensões: mestiçagens na arte contemporânea no Brasil e no Canadá. Interfaces Brasil/Canadá, Pelotas, v. 6, n. 1, p. 109-130, 2006. Disponível em:
https://periodicos.ufpel.edu.br/ojs2/index.php/interfaces/article/view/6920. Acesso em: 19 fev. 2019. CATTANI, I. B. Cruzamentos e tensões: mestiçagens na arte contemporânea no Brasil e no Canadá. Interfaces Brasil/Canadá, Pelotas, v. 6, n. 1, p. 109-130, 2006. Disponível em:
https://periodicos.ufpel.edu.br/ojs2/index.php/interfaces/article/view/6920. Acesso em: 19 fev. 2019. M, R. M. Sensibilidades biográficas e percepções temporais na obra de Paulo Gaiad. In:
Ó CHEREM, R. M. Sensibilidades biográficas e percepções temporais na obra de Paulo Gaiad. In:
SIMPÓSIO NACIONAL DE HISTÓRIA, 28., 2015, Florianópolis. Anais eletrônicos [...] Florianópolis:
UFSC/UDESC, 2015. p. 1-16. Disponível em: CHEREM, R. M. Sensibilidades biográficas e percepções temporais na obra de Paulo Gaiad. In:
SIMPÓSIO NACIONAL DE HISTÓRIA, 28., 2015, Florianópolis. Anais eletrônicos [...] Florianópolis:
UFSC/UDESC, 2015. p. 1-16. Disponível em: CHEREM, R. M. Sensibilidades biográficas e percepções temporais na obra de Paulo Gaiad. In:
SIMPÓSIO NACIONAL DE HISTÓRIA, 28., 2015, Florianópolis. Anais eletrônicos [...] Florianópolis:
UFSC/UDESC, 2015. p. 1-16. Disponível em: SIMPÓSIO NACIONAL DE HISTÓRIA, 28., 2015, Florianópolis. Anais eletrônicos [...] Florianópolis:
UFSC/UDESC, 2015. p. 1-16. Disponível em: http://www.snh2015.anpuh.org/resources/anais/39/1434396861_ARQUIVO__Sensibilidadesbiogra
ficasepercepcoestemporaisnaobradePauloGaiad.pdf. Acesso em: 25 jan. 2019. http://www.snh2015.anpuh.org/resources/anais/39/1434396861_ARQUIVO__Sensibilidadesbiogra
ficasepercepcoestemporaisnaobradePauloGaiad.pdf. Acesso em: 25 jan. 2019. CHIARELLI, T. Considerações sobre o uso de imagens de segunda geração na arte contemporânea. In: BASBAUM, R. (org.). Arte contemporânea brasileira: texturas, dicções, ficções, estratégias. Rio
de Janeiro: Rios Ambiciosos, 2001. p. 257-270. Disponível em: CHIARELLI, T. Considerações sobre o uso de imagens de segunda geração na arte contemporânea. In: BASBAUM, R. (org.). Arte contemporânea brasileira: texturas, dicções, ficções, estratégias. Rio
de Janeiro: Rios Ambiciosos, 2001. p. 257-270. Disponível em: CHIARELLI, T. Considerações sobre o uso de imagens de segunda geração na arte contemporânea. In: BASBAUM, R. (org.). Arte contemporânea brasileira: texturas, dicções, ficções, estratégias. Rio
de Janeiro: Rios Ambiciosos, 2001. p. 257-270. Disponível em: DAPesquisa, Florianópolis, v. 16, p. 01-22, jul. 2021. 7 CONSIDERAÇÕES FINAIS E como na série A divina comédia, a
imagem fotográfica ganhou outra tessitura depois de submetida aos recursos pictóricos. Nessa perspectiva, além de orientado pelo tema da memória como recriação, o projeto
poético de Gaiad associou-se às práticas artísticas ditas “contemporâneas”, isto é, aquelas que se
afastam do preceito segundo o qual cada arte caminharia progressivamente para uma depuração
dos elementos que não lhe são essenciais. Quando se trata da produção de pintores
contemporâneos, como a de Paulo Gaiad, é possível encontrar uma desconcertante profusão de
programas de arte e nos quais não há nenhum material que desfrute da prerrogativa de ser
prontamente reconhecido como material para a pintura. A contaminação da pintura por outros
meios, como a fotografia, produziu um novo cenário artístico, abalizado por relações de
comunidade e não de exclusão. COUTINHO, W. Festa e democracia na arte do Parque Lage. Jornal do Brasil, Rio de Janeiro, 23 jul.
1984. Caderno B, p. 6. DIDEROT, D. Salão de 1763. In: LICHTENSTEIN, Jacqueline (org.). Os Gêneros pictóricos. São Paulo:
Ed. 34, 2006. p. 89-91. ( A Pintura: textos essenciais, 10) EPSTEIN, J. A inteligência de uma máquina - excertos. In: XAVIER, I. (org.). A experiência do cinema:
antologia. Rio de Janeiro: Graal, 1983. p. 283-292. (Coleção Arte e Cultura, 5). Disponível em:
https://www.academia.edu/7047622/38145313_Ismail_Xavier_A_Experiencia_Do_Cinema. Acesso
em: 21 jan. 2019. GAIAD, P. A Cor da nossa tela: Paulo Gaiad. [Entrevista cedida a] TV UFSC. Direção: Zeca Pires. Florianópolis, 2015. 1 vídeo (4min.27s). Publicado pelo canal TV UFSC no YouTube. Disponível
em: https://www.youtube.com/watch?v=4ek53mFmR08. Acesso em: 25 jan. 2019. GAIAD, P . Mariana de Abreu entrevista o artista plástico Paulo Gaiad. [Entrevista cedida a]
Mariana de Abreu Bastian , 20vê, TVCOM/RBS, Florianópolis, 2007. 1 vídeo (12min.49s). Publicado
pelo canal Mariana de abreu bastian no YouTube. Disponível em:
https://www.youtube.com/watch?v=s19aQo41fSY. Acesso em: 25 jan. 2019. EXPOSIÇÃO Impossibilias: arquivo e memória em Paulo Gaiad. Florianópolis : Fundação Cultural
Badesc, 26 nov. 2015. Disponível em: https://fundacaoculturalbadesc.com/exposicao-de-paulo-
gaiad-revisita-sua-trajetoria-artistica-e-ocupa-pela-primeira-vez-todos-os-espacos-da-fundacao-
cultural-badesc/. Acesso em: 25 jan. 2019. GOMBRICH, E. H. Arte e ilusão: um estudo da psicologia da representação pictórica. São Paulo:
WMF Martins Fontes, 2007. GONZÁLEZ FLORES, L. Fotografia e pintura: dois meios diferentes? São Paulo: WMF Martins Fontes,
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Wagner Jonasson da Costa Lima https://esgotadoarquivo.files.wordpress.com/2018/05/282646798-arte-contemporanea-
brasileira.pdf. Acesso em 26 jan. 2019. HUYSSEN, A. Memórias do modernismo. Rio de Janeiro: UFRJ, 1996. HUYSSEN, A. Memórias do modernismo. Rio de Janeiro: UFRJ, 1996. LIMA, F. Paulo Gaiad. Florianópolis: Tempo Editorial, 2010. LIMA, F. Paulo Gaiad. Florianópolis: Tempo Editorial, 2010. LIMA, F. Paulo Gaiad. Florianópolis: Tempo Editorial, 2010. CLARK, K. Paisagem na arte. Lisboa: Ulisseia, 1961. CLARK, K. Paisagem na arte. Lisboa: Ulisseia, 1961. CLARK, K. Paisagem na arte. Lisboa: Ulisseia, 1961. COSTA, M. L. A Bela enfurecida. In: MemoriaLage. Coleção Marcus Lontra (1983-1987) .Rio de Janeiro :
Escola de Artes Visuais do Parque Lage, 14 jul. 1984. 1 Cartaz, color., 43,9 x 31,0 cm . Disponível
em: https://www.memorialage.com.br/marcus-lontra/. Acesso em: 10 jan. 2019. COUTINHO, W. Festa e democracia na arte do Parque Lage. Jornal do Brasil, Rio de Janeiro, 23 jul. 1984. Caderno B, p. 6. DAPesquisa, Florianópolis, v. 16, p. 01-22, jul. 2021.
DOI: https://doi.org/10.5965/18083129152021e0024 LUCIE-SMITH, E. Dicionário de termos da arte. Lisboa: Dom Quixote, 1990. DAPesquisa, Florianópolis, v. 16, p. 01-22, jul. 2021. DOI: https://doi.org/10.5965/18083129152021e0024 21 Pintura, fotografia e memória na produção artística de Paulo Gaiad
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/ MAKOWIECKY, S. Paulo Gaiad – Artista – Viajante. In: ENCONTRO ASSOCIAÇÃO NACIONAL DE
PESQUISADORES EM ARTES PLÁSTICAS, 18., 2009, Salvador. Anais eletrônicos [...] Salvador:
EDUFBA, 2009. p. 2717-2731. Disponível em:
http://anpap.org.br/anais/2009/pdf/chtca/sandra_makowiecky.pdf. Acesso em: 25 jan. 2019. MATTOS, C. V. de (org.). Goethe e Hackert: sobre a pintura de paisagem: quadros na natureza na
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SCHAPIRO, M. A arte moderna: séculos XIX e XX : ensaios escolhidos. São Paulo: Editora da
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XX. São Paulo: Cosac Naify, 2008. p. 79-125. DAPesquisa, Florianópolis, v. 16, p. 01-22, jul. 2021. DOI: https://doi.org/10.5965/18083129152021e0024 22 22
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Microneedle Array Design Determines the Induction of Protective Memory CD8+ T Cell Responses Induced by a Recombinant Live Malaria Vaccine in Mice
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Abstract Copyright: 2011 Carey et al. This is an open-access article distributed under the terms of the Creative Commons Attributi
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright: 2011 Carey et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was funded by Enterprise Ireland (CFTD 07/117, http://www.enterprise-ireland.com), Science Foundation Ireland (NAP156, NAP170, www.sfi. ie) and the Medical Research Council, United Kingdom (G0600311, www.mrc.ac.uk). The funders had no role in study design, data collection and analysis, decision
to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: anne.moore@ucc.ie * E-mail: anne.moore@ucc.ie Microneedle Array Design Determines the Induction of
Protective Memory CD8+ T Cell Responses Induced by a
Recombinant Live Malaria Vaccine in Mice John B. Carey1, Frances E. Pearson2, Anto Vrdoljak1, Marie G. McGrath1, Abina M. Crean1, Patrick T. Walsh3, Timothy Doody1, Conor O’Mahony4, Adrian V. S. Hill2, Anne C. Moore1,5* 1 School of Pharmacy, University College Cork, Cork, Ireland, 2 The Jenner Institute, University of Oxford, Oxford, United Kingdom, 3 National Childrens’ Research Centre,
Our Lady’s Childrens’ Hospital Crumlin, Dublin, Ireland, 4 Tyndall National Institute, Lee Maltings, University College Cork, Cork, Ireland, 5 Department of Pharmacology,
University College Cork, Cork, Ireland Abstract Background: Vaccine delivery into the skin has received renewed interest due to ease of access to the immune system and
microvasculature, however the stratum corneum (SC), must be breached for successful vaccination. This has been achieved
by removing the SC by abrasion or scarification or by delivering the vaccine intradermally (ID) with traditional needle-and-
syringes or with long microneedle devices. Microneedle patch-based transdermal vaccine studies have predominantly
focused on antibody induction by inactivated or subunit vaccines. Here, our principal aim is to determine if the design of a
microneedle patch affects the CD8+ T cell responses to a malaria antigen induced by a live vaccine. Methodology and Findings: Recombinant modified vaccinia virus Ankara (MVA) expressing a malaria antigen was
percutaneously administered to mice using a range of silicon microneedle patches, termed ImmuPatch, that differed in
microneedle height, density, patch area and total pore volume. We demonstrate that microneedle arrays that have small
total pore volumes induce a significantly greater proportion of central memory T cells that vigorously expand to secondary
immunization. Microneedle-mediated vaccine priming induced significantly greater T cell immunity post-boost and
equivalent protection against malaria challenge compared to ID vaccination. Notably, unlike ID administration, ImmuPatch-
mediated vaccination did not induce inflammatory responses at the site of immunization or in draining lymph nodes. Conclusions/Significance: This study demonstrates that the design of microneedle patches significantly influences the
magnitude and memory of vaccine-induced CD8+ T cell responses and can be optimised for the induction of desired
immune responses. Furthermore, ImmuPatch-mediated delivery may be of benefit to reducing unwanted vaccine
reactogenicity. In addition to the advantages of low cost and lack of pain, the development of optimised microneedle array
designs for the induction of T cell responses by live vaccines aids the development of solutions to current obstacles of
immunization programmes. Citation: Carey JB, Pearson FE, Vrdoljak A, McGrath MG, Crean AM, et al. (2011) Microneedle Array Design Determines the Induction of Protective Memory CD8+ T
Cell Responses Induced by a Recombinant Live Malaria Vaccine in Mice. PLoS ONE 6(7): e22442. doi:10.1371/journal.pone.0022442 Editor: Erika Martins Braga, Universidade Federal de Minas Gerais, Brazil Received January 24, 2011; Accepted June 27, 2011; Published July 25, 2011 ey et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
tion, and reproduction in any medium, provided the original author and source are credited. PLoS ONE | www.plosone.org Microneedle array design influences the magnitude and
phenotype of vaccine-induced CD8+ T cell response The microneedle vaccine delivery field has predominantly
focussed on antibody induction by subunit vaccines [9]. It was
previously concluded, using a subunit vaccine that induction of
serum antibody responses was independent of depth of delivery,
density of microneedles or area of application [10], thus proposing
that the design of the microneedle array has little impact on the
induction of immunity. Furthermore, efficacy studies of micro-
needle-delivered vaccines have centred on mucosal protection,
generally of influenza virus respiratory challenge. Using a mouse malaria model of recombinant MVA-PbCSP-
induced immunity and protection [14], we determined if the
dimensions of microneedle arrays affects the phenotype and mag-
nitude of the vaccine-induced immunity. A range of ImmuPatch
microneedle arrays were fabricated that differed in the area of the
patch, the density of the microneedles, the height of each micro-
needle and the total pore volume of each patch. The total pore
volume of the microneedle array refers to the maximum volume of
the conduits that can be created in the skin after application of a
microneedle array and has been arbitrarily defined as small,
intermediate or large. Nine array types with increasing total pore
volume that differ in microneedle density or height were fabricated
(Table 1). Live recombinant viral vectors that induce T cell responses are
demonstrating potential as vaccine platforms against diseases such
as malaria, tuberculosis HIV and cancer [11]. The investigation of
microneedle-delivery of live vaccines has to date been limited to
antibody induction by a Japanese Encephalitis virus vaccine and
guinea pig CD4+ T cell responses to BCG vaccination [12,13]. Induction of protective, systemic, anti-malarial cellular immunity,
particularly mediated by CD8+ T cells, has not been investigated
in any microneedle live vaccine delivery study, to our knowledge. In this study we examined the use of microneedle arrays to
administer recombinant MVA to induce this CD8+ T cell re-
sponse. This replication incompetent poxvirus is demonstrating
strong clinical potential in malaria and TB immunization regimes
where the intradermal route has been a traditional route of choice
[11]. The present study was designed with two main objectives. Firstly, we aimed to determine if microneedle array design
impacted on the induction of primary and secondary T cell
responses by a live vaccine. The second objective was to determine
if microneedle-mediated immunization could increase the immu-
nogenicity and efficacy of homologous, repeated immunization
with the same virus vector. Introduction immunization with needle–and-syringe is technically challenging
to administer and inaccurate administration can lead to increased
side effects [3,4]. Skin scarification administration of poxviruses is
efficacious, as evidenced by the smallpox eradication campaign
and the recent demonstration in mice that delivery of the poxvirus,
modified vaccinia virus Ankara (MVA), by skin scarification
induced greater protective efficacy against vaccinia virus com-
pared to systemic immunization [5]. However this vaccine delivery
method also requires specialised training and equipment and
cannot be self-administered [5]. Alternative epidermal abrasion
vaccination techniques are being developed, however these can
result in increased adverse events compared to intradermal (ID)
delivery [6]. Microneedles are micron-scale needles that penetrate
the stratum corneum (SC), creating temporary conduits for Vaccination represents the primary public health measure to
combat infectious diseases. However estimates of the cost of global
immunization programmes to 2015 demonstrate that up to 60% of
the total cost of administering some vaccines will be due to systems
costs, predominantly cold chain, personnel and training [1]. Use of
a simpler vaccine delivery device, which is preferably pain-free,
that eliminated sharps waste and reduces the requirements for
training in immunization technique and logistics should have a
significant positive outcome on the cost and effectiveness of
immunization programmes. The intradermal route has demon-
strated significant advantages with respect to dose-sparing and
immunogenicity in comparison to other routes [2], however ID July 2011 | Volume 6 | Issue 7 | e22442 1 July 2011 | Volume 6 | Issue 7 | e22442 PLoS ONE | www.plosone.org ImmuPatch Mediated MVA Vaccination percutaneous drug or vaccine administration to desired depths in
the skin or underlying tissue. Due to their micron sized height,
they do not stimulate underlying pain receptors [7]. We developed
a method of manufacturing ‘wet-etch’ solid silicon microneedle
array patches [8] that produces microneedles with an outer
pyramidal profile (Fig. 1), termed ImmuPatch. By virtue of their
smooth surface and ultrasharp tips, these ImmuPatch devices go
cleanly in and out of tissue at very low insertion forces. Thus,
ImmuPatch arrays do not abrade the skin, instead they create
temporary pores through the stratum corneum (SC) through
which vaccine enters the skin. using a live recombinant vaccine. Secondly, unlike previous studies
examining antibody induction, the design of the microneedle array
has a significant impact on the magnitude and the memory phe-
notype of the vaccine-induced CD8+ T cell response. Introduction Finally, we
demonstrate that ImmuPatch delivery of a live vaccine does not
induce a pro-inflammatory response, in contrast to ID immuni-
zation and therefore may be of particular benefit to reducing
vaccine reactogenicity. Microneedle array design influences the magnitude and
phenotype of vaccine-induced CD8+ T cell response Subsequent to a 6 hour stimulation
with the Pb9 epitope, examination of the memory phenotype of all
antigen-specific, cytokine-secreting T cells demonstrated that, in
contrast to ID immunization, the use of a small volume micro-
needle array induced a significantly higher proportion of central
memory (TCM) CD8+ T cells and significantly reduced effector
memory (TEM) response (Fig. 2B, Figure S1). Therefore, significantly decreased frequencies of antigen-specific single and
dual cytokine-secreting CD8+ T cells, compared to intradermal
needle-and-syringe
vaccination
(ID)
(Fig. 2A). In
contrast,
immunization with larger pore volume microneedle arrays in-
duced a T cell response that was equivalent in magnitude and
multi-functionality to ID. Vaccination using a flat silicon patch,
with no microneedles, resulted in a background T cell response
(data not shown), demonstrating that breaching the SC is required
to induce immunity. We used a conventional gating strategy to determine the
memory phenotype of antigen-specific CD8+ T cells, using the Figure 2. Magnitude and phenotype of the antigen-specific CD8+ T cell response after a single immunization. Groups of 5 BALB/c mice
were immunized with MVA-PbCSP by the intradermal (id) route or using an ImmuPatch device of increasing pore volume as outlined in Table 1. (A)
Antigen-specific polyfunctional CD8+ T cell responses in spleen cells were quantified after intracellular cytokine staining (ICS) of IFN-c, TNF-a, and IL-2
subsequent to stimulation with the immunodominant Pb9 peptide. Data are expressed as the frequencies of single cytokine secreting IFN-c+ and
dual-secreting IFN-c+TNF-a+ CD8+ T cells, +/2 standard error of the mean (+/2SEM), 2 weeks after immunization. No IL-2 production was observed. (B) Memory phenotype of the antigen-specific response. Splenocytes were also stained for CD62L and CD127 in addition to CD8 and ICS of cytokines. The percentage of cytokine-producing CD8+ T cells that were central memory TCM (blue pie section), effector T cells, TE (red pie section), effector
memory T cells, TEM,: (green pie section) and intermediate phenotype, Tint, (orange pie sections) were determined. Each dot represents one mouse. Graphs were generated after performing a Boolean analysis in FlowJo and data analysis in SPICE software. Pie charts display relative percentages of
CD8+ T cells that are TCM, TEM and TE. * p,0.05, ** p,0.01, ***p,0.001 compared with ID vaccinated mice in (A) by one way ANOVA and in (B) by
unpaired student t test. Similar results were obtained in four independent experiments. doi:10.1371/journal.pone.0022442.g002 Figure 2. Microneedle array design influences the magnitude and
phenotype of vaccine-induced CD8+ T cell response Subsequent to a 6 hour stimulation
with the Pb9 epitope, examination of the memory phenotype of all
antigen-specific, cytokine-secreting T cells demonstrated that, in
contrast to ID immunization, the use of a small volume micro-
needle array induced a significantly higher proportion of central
memory (TCM) CD8+ T cells and significantly reduced effector
memory (TEM) response (Fig. 2B, Figure S1). Therefore,
Microneedle Array
Microneedle height
Number of Microneedles per Array
Array Area (mm2)
Total volume mm3
Pore volume range
A
100 mm
16
29.16
0.0018
Small
F
100 mm
100
29.16
0.0113
Small
B
200 mm
16
29.16
0.0145
Intermediate
D
200 mm
25
29.16
0.0227
Intermediate
E
200 mm
36
29.16
0.0326
Intermediate
C
300 mm
16
29.16
0.0499
Intermediate
G
200 mm
81
54.76
0.0734
Large
T125
125 mm
400
100
0.0902
Large
H
300 mm
36
54.76
0.1123
Large
doi:10.1371/journal.pone.0022442.t001 Microneedle Array
Microneedle height
Number of Microneedles per Array
Array Area (mm2)
Total volume mm3
Pore volume range
A
100 mm
16
29.16
0.0018
Small
F
100 mm
100
29.16
0.0113
Small
B
200 mm
16
29.16
0.0145
Intermediate
D
200 mm
25
29.16
0.0227
Intermediate
E
200 mm
36
29.16
0.0326
Intermediate
C
300 mm
16
29.16
0.0499
Intermediate
G
200 mm
81
54.76
0.0734
Large
T125
125 mm
400
100
0.0902
Large
H
300 mm
36
54.76
0.1123
Large
doi:10.1371/journal.pone.0022442.t001 expression of CD62L and CD127 on TAPI-2 treated cells to
identify central memory T cells (TCM: CD62L+ CD127+), effector
memory T cells (TEM: CD62L2 CD127+), effector T cells (TE:
CD62L2 CD1272) and intermediate phenotype T cells (Tint,
CD62L+ CD1272 ) [16,17]. Subsequent to a 6 hour stimulation
with the Pb9 epitope, examination of the memory phenotype of all
antigen-specific, cytokine-secreting T cells demonstrated that, in
contrast to ID immunization, the use of a small volume micro-
needle array induced a significantly higher proportion of central
memory (TCM) CD8+ T cells and significantly reduced effector
memory (TEM) response (Fig. 2B, Figure S1). Therefore, expression of CD62L and CD127 on TAPI-2 treated cells to
identify central memory T cells (TCM: CD62L+ CD127+), effector
memory T cells (TEM: CD62L2 CD127+), effector T cells (TE:
CD62L2 CD1272) and intermediate phenotype T cells (Tint,
CD62L+ CD1272 ) [16,17]. Microneedle array design influences the magnitude and
phenotype of vaccine-induced CD8+ T cell response Here, we demonstrate that micro-
needle percutaneous immunization induces increased post-boost
CD8+ T cell response, compared to intradermal immunization, Female BALB/c mice were immunized with MVA-PbCSP by
the intradermal (ID) route or by ImmuPatch-based immunization. The ID route was chosen as it has been repeatedly used in clinical
studies and, similar to microneedles, the vaccine is delivered to
skin. Two weeks after priming, the frequency, multi-functional
quality [15] and memory phenotype [16,17] of CD8+ T cells in the
spleen that recognised the dominant, nine-mer, MHC class I
epitope in PbCSP, termed Pb9, was determined. Flow cytometry
combined with Boolean analysis revealed that two populations of
polyfunctional
antigen-specific
CD8+
T-cell
responses
were
primed by MVA-PbCSP immunization, namely CD8+IFN-c+,
single cytokine-secreting cells and CD8+IFN-c+TNF-a+, dual
cytokine producing T cells. These populations were approximately
equivalent in frequency in each group (Fig. 2A, compare
IFN+TNF+ with TNF+, Figure S1). No other multi-functional
phenotype was observed. Of interest, immunization using arrays A
and F, that possess the smallest total pore volume, induced Figure 1. ImmuPatch microneedle arrays. Left: Scanning electron micrograph (SEM) that compares the bevel of a 26G needle to microneedles
on an ImmuPatch device. Right: SEM image of individual microneedle. doi:10.1371/journal.pone.0022442.g001 Figure 1. ImmuPatch microneedle arrays. Left: Scanning electron micrograph (SEM) that compares the bevel of a 26G needle to microneedles
on an ImmuPatch device. Right: SEM image of individual microneedle. doi:10.1371/journal.pone.0022442.g001 July 2011 | Volume 6 | Issue 7 | e22442 PLoS ONE | www.plosone.org 2 ImmuPatch Mediated MVA Vaccination Table 1. Microneedle Array Designs. significantly decreased frequencies of antigen-specific single and
dual cytokine-secreting CD8+ T cells, compared to intradermal
needle-and-syringe
vaccination
(ID)
(Fig. 2A). In
contrast,
immunization with larger pore volume microneedle arrays in-
duced a T cell response that was equivalent in magnitude and
multi-functionality to ID. Vaccination using a flat silicon patch,
with no microneedles, resulted in a background T cell response
(data not shown), demonstrating that breaching the SC is required
to induce immunity. We used a conventional gating strategy to determine the
memory phenotype of antigen-specific CD8+ T cells, using the
expression of CD62L and CD127 on TAPI-2 treated cells to
identify central memory T cells (TCM: CD62L+ CD127+), effector
memory T cells (TEM: CD62L2 CD127+), effector T cells (TE:
CD62L2 CD1272) and intermediate phenotype T cells (Tint,
CD62L+ CD1272 ) [16,17]. Microneedle array design influences the magnitude and
phenotype of vaccine-induced CD8+ T cell response Thus delivering a vaccine with a small volume microneedle array
induces a primary T cell response, with a higher proportion of
antigen-specific T cells that are of a TCM memory phenotype that
responds vigorously to secondary immunization. Analysis of the
total number of antigen-specific CD8+ T cells post-prime and post-
boost demonstrated a similar pattern as that observed for the
percentage of antigen-specific CD8+ T cells, for example, priming
with the smallest pore volume microneedle arrays induced a
significantly decreased number of antigen-specific CD8+ T cells
post-prime and a significantly increased number of antigen-specific
CD8+ T cells subsequent to ID boosting (Figure S3). boosting immunization. All groups were boosted in the same
manner so that post-boost immunity reflected differences in the
existing primary response. Priming using small pore volume
microneedle arrays (arrays A and F) resulted in significantly
increased post-boost T cell response compared to ID immuniza-
tion (Fig. 3A, Figure S2). No significant increase in the
magnitude of the T cell response was observed in any other
group compared to priming with a flat microneedle patch. The
post-boost response was dominated by IFN-c+ CD8+ T cells,
compared to an equivalent frequency of both cytokine-secreting
populations post-prime. A significant negative correlation between
pore volume and antigen-specific T cell responses was observed for
post-boost total CD8+ T cell responses (p,0.0001 r = 20.4786). Thus delivering a vaccine with a small volume microneedle array
induces a primary T cell response, with a higher proportion of
antigen-specific T cells that are of a TCM memory phenotype that
responds vigorously to secondary immunization. Analysis of the
total number of antigen-specific CD8+ T cells post-prime and post-
boost demonstrated a similar pattern as that observed for the
percentage of antigen-specific CD8+ T cells, for example, priming
with the smallest pore volume microneedle arrays induced a
significantly decreased number of antigen-specific CD8+ T cells
post-prime and a significantly increased number of antigen-specific
CD8+ T cells subsequent to ID boosting (Figure S3). immunization with ImmuPatch arrays with small volumes induces
an immune response that has a lower frequency of cytokine-
producing T cells, which have differentiated to a TCM memory
phenotype. p
yp
We demonstrate that microneedle height is not the predominant
factor responsible for T cell response. Post-boost CD8+ T cell responses The
ability
of
T
cells
induced
by
ID
or
ImmuPatch
immunization to re-expand to a secondary homologous immuni-
zation with MVA-PbCSP was examined two weeks after an ID Figure 3. Magnitude and phenotype of the antigen-specific CD8+ T cell response after an ID boosting immunization. Groups of 5
BALB/c mice were immunized with MVA-PbCSP by the ID route or using an ImmuPatch device of increasing pore volume as outlined in Table 1. Alternatively vaccine was administered to mice with a silicon patch that was not etched with microneedles, termed ‘Flat’. Two weeks after priming, all
mice were immunized with MVA-PbCSP by the intradermal (id) route. (A) Antigen-specific polyfunctional CD8+ T cell responses in spleen cells were
quantified as described in Figure 2, two weeks after the boosting immunization. Data are expressed as the frequencies of single cytokine secreting
IFN-c+ and dual-secreting IFN-c+TNF-a+ CD8+ T cells, +/2 standard error of the mean (+/2SEM), 2 weeks after immunization. (B) Memory phenotype
of the antigen-specific response was determined as described in Fig. 2B. Pie charts display relative percentages of CD8+ T cells that are TCM, TEM and
TE. * p,0.05, ** p,0.01, ***p,0.001 compared with ID vaccinated mice in (A) by one way ANOVA and in (B) by unpaired student t test. Data is pooled
from four independent experiments (n = 5 mice/group per experiment). doi:10.1371/journal.pone.0022442.g003 Figure 3. Magnitude and phenotype of the antigen-specific CD8+ T cell response after an ID boosting immunization. Groups of 5
BALB/c mice were immunized with MVA-PbCSP by the ID route or using an ImmuPatch device of increasing pore volume as outlined in Table 1. Alternatively vaccine was administered to mice with a silicon patch that was not etched with microneedles, termed ‘Flat’. Two weeks after priming, all
mice were immunized with MVA-PbCSP by the intradermal (id) route. (A) Antigen-specific polyfunctional CD8+ T cell responses in spleen cells were
quantified as described in Figure 2, two weeks after the boosting immunization. Data are expressed as the frequencies of single cytokine secreting
IFN-c+ and dual-secreting IFN-c+TNF-a+ CD8+ T cells, +/2 standard error of the mean (+/2SEM), 2 weeks after immunization. (B) Memory phenotype
of the antigen-specific response was determined as described in Fig. 2B. Pie charts display relative percentages of CD8+ T cells that are TCM, TEM and
TE. Microneedle array design influences the magnitude and
phenotype of vaccine-induced CD8+ T cell response Vaccine delivery using T125
arrays, which have microneedles of height 125 mm, similar to
patches A and F, but with a pore volume similar to patches G and
H, induced a response that was significantly different to A and F
but equivalent to patches G and H (Fig. 2). This demonstrates
that, compared to administration depth, the total volume of a
microneedle array is a dominant feature that influences the
magnitude and phenotype of the vaccine-induced T cell response. The most significant and strongest positive correlation (Spear-
man’s rho = 0.6859 p,0.0001) was observed between total volume
and cytokine producing CD8+ T cell responses post-prime. In
contrast, other design parameters, such as microneedle height
were either not as strongly correlated (Spearman’s rho = 0.516,
p,0.001) or, in the case of the number of microneedles per array,
did not correlate (Spearman’s rho = 0.289, not significant) with
antigen-specific CD8+ T cell responses. Therefore, we demon-
strate that total volume of the microneedle array and not
microneedle length is a dominant design feature that impacts the
type and magnitude of a CD8+ T cell response that is induced by a
live vaccine. The memory phenotype in all groups subsequent to an ID boost
was predominantly TEM (Fig. 3B, Figure S2). Priming with
ImmuPatch array F, but not other small volume arrays, induced
a significantly increased proportion of TEM and significantly
decreased TCM CD8+ T cell phenotype compared to ID immu-
nization post-boost. Microneedle array design influences the magnitude and
phenotype of vaccine-induced CD8+ T cell response Magnitude and phenotype of the antigen-specific CD8+ T cell response after a single immunization. Groups of 5 BALB/c mice
were immunized with MVA-PbCSP by the intradermal (id) route or using an ImmuPatch device of increasing pore volume as outlined in Table 1. (A)
Antigen-specific polyfunctional CD8+ T cell responses in spleen cells were quantified after intracellular cytokine staining (ICS) of IFN-c, TNF-a, and IL-2
subsequent to stimulation with the immunodominant Pb9 peptide. Data are expressed as the frequencies of single cytokine secreting IFN-c+ and
dual-secreting IFN-c+TNF-a+ CD8+ T cells, +/2 standard error of the mean (+/2SEM), 2 weeks after immunization. No IL-2 production was observed. (B) Memory phenotype of the antigen-specific response. Splenocytes were also stained for CD62L and CD127 in addition to CD8 and ICS of cytokines. The percentage of cytokine-producing CD8+ T cells that were central memory TCM (blue pie section), effector T cells, TE (red pie section), effector
memory T cells, TEM,: (green pie section) and intermediate phenotype, Tint, (orange pie sections) were determined. Each dot represents one mouse. Graphs were generated after performing a Boolean analysis in FlowJo and data analysis in SPICE software. Pie charts display relative percentages of
CD8+ T cells that are TCM, TEM and TE. * p,0.05, ** p,0.01, ***p,0.001 compared with ID vaccinated mice in (A) by one way ANOVA and in (B) by
unpaired student t test. Similar results were obtained in four independent experiments. doi:10.1371/journal.pone.0022442.g002 July 2011 | Volume 6 | Issue 7 | e22442 3 PLoS ONE | www.plosone.org ImmuPatch Mediated MVA Vaccination boosting immunization. All groups were boosted in the same
manner so that post-boost immunity reflected differences in the
existing primary response. Priming using small pore volume
microneedle arrays (arrays A and F) resulted in significantly
increased post-boost T cell response compared to ID immuniza-
tion (Fig. 3A, Figure S2). No significant increase in the
magnitude of the T cell response was observed in any other
group compared to priming with a flat microneedle patch. The
post-boost response was dominated by IFN-c+ CD8+ T cells,
compared to an equivalent frequency of both cytokine-secreting
populations post-prime. A significant negative correlation between
pore volume and antigen-specific T cell responses was observed for
post-boost total CD8+ T cell responses (p,0.0001 r = 20.4786). Homologous vaccination using patches in prime and
boost compared to that of the naı¨ve group, were determined (far right
column). This demonstrated that immunization with A/ID, F/ID,
F/G and ID/ID induced a significantly greater reduction in the
risk of becoming parasitaemic, compared to unimmunized control
mice. In contrast, other immunization groups had a similar risk as
the naive mice of developing blood stage parasitaemia following
sporozoite infection. There are no significant differences between
ID/ID and the protective ImmuPatch regimes (A/ID, F/ID and
F/G), however using ImmuPatch-mediated priming and boosting
(F/G) resulted in the most reduced risk of becoming parasitemic
(hazard ratio = 0.18). Overall, these challenge studies demonstrate
that ImmuPatch priming or ImmuPatch prime/boosting is as
efficacious as needle-and-syringe delivery of repeated MVA-
PbCSP immunizations, resulting in protection against infection
or a delay in the progression to blood-stage malaria i.e., partial
protection. In a clinical context, it would be preferable, from a cost and
logistic viewpoint, if the same route or delivery device is used in
repeated immunizations. To determine if microneedle-mediated
vaccination could completely replace needle-and-syringe based
delivery, naı¨ve mice were immunized with the leading priming
ImmuPatch arrays, A or F, and boosted using the same array or
using array G. This microneedle array was chosen as a boosting
device as it induced immune responses with similar memory and
functional phenotype to needle-and-syringe post-prime (Fig. 2). The magnitude and multi-functionality of the T cell response
induced by a patch/patch prime-boost regime was equivalent to
that induced by repeated ID immunizations (Fig. 4). An immune
response that was primed using array A or F and boosted with
microneedle array G resulted in increased T cell responses,
however this was not significantly greater than the ID/ID group. Therefore, the repeated use of a needle-and-syringe can be
eliminated by microneedle arrays that can induce equivalent or
higher CD8+ T cell responses to vaccine antigen post-boost. Protective efficacy against a malaria sporozoite challenge Protective efficacy against a malaria sporozoite challenge
To assess whether the vaccination method impacted on the
protective efficacy of a homologous MVA-PbCSP immunization
regime, immunized mice were challenged with P. berghei sporo-
zoites two weeks after the second immunization (Table 2). The
level of sterile protection and the time to 0.5% parasitemia were
determined. Two separate challenge studies were pooled in this
analysis (n = 5 mice/group per challenge study). We first deter-
mined if the immunization regimes responded in a similar manner
in repeated challenge studies. No significant differences were
observed between the two challenge studies for any group except
group H/id, thus this group was removed from evaluation of
protective efficacy. Significant differences in the hazard ratio Figure 4. Priming and boosting with ImmuPatch devices
induces equivalent or higher T cell responses to homologous
ID immunization. Mice were immunized by the ID route or with
ImmuPatch arrays A, F or G. Two weeks after boosting, mice were
boosted by the ID route (id/id) or boosted using the same array used in
the prime (A/A, F/F or G/G) or with array G (A/G, F/G, G/G). Antigen-
specific polyfunctional CD8+ T cell responses in spleen cells were
quantified as described in Figure 2, two weeks after the boosting
immunization. Data are expressed as the frequencies of single cytokine
secreting IFN-c+ and dual-secreting IFN-c+TNF-a+ CD8+ T cells, +/2
standard error of the mean (+/2SEM, n = 4 mice/group), 2 weeks after
immunization. Similar results were obtained in two independent
experiments. doi:10.1371/journal.pone.0022442.g004 Figure 4. Priming and boosting with ImmuPatch devices
induces equivalent or higher T cell responses to homologous
ID immunization. Mice were immunized by the ID route or with
ImmuPatch arrays A, F or G. Two weeks after boosting, mice were
boosted by the ID route (id/id) or boosted using the same array used in
the prime (A/A, F/F or G/G) or with array G (A/G, F/G, G/G). Antigen-
specific polyfunctional CD8+ T cell responses in spleen cells were
quantified as described in Figure 2, two weeks after the boosting
immunization. Data are expressed as the frequencies of single cytokine
secreting IFN-c+ and dual-secreting IFN-c+TNF-a+ CD8+ T cells, +/2
standard error of the mean (+/2SEM, n = 4 mice/group), 2 weeks after
immunization. Similar results were obtained in two independent
experiments. Post-boost CD8+ T cell responses * p,0.05, ** p,0.01, ***p,0.001 compared with ID vaccinated mice in (A) by one way ANOVA and in (B) by unpaired student t test. Data is pooled
from four independent experiments (n = 5 mice/group per experiment). doi:10.1371/journal.pone.0022442.g003 July 2011 | Volume 6 | Issue 7 | e22442 PLoS ONE | www.plosone.org 4 ImmuPatch Mediated MVA Vaccination Microneedle mediated immunization does not induce
pro-inflammatory innate responses We wished to determine some underlying mechanisms respon-
sible for the microneedle-mediated modulation of antigen-specific
CD8+ T cell immunity. In contrast to other transcutaneous
immunization studies, ImmuPatch delivery did not rely on skin
pre-treatment, such as shaving, as we delivered vaccine to the part
of the ear that lacks fur, or on the use of high velocity applicators
that stress the skin. We hypothesised that ImmuPatch-mediated
vaccination did not induce a pro-inflammatory response post-
immunization, thereby permitting the generation of a strong TCM
response [18,19]. We examined the induction of cytokine and
chemokine mRNA in the lymph nodes and at the site of
immunization, up to 48 hours post-immunization with MVA-
PbCSP, as a marker of local inflammatory responses to immu-
nization (Fig. 5). Pro-inflammatory cytokines such as IL-1 and
IL-6 were induced to a significantly higher level, compared to
ImmuPatch-treated mice, in lymph nodes of ID immunized mice
at 6 and 18 hrs post immunization. A second wave of gene
transcription of pro-inflammatory cytokines and chemokines,
specifically TNF-a, CCL4 and IL-12p40, occurred at 18 hrs
post-ID immunization that, for the first two molecules were
significantly greater than lymph nodes from ImmuPatch-treated or
naive mice. Apart from RANTES, no significant changes in pro-
inflammatory cytokine induction were observed when vaccine was
delivered with ImmuPatch compared to lymph nodes of naı¨ve
mice (Fig. 5A). A similar pattern of early and strong pro-
inflammatory mRNA induction in the skin was observed for most
cytokines in ID immunized compared to patch-immunized mice
(Fig. 5B). However, ImmuPatch mediated immunization resulted
in significantly higher IL-1 and IL-10 and equivalent TNF-a
mRNA in the skin compared to ID immunization at 6 hours. No
mRNA transcript for IL-12p40 or type I IFN could be detected at
the site of immunization. Furthermore, higher levels of TNF-a
protein was detected by standard cytokine ELISA in the serum at
18 hrs after ID (5.8+/23.3 pg/ml) compared to ImmuPatch
immunization (1.4+/21.3 pg/ml, mean +/2 SEM, n = 4 mice
per group). Analysis of other cytokine proteins, such as IL-1 and
IL-6, in serum was at the limit of detection of ELISA. These data
demonstrate that microneedle-mediated immunization did not
induce a classical pro-inflammatory response that was observed
subsequent to ID immunization. Protective efficacy against a malaria sporozoite challenge We next examined if the observed differences in cytokine and
chemokine induction at the site of immunization and lymph node
impacted on trafficking and activation of the CD11c+ or CD11c-
subset of antigen presenting cells (APC). The total number of cells
in the lymph node was consistently three-fold higher in ID doi:10.1371/journal.pone.0022442.g004 July 2011 | Volume 6 | Issue 7 | e22442 July 2011 | Volume 6 | Issue 7 | e22442 5 PLoS ONE | www.plosone.org ImmuPatch Mediated MVA Vaccination Table 2. Microneedle-mediated immunization induces efficacy against P. berghei sporozoite infection. Immunization Regime
(Prime/Boost)
No. protected/total
Protection (%)
Median time to 0.5%
parasiteamia in days
Hazard Ratio (95% CI)a
p-valueb
Naı¨ve
0/10
0
6.52
-
-
ID/ID
4/10
40
6.91
0.25 (0.08–0.81)
0.020*
A/ID
3/10
30
6.76
0.33 (0.11–1.00)
0.050*
F/ID
5/10
50
7.46
0.25 (0.08–0.80)
0.020*
F/G
5/10
50
6.88
0.18 (0.05–0.68)
0.011*
B/ID
4/10
40
6.49
0.39 (0.13–1.16)
0.090
D/ID
2/10
20
6.61
0.44 (0.16–1.22)
0.120
E/ID
3/10
30
6.38
0.59 (0.22–1.59)
0.300
C/ID
4/10
40
6.55
0.61 (0.22–1.74)
0.360
G/ID
4/10
40
6.47
0.39 (0.13–1.17)
0.090
H/ID
6/10
60
ndc
0.04 (0.005–0.32)c
0.002c
Mice were immunized and challenged and protection results analysed as described in Methods. aCompared to Naı¨ve, unimmunized mice (95% confidence interval);
bcompared to Naı¨ve,
*p#0.05. cNot determined or unable to determine significance compared to naive group as repeated challenge studies were significantly different. doi:10.1371/journal.pone.0022442.t002 p#0.05. Not determined or unable to determine significance compared to naive group as repeated challenge studies were significantly different. oi:10.1371/journal.pone.0022442.t002 compared to ImmuPatch treated and naı¨ve mice (Figure S4). Modest increases were observed in the numbers of CD11c+ cells
and MHC class II expression in the lymph node after ImmuPatch
immunization compared to naı¨ve mice at 24 hours (Fig. 6). In
contrast, ID administration of MVA caused a more substantial
increase in CD11c+MHCII+ and CD11c+MHCII- cells in the
lymph node at both 24 and 96 hours post-immunization. A similar
effect was noted at the site of immunization; ImmuPatch delivery
of MVA resulted in a transient increase in CD11c+MHCII- cells
at 24 hrs and CD11c-MHCII+ cells at 24 and 96 hrs, while the
proportion of CD11c+MHCII+ cells was similar to naı¨ve at both
times. In contrast, ID delivery induced a substantial increase in
CD11c+MHCII+ cells that were maintained in the ear up to
96 hrs post-vaccination. odistribution due to ID or ImmuPatch delivery Next we wished to determine if the administration route
impacted on the biodistribution of the delivered material. Firstly,
we histologically determined that microneedle-delivered material
is distributed mainly in the epidermis, with some delivery to the
dermis, 30 minutes after administration of red fluorescent beads
(100nm diameter) to mice ears using Array F (Figure S5). A
similar distribution was observed when MVA expressing red
fluorescent protein (MVA-RFP) was administered to ex vivo
porcine skin. In comparison to ID injection of MVA-RFP, where
transfected cells were observed in a dermal bolus, a tract of mostly
epidermal infected cells was observed subsequent to ImmuPatch
Array G administration (Figure S6). Therefore, although the proportion of cells that are FITC+ in
the lymph node is lower in ImmuPatch versus ID-treated mice, the
frequency of FITC+ cells capable of delivering signal 1 and 2 from
the same cell is equivalent in both groups and ImmuPatch treated
mice have fewer cells incapable of delivering either signal 1 or 2 to
T cells. Protective efficacy against a malaria sporozoite challenge These changes in APC numbers and
MHCII expression are consistent with increased inflammatory
chemokine and cytokine expression (Fig. 5). Finally, in the absence of having an MVA that highly expressed
a fluorescent marker that could be easily detected by flow
cytometry, we administered FITC mixed with the vaccine. A
similar frequency of live cells in the ear contained FITC at 24 and
96 hours in both groups (Fig. 7). However, ID delivery resulted in
higher levels of FITC in the lymph node compared to ImmuPatch. Therefore, both ID and ImmuPatch administration result in
sustained presence of the delivered cargo up to 96 hrs in cells at
the site of administration, however ID delivery resulted in greater
transport to the lymph node. We next examined the frequency of cells that had taken up
FITC that were capable of delivering signal 1 (MHC class II) and
signal 2 (CD80) to T cells in the lymph node (Fig. 8). We gated
out CD3+ T cells and CD19+ B cell and focused on live FITC+
cells that were MHCII+ and/or CD80+ that can be viewed as
capable of antigen presentation and co-stimulation. Similar
frequencies of FITC+MHCIIhiCD80hi cells were detected in both
groups at both times. ImmuPatch treated mice had twice the
frequency of MHCII+CD80+ cells compared to ID at 24 hours
(35% compared to 16.8%). A higher proportion of FITC+ cells
that were negative for both CD80 and MHC class II were detected
in ID treated compared to ImmuPatch treated animals (24%
compared to 6% at 24 hours). Discussion Secondly, ID immunization resulted in a
marked, sustained increase in level of CD11c+MHCII+ cells at the
site of immunization, whereas ImmuPatch immunization did not. We propose that this continued presence of activated APC, that
are capable of sending signals 1 and 2 to T cells in the skin may
influence the induction of more TEM and TE cells as antigen
persistence is known to maintain CD8+ T cells in an effector state
[28,29]. vaccine-induced CD8+ T cell response. We demonstrate that
administration of a live recombinant vaccine using these wet-
etched silicon microneedles induces equivalent or significantly
greater CD8+ T cell responses compared to needle-and-syringe
intradermal delivery. In contrast to previous studies that examined
microneedle array design in the context of antibody induction
[10], we demonstrate that microneedle design significantly impacts
on the magnitude and memory phenotype of the vaccine-induced
CD8+ T cell response and subsequent protection against liver-
stage malaria challenge. We also demonstrate that in contrast to
needle-and-syringe delivery, microneedle-mediated vaccination
does not induce classical inflammatory responses, suggesting that
ImmuPatch vaccine delivery could potentially eliminate unwanted
reactogenicity. vaccine-induced CD8+ T cell response. We demonstrate that
administration of a live recombinant vaccine using these wet-
etched silicon microneedles induces equivalent or significantly
greater CD8+ T cell responses compared to needle-and-syringe
intradermal delivery. In contrast to previous studies that examined
microneedle array design in the context of antibody induction
[10], we demonstrate that microneedle design significantly impacts
on the magnitude and memory phenotype of the vaccine-induced
CD8+ T cell response and subsequent protection against liver-
stage malaria challenge. We also demonstrate that in contrast to
needle-and-syringe delivery, microneedle-mediated vaccination
does not induce classical inflammatory responses, suggesting that
ImmuPatch vaccine delivery could potentially eliminate unwanted
reactogenicity. We observed a negative correlation between pore volume and
TCM suggesting that antigen load can be optimised for the
induction of memory CD8+ T cell responses. Of note, total pore
volume and not microneedle length was the dominant feature that
influenced the magnitude and phenotype of the induced T cell
response. This was particularly evident when arrays A and F are
compared to array T125. Total array volume is determined by
height (as a variable in the equation of a circular cone) and
number of microneedles per array and therefore permits the
determination of these combined parameters on CD8+ T cell
vaccine-induced immunity. Discussion We propose that the total microneedle
array volume is the key design feature that should be examined for
specific T cell responses post-vaccination. We propose that total
pore volume most accurately reflects the total amount of vaccine
that is available to the immune system. We speculate that a lower
live virus vaccine dose, delivered by small pore volume patches,
favours a TCM response, however as the availability of antigen
increases with increasing total pore volume, the response begins to
more resemble a bolus administration; as is observed in Figure 2. Further studies are required to determine how such subtle
differences in microneedle design are involved in the observed
differences in memory induction by different ImmuPatch devices. g
y
Here
we
demonstrate
that
ImmuPatch
vaccine
delivery
eliminates most of the skin inflammatory response and significantly
reduces inflammatory markers in the draining lymph node that is
observed by ID vaccination. We propose that this is likely due to
the lack of abrasion, irritation and stress of the skin, features that
are common to previous epidermal drug or vaccine targeting, such
as skin scarification [5], or stripping [6,20] that tear the SC from
the skin and expose the underlying epidermis, or alternatively,
biolistic targeting using gene guns or spring-loaded applicators. In
contrast to these disruptive methods that can result in systemic
adverse events [6], microneedle administration creates temporary
channels through the SC [7,21,22]. Furthermore ImmuPatch
delivery does not cause shear stress during application as no high
velocity applicators are required for the microneedles to penetrate
the SC. We propose that increased IL-1 and equivalent TNF-a
mRNA expression in the skin of ImmuPatch vaccinated mice
could be due to MVA infection of keratinocytes that constitute the
majority of the epidermis and are major producers of these
cytokines [2,23]. The delivery method also impacted on bio-
distribution. Injection of a bolus fluid into the dermis causes
increased local pressure and capillary permeability and permits
efficient antigen drainage into lymphatic capillaries that form an
extensive network in the dermis [2,24,25,26]. In contrast,
ImmuPatch delivery resulted in equally efficient uptake of the
marker in the skin, however the fluorescent label did not drain as
effectively to lymph. Also, in contrast to ID delivery, ImmuPatch-
administered cargo is delivered equally to the epidermis and to the
dermis (Figure S5, Figure S6). Discussion Secondly, we determined the amount of red fluorescent beads
that can be recovered from inside the skin of mouse ears
subsequent to ImmuPatch administration. To lessen the effects
of biodistribution, we harvested ears 30 minutes after treatment. Approximately 3% of the original material was delivered into the
ear (Figure S7). No significant difference in the percentage
recovered was observed between microneedle designs in this study,
at this early timepoint. Development of low cost, needle-free, painless, safe, efficacious
immunization strategies is an important goal in global health care. ImmuPatch is a microneedle-based technology that is being
developed as an easy-to-use, pain-free, patch that, by creating
temporary channels through the impermeable stratum corneum,
delivers vaccine through the skin. Here we aimed to determine if
the patch design impacted on the magnitude and phenotype of the PLoS ONE | www.plosone.org July 2011 | Volume 6 | Issue 7 | e22442 July 2011 | Volume 6 | Issue 7 | e22442 6 ImmuPatch Mediated MVA Vaccination PLoS ONE | www.plosone.org
7
July 2011 | Volume 6 | Issue 7 | e2244 PLoS ONE | www.plosone.org July 2011 | Volume 6 | Issue 7 | e22442 PLoS ONE | www.plosone.org PLoS ONE | www.plosone.org 7 ImmuPatch Mediated MVA Vaccination Figure 5. Induction of cytokine and chemokine mRNA in the skin and draining lymph node by ID or ImmuPatch vaccine delivery. Mice were immunized with MVA-PbCSP by the ID route or using ImmuPatch array F at time 0. At 6, 18 and 48 hours after immunization, 5 mice per
vaccinated group were culled and the skin site of immunization and the draining lymph nodes were harvested and snap frozen. Skin and lymph
nodes from 2 naı¨ve mice were harvested to determine background levels of expression. Gene expression values relative to GAPDH were calculated as
22DCt. The mean (+/2 SEM) values for each group are plotted; cytokine and chemokine induction due to ID immunization is in red; due to ImmuPatch
immunization is in blue and naives is in black. * p,0.05, ** p,0.01, ***p,0.001 by unpaired student t test of the two immunized groups, {p,0.05
compared to naı¨ve mice. Similar results were obtained in two independent experiments. doi:10.1371/journal.pone.0022442.g005 generation [27] and that the default differentiation memory
generation pathway for CD8+ T cells is deflected by sustained
inflammation [18]. PLoS ONE | www.plosone.org Discussion We suggest that microneedle
array targeting to individually created pores in the epidermis and
upper dermis does not result in increased interstitial pressure and
subsequent lymphatic drainage. However, despite decreased
lymph node drainage subsequent to ImmuPatch delivery, equi-
valent frequencies of FITC+ cells capable of delivering stimulatory
TCR and co-stimulatory signals to naı¨ve T cells were observed in
draining lymph nodes in both immunized groups. Overall this
demonstrates that antigen presentation occurs in ImmuPatch-
immunized animals in the absence of strong inflammatory
responses. Previous efficacy studies involving microneedle-delivered vac-
cines have focussed on mucosal protection, predominantly as-
sessing influenza virus respiratory challenge [9,30]. In contrast,
here we are assessing systemic cell mediated immunity against
liver-stage malaria as the protective mechanism [31]. Defining the
optimal microneedle design that promotes systemic cell-mediated
efficacy is a novel area of investigation that has future clinical
consequences. Homologous MVA-PbCSP immunization induces
weak efficacy and as such, represents a vaccine model that can be
used to design methods that improve both immunogenicity and
efficacy [14]. Despite significantly greater induction of CD8+ T
cell responses in the spleen by small pore volume ImmuPatch
microneedle arrays, no significant differences in efficacy were
observed between ID and ImmuPatch immunization when mice
were challenged 2 weeks after boosting. This may reflect dif-
ferences in immunity in the spleen compared to the liver or the
phenotype of a protective immune response. It highlights our
incomplete understanding of correlates of protective immunity
against liver stage Plasmodium infection and the necessity of looking
at efficacy in addition to immunogenicity. Overall, the result
demonstrates that a small pore volume ImmuPatch device, that
induces a higher proportion of CD8+ T cells with a TCM responses
post-prime (compared to ID), should be chosen to maximise
efficacy against liver-stage malaria. We propose that the increased proportion of TCM is due to the
lack of inflammation post-ImmuPatch immunization. It has been
previously demonstrated that curtailment of inflammation during
the initiation of immunity results in accelerated T cell memory PLoS ONE | www.plosone.org July 2011 | Volume 6 | Issue 7 | e22442 8 ImmuPatch Mediated MVA Vaccination Figure 6. Cell trafficking at the site of immunization and lymph node subsequent to ImmuPatch or ID v
from the site of immunization (A), or lymph node (B), from naı¨ve mice (upper left panel) or mice immunized with MVA
F (upper middle and upper right panels) or ID (lower middle and lower right panels) at 24 (middle panels) o
administration. The expression of CD11c and MHC class II was assessed on live CD3- and CD19- cells. Each plot is r
group. Similar results were obtained in two independent experiments. doi:10.1371/journal.pone.0022442.g006 Figure 6. Cell trafficking at the site of immunization and lymph node subsequent to ImmuPatch or ID vaccination. Cells were isolated
from the site of immunization (A), or lymph node (B), from naı¨ve mice (upper left panel) or mice immunized with MVA-PbCSP using ImmuPatch array
F (upper middle and upper right panels) or ID (lower middle and lower right panels) at 24 (middle panels) or 96 hours (right panels) post-
administration. The expression of CD11c and MHC class II was assessed on live CD3- and CD19- cells. Each plot is representative of 3 samples per
group. Similar results were obtained in two independent experiments. doi:10.1371/journal.pone.0022442.g006 Figure 6. Cell trafficking at the site of immunization and lymph node subsequent to ImmuPatch or ID vaccination. Cells were isolated
from the site of immunization (A), or lymph node (B), from naı¨ve mice (upper left panel) or mice immunized with MVA-PbCSP using ImmuPatch array
F (upper middle and upper right panels) or ID (lower middle and lower right panels) at 24 (middle panels) or 96 hours (right panels) post-
administration. The expression of CD11c and MHC class II was assessed on live CD3- and CD19- cells. Each plot is representative of 3 samples per
group. Similar results were obtained in two independent experiments. doi:10.1371/journal.pone.0022442.g006 July 2011 | Volume 6 | Issue 7 | e22442 PLoS ONE | www.plosone.org 9 ImmuPatch Mediated MVA Vaccination Figure 7. Biodistribution and clearance subsequent to ID or ImmuPatch delivery. FITC (1mg/ml) was added to MVA-PbCSP and
administered to mice by the ID route or using ImmuPatch array F. At 24 and 96 hours ears and lymph nodes were harvested from 3 mice per group. The level of FITC detected in live cells from the skin and lymph nodes of ImmuPatch (top panels) or ID (lower panels) treated animals at 24 and
96 hours post-immunization. Vaccinated mice are represented by the bold line, naive mice by the shaded histogram. The plots are representative of 3
mice per group and of 3 repeated experiments. doi:10.1371/journal.pone.0022442.g007 Figure 7. Biodistribution and clearance subsequent to ID or ImmuPatch delivery. FITC (1mg/ml) was added to MVA-PbCSP and
administered to mice by the ID route or using ImmuPatch array F. At 24 and 96 hours ears and lymph nodes were harvested from 3 mice per group. The level of FITC detected in live cells from the skin and lymph nodes of ImmuPatch (top panels) or ID (lower panels) treated animals at 24 and
96 hours post-immunization. Vaccinated mice are represented by the bold line, naive mice by the shaded histogram. The plots are representative of 3
mice per group and of 3 repeated experiments. doi:10.1371/journal.pone.0022442.g007 Figure 8. Antigen presentation capability subsequent to ID or ImmuPatch delivery. FITC and MVA-PbCSP was administered to mice and
monitored as described in Figure 7. The expression of MHC class II and CD80 on FITC+ CD32CD192 live cells was determined in the lymph node of
treated animals. Numbers in the upper right hand of each plot represent the percentage of live CD32CD192 cells that express high or positive levels
or are negative for CD80 and MHC class II. The plots are representative of 3 mice per group and of 3 repeated experiments. doi:10.1371/journal.pone.0022442.g008 Figure 8. Antigen presentation capability subsequent to ID or ImmuPatch delivery. FITC and MVA-PbCSP was administered to mice and
monitored as described in Figure 7. The expression of MHC class II and CD80 on FITC+ CD32CD192 live cells was determined in the lymph node of
treated animals. Numbers in the upper right hand of each plot represent the percentage of live CD32CD192 cells that express high or positive levels
or are negative for CD80 and MHC class II. The plots are representative of 3 mice per group and of 3 repeated experiments. doi:10.1371/journal.pone.0022442.g008 PLoS ONE | www.plosone.org July 2011 | Volume 6 | Issue 7 | e22442 10 ImmuPatch Mediated MVA Vaccination and flow cytometry (ICS) in the same method as previously
described [17,33]. Briefly, ACK-treated splenocytes were pre-
incubated for 1 hour with TAPI-2 (4 mg/well, equivalent to
100 mM peptide, Peptides International [34] and subsequently
incubated for 5 h in the presence of 1 mg/mL Pb9, 100 mM TAPI-
2 and 2 mL/mL Golgi-Plug (BD Biosciences). Staining antibodies
were specific for mouse and purchased from eBioscience. After
blocking Fc receptors with anti-CD16/CD32, cells were surface
stained for 30 min at 4uC with Pacific Blue-labeled anti-CD8a and
APC-Alexa Fluor 700-labeled anti-CD4, PE-Cy7-labeled CD127
and PerCpCy5.5-labeled CD62L. Cells were permeabilized in
Cytofix/Cytoperm solution as per manufacturer’s instructions (BD
Biosciences). Intracellular cytokines were stained with APC-
labeled anti-IFN-c, FITC-labeled anti-TNF-a, and PE-labeled
anti-IL-2. Flow cytometric analyses were performed using an
LSRII (BD Biosciences) and data were analyzed with FlowJo (Tree
Star) software. One million events per sample were acquired. Analysis of multifunctional T cell responses was performed by
using Boolean analysis in FlowJo software and SPICE 4.0 (M. Roederer NIH, Bethesda). Three major subsets of cytokine-
expressing, antigen-specific T cells were defined according to their
expression of CD62L and CD127 [16,17]. These markers are
associated with central memory T cells (TCM: CD62L+ CD127+),
effector memory T cells (TEM: CD62L2 CD127+), effector T cells
(TE: CD62L2 CD1272) and intermediate phenotype T cells (Tint,
CD62L+ CD1272 ). Vaccines The construction, design and preparation of Modified Vaccinia
Virus Ankara (MVA) expressing P. berghei CSP (MVA-PbCSP) and
red
fluorescent
protein
(MVA-RFP))
have
been
previously
described [32]. All viruses were resuspended in endotoxin-free
PBS for immunization. ImmuPatch microneedle patch design Silicon microneedles were fabricated using wet-etch technology
as previously described [8]. The area of each microneedle patch
and the length and number of microneedles per patch were
designed to produce a microneedle patch that created specific total
pore volumes when inserted into skin. The pore volume of these
pyramidal silicon microneedles was determined using the formula
to calculate the volume of a right circular cone. The total pore
volume per array is the sum of the volume of each pore on the
array. The specific dimensions of each patch is detailed in
Table 1. Sporozoite challenge and survival analysis Mice were challenged intravenously with 1000 Plasmodium berghei
sporozoites (ANKA 234) as described previously [14,17]. Giemsa-
stained blood smears were screened to day 20 post-challenge and
% parasitaemia ascertained [35]. Linear regression analysis was
used to determine the timepoint at which parasiteamia would
reach 0.5% in parasitic mice. A Cox’s Proportional Hazards
Regression model was used to test for significant differences in the
chance of reaching the 0.5% parasitaemia threshold at any one
timepoint (assuming that this ratio is the same at each time point)
as compared to the naı¨ve control group during the monitoring
period. RT-PCR assessment of cytokine and chemokine induction RT-PCR assessment of cytokine and chemokine induction
Ears and lymph nodes were snap-frozen in liquid nitrogen
immediately after harvesting. Tissues were then disrupted using
MagNA Lyser Green Beads and total RNA purified using High Therefore, ImmuPatch mediated immunization is a viable
alternative to needle-and-syringe based administration of a T cell
inducing live vaccine that reduces innate inflammation, with
potentially reduced reactogenicity. The design of the microneedle
device impacts on the magnitude and phenotype of the induced
immunity and efficacy and should be optimised for use with a
CD8+ T cell inducing live vaccine. This finding underlies future
studies to develop coated or dissolvable microneedle systems
incorporating vaccines that induce cell-mediated immunity. In
human studies to date (data not shown and refs [7,22]) no adverse
events or pain have been reported using these microneedle arrays. In combination with other advantages of this system, including the
lack of pain, the elimination of sharps waste and the capacity to
affordably mass manufacture these microneedles, ImmuPatch
mediated vaccination demonstrates potential as a feasible needle-
free approach to vaccination that aims to overcome several cost
and logistic obstacles of immunization programmes. FITC uptake and cell trafficking
FITC (1mg/ml solution in PBS) Female BALB/c mice 4–6 weeks old (Harlan UK) were used in
all experiments which were conducted in strict accordance with the
terms of licences from the Irish Department of Health and Children,
under the Cruelty to Animals Act (licence numbers B100/4034 and
B100/3157) and the UK Home Office, under the terms of the
Animals (Scientific Procedures) Act 1986 (licence numbers 30/7793
and 30/2414)and according to the approval of the UCC AECC and
University of Oxford Animal Ethics Commitees. Mice were
immunized with 16106pfu MVA-PbCSP in phosphate buffered
saline (PBS) (Sigma). Vaccine was administered with a conventional
28G needle and syringe intradermally (ID) into the ear (50 ml of
16106pfu per mouse). Alternatively, 5 ml of vaccine was placed on
the dorsal surface of the ear and administered to the anaesthetised
mouse by pressing a microneedle array onto the ear, using a force of
approximately 10–20N (16106pfu in 10 ml per mouse). Mice were
primed on day 0. Post-prime T cell responses were analysed in the
spleen on day 14 after immunization. Mice were boosted by the ID
route or using a microneedle array at day 14 post-immunization. Vaccine-induced immunity was tested in the spleen of all groups 14
days after boosting. p
g
FITC (1mg/ml solution in PBS) was added to MVA-PbCSP
and administered to mice by the ID route or using ImmuPatch
array F in the same manner as used for immunization. At 24 and
96 hours after delivery ears and lymph nodes were harvested from
3 mice per group per timepoint. Cells were isolated from ears by
modifying a previously described method [36]. Briefly ears were
separated into dorsal and ventral leaflets, placed dermal side down
in supplemented RPMI1640 media, scored with a blade and
scraped to encourage release of cells from the tissue. Following one
hour incubation at 37uC, 5% CO2, cells were collected and passed
through a 70 mm strainer, centrifuged and re-suspended in PBS. Similarly, the LNs were homogenised through a 70 mm cell
strainer, centrifuged and re-suspended in PBS. Sample cell
suspensions were blocked with Fc-block (BD) and subsequently
stained with anti-mouse CD3 and CD19 to gate T and B cells,
CD11c, MHC class II (IA/IE) and CD80 (ebioscience). A live/
dead cell stain (Invitrogen) was included. Cells within the live cell
gate were then analysed for levels of FITC or surface markers. PLoS ONE | www.plosone.org Immunogenicity Studies T cell responses to the dominant MHC class I epitope Pb9
(SYIPSAEKI) [14] were analysed by intracellular cytokine staining PLoS ONE | www.plosone.org July 2011 | Volume 6 | Issue 7 | e22442 11 ImmuPatch Mediated MVA Vaccination Pure RNA tissue kit following the manufacturer’s protocol (Roche,
Germany). cDNA was prepared from isolated RNA using The
High
Capacity
cDNA
Reverse
Transcription
Kit
(Applied
Biosystems, USA). Real-time quantitative RT-PCR analysis of
cDNA samples, prepared from isolated RNA, for selected genes
were performed using the TaqManH Gene Expression Assays and
ABI7300 Real time PCR System instrument and software (Applied
Biosystems) following the manufacturer’s protocols. The relative
expression of the following genes was measured: TNF-a (ID:
Mm00443258_m1), IL1-a (ID: Mm00439620_m1), IL-1b (ID:
Mm00434228_m1), IL-6 (ID: Mm00446190_m1), IL-10 (ID:
Mm00439616_m1), TGF-b1 (ID: Mm00441726_m1), Ccl4 (ID:
Mm00443112_m1), Ccl5 (ID: Mm01302428_m1), IL-12b (ID:
Mm00434174_m1), IFN-b (ID: Mm00439552_s1). The house-
keeping gene used was GAPDH (ID: 4352932E). Real-time PCR
data were analyzed as follows: cycle numbers at threshold crossing
(Ct) values were subtracted from Ct values for a control house-
keeping gene, GAPDH, to generate DCt values. Gene expression
values relative to GAPDH were calculated as 22DCt. Pure RNA tissue kit following the manufacturer’s protocol (Roche,
Germany). cDNA was prepared from isolated RNA using The
High
Capacity
cDNA
Reverse
Transcription
Kit
(Applied
Biosystems, USA). Real-time quantitative RT-PCR analysis of
cDNA samples, prepared from isolated RNA, for selected genes
were performed using the TaqManH Gene Expression Assays and
ABI7300 Real time PCR System instrument and software (Applied
Biosystems) following the manufacturer’s protocols. The relative
expression of the following genes was measured: TNF-a (ID:
Mm00443258_m1), IL1-a (ID: Mm00439620_m1), IL-1b (ID:
Mm00434228_m1), IL-6 (ID: Mm00446190_m1), IL-10 (ID:
Mm00439616_m1), TGF-b1 (ID: Mm00441726_m1), Ccl4 (ID:
Mm00443112_m1), Ccl5 (ID: Mm01302428_m1), IL-12b (ID:
Mm00434174_m1), IFN-b (ID: Mm00439552_s1). The house-
keeping gene used was GAPDH (ID: 4352932E). Real-time PCR
data were analyzed as follows: cycle numbers at threshold crossing
(Ct) values were subtracted from Ct values for a control house-
keeping gene, GAPDH, to generate DCt values. Gene expression
values relative to GAPDH were calculated as 22DCt. Figure S4
Total cell counts in draining lymph nodes
post-immunization. BALB/c mice were immunized with
MVA-PbCSP by the ID route or using microneedle array F
(‘Patch’). Naive mice were untreated. Acknowledgments The authors wish to thank Sarah Gilbert and Ali Turner at the Jenner
Institute for support with virus vectors, Hilary Watt at the Jenner Institute
for statistical support and James Birchall, School of Pharmacy, University
of Cardiff for support in establishing the ex vivo skin culture model. Supporting Information Data are expressed as the total number of cytokine-
secreting CD8+ T cells, +/2 standard error of the mean (+/
2SEM), 2 weeks after a single (A) or prime-boost (B) immuniza-
tion. * p,0.05, ** p,0.01, ***p,0.001 compared with ID
vaccinated mice in by one way ANOVA. (PDF) Supporting Information Figure
S1
Sample FACS plots Figure 2. Mice were
immunized by the ID route (top panels) or using microneedle
Array F (middle panels) or Array G (lower panels). (A) Magnitude
of the multi-functional TNF-a (Y-axis), IFN-c (X-axis) response in
gated live CD8+ T cells that were unstimulated (left panels) or
stimulated with Pb9 epitope (right panels). (B) Memory phenotype
of antigen-specific CD8+ T cells; CD62L (Y-axis) CD127 (X-axis). (PDF) Figure
S7
Delivery
efficiency
of
nanospheres
into
murine skin. Red fluorescent nanospheres were administered
to mice in the same manner as described in Figure S4, using arrays
F, C or H or using a flat silicon patch with no microneedles (‘flat’). After 30 minutes, animals were sacrificed and the outside of the
ears were swabbed with wet cotton wool to remove beads that
were on the skin surface. Ears were homogenised in a HCl/
Tween80/PBS solution (1.0:0.1:0.07 v/v/v). The homogenate
was centrifuged at 1400rpm for 3 minutes and the fluorescence in
the supernatant was determined and compared to the fluorescence
present in the original nanosphere solution administered to mice. Mean (+/2 SEM) with individual percentage fluorescence
recovered from inside the ear is represented for each group. Mean (+/2 SEM) for Flat patch = 0.96 (0.14)%; Patch F = 3.17
(0.46)%; Patch C = 2.58 (0.60)%; Patch H = 2.87 (0.30)%. (PDF) Figure S2
Sample FACS plots Figure 3. Mice were primed
by the ID route (top panels) or using microneedle Array F (middle
panels) or Array G (lower panels) and boosted by the ID route. (A)
Magnitude of the multi-functional TNF-a (Y-axis), IFN-c (X-axis)
response in gated live CD8+ T cells that were unstimulated (left
panels) or stimulated with Pb9 epitope (right panels). (B) Memory
phenotype of antigen-specific CD8+ T cells; CD62L (Y-axis)
CD127 (X-axis). (PDF) Figure S3
Total number of antigen-specific CD8+ T cells
after a prime and prime-boost immunization. BALB/c
mice were immunized with MVA-PbCSP by the intradermal (id)
route or using an ImmuPatch device of increasing pore volume
and were examined after priming or after and ID boost. The total
number of antigen-specific CD8+ T cells in spleens were quantified
after intracellular cytokine staining (ICS) of IFN-c, TNF-a, and
IL-2 subsequent to stimulation with the immunodominant Pb9
peptide. Statistical Analysis Figure S6
Recombinant MVA infects different skin
layers when administered by ID or ImmuPatch. Trans-
gene expression (red fluorescent protein RFP) detected in ex vivo
pig skin cultures when MVA-RFP (16106pfu) is delivered by ID or
microneedle array G. Freshly excised pig skin was setup in a short-
term ex vivo culture. MVA-RFP was injected intradermally or
administered using microneedle array G and skin was cultured
for 14 hours at 37uC to permit virus infection and transgene
expression. Skin was then snap frozen and cryo-sectioned into
10 mm sections. Samples were examined by light microscopy
(406). The site of MVA administration is indicated by arrows. Similar results were obtained in four independent experiments. (PDF) Data were analyzed using GraphPad Prism version 5 for
Windows (GraphPad Software, San Diego, California, USA). Normality of was assessed by Kolmogorov-Smirnov test. Unpaired
two-tailed Student’s t-test or one way ANOVA were performed, as
appropriate, to compare the responses between groups. Cox’s
Proportional Hazards Regression analysis was preformed using
STATA. Immunogenicity Studies Mean with individual cell
counts in homogenised lymph nodes that were harvested from all
mice at 24 hours (top panel) or 48 hours (bottom panel) after
immunization was determined using a Coulter counter. * p,0.05,
** p,0.01 compared with ID vaccinated mice in by one way
ANOVA. (PDF) Figure S5
Distribution of microneedle-delivered fluo-
rescent nanospheres in mouse ear. A 5 ml solution of red
fluorescent nanospheres, 100nm in diameter (Invitrogen) were
administered to the ears of anaesthetised BALB/c mice using
microneedle array F. Thirty minutes post-administration, animals
were culled and ears were removed, preserved and cryosectioned
into 10 mm sections. Cell nuclei were stained using DAPI. Samples
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data: JBC FEP AV PTW ACM. Contributed reagents/materials/analysis
tools: AMC PTW COM AVSH. Wrote the paper: FEP ACM. Conceived and designed the experiments: JBC FEP AV AVSH ACM. Performed the experiments: JBC FEP AV PTW MGM TD. Analyzed the
data: JBC FEP AV PTW ACM. Contributed reagents/materials/analysis
tools: AMC PTW COM AVSH. Wrote the paper: FEP ACM. PLoS ONE | www.plosone.org July 2011 | Volume 6 | Issue 7 | e22442 12 ImmuPatch Mediated MVA Vaccination References Darrah PA, Patel DT, De Luca PM, Lindsay RW, Davey DF, et al. (2007)
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Single-dose immunogenicity and protective efficacy of simian adenoviral vectors
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Effective induction of high-titer antibodies by viral vector vaccines. Nat Med 14:
819–821. 18. Pham NL, Badovinac VP, Harty JT (2009) A default pathway of memory CD8
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directs memory precursor and short-lived effector CD8(+) T cell fates via the
graded expression of T-bet transcription factor. Immunity 27: 281–295. PLoS ONE | www.plosone.org July 2011 | Volume 6 | Issue 7 | e22442 13
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English
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As-cast titanium aluminides microstructure modification
|
Materials research
| 1,999
|
cc-by
| 2,995
|
Received: August 15, 1998; Revised: March 30, 1999 Two cast γ titanium aluminides, Ti47Al and Ti50Al (at. %), were obtained by arc melting under
an argon atmosphere. The as-cast microstructure modification by annealing under protective argon
atmosphere and hot isostatic pressing (HIP) was analysed by optical and scanning electron micros-
copy with microanalysis facilities. As-cast structures presented strong microsegregations, being
composed of primary α dendrites (with low Al content), which transformed into lamellar α2 + γ
during solid state cooling, and interdendritic γ phase (with high Al content). Annealing in the α + γ
field was responsible for a partial reduction of microsegregation, a decreasing of the volume fraction
of α2 + γ lamellar dendrites, an increasing of γ volume fraction and the occurrence of a small fraction
of α2 particles and plates in some of the γ grains. Annealing in the α field at 1400 °C for one hour
was insufficient to eliminate the as-cast microstructure in the Ti50Al alloy and produced large
equiaxed lamellar colonies of α2+γ in the Ti47Al alloy. HIP in the α + γ field (to avoid excessive
grain growth) was responsible for microstructure modifications similar to the ones obtained by heat
treating under similar soaking conditions. high cooling rates or melt undercooling inhibit the forma-
tion of the pro-peritectic solid phase; the incompleteness of
the peritectic reactions will cause the remaining liquid to
progressively enrich in aluminium and solidify, at lower
temperatures, as interdendritic γ phase. The as-cast struc-
ture presents strong microsegregation and texture needing
thermomechanical treatments to be eliminated3,4,5. Mi-
crosegregation is responsible for aluminium contents vary-
ing from 44.7 at. % in α2 + γ dendrites to 48.9 at. % in
interdendritic γ for a Ti48Al2Cr2Nb alloy4, while these
figures attain 51 and 56 %, respectively, for a Ti50Al alloy3. During solid state cooling α-dendrites (hcp structure) trans-
form into lamellar structures, composed of α + γ phases
(this last phase has an ordered fct - L10 structure) and, at
lower temperatures, to α2 + γ phases (α2 has an ordered hcp
- DO19 structure)3,6,7. email: fviana@fe.up.pt © 1999 © 1999 Materials Research, Vol. 2, No. 3, 191-195, 1999. Keywords: titanium aluminides, microstructure, heat treatment 1. Introduction Titanium aluminides with low density, oxidation resis-
tance up to 800 °C and good creep resistance are promising
alloys for high temperature structural applications, the lack
of room temperature ductility being the main obstacle to its
utilization as engineering materials1,2. Mechanical properties of these alloys are strongly mi-
crostructure dependent. Structures with fine mixtures of γ
grains and α2 plates or particles, called “duplex” or “near-
gamma”, conduct to a reasonable balance of properties but
at relatively low levels; refined lamellar structures are
responsible for properties, up to 760 °C, comparable or
better than those of Ni-based superalloys. Several γ-TiAl
based alloys with small amounts of refractory elements, for
increased high temperature properties, and grain refiners
have been studied to attain well-balanced room tempera-
ture ductility and high temperature strength1. Homogenizing treatments conducted in the α + γ field
are generally inefficient, higher temperatures, in the α field,
being usually needed8. Treatments in the α field are respon-
sible for excessive grain growth, so the temperature has to
be kept low9. Microstructures obtained after cooling from
the α phase field are sensitive to the cooling rate: low
cooling rates, typically in the furnace, lead to lamellar The solidification of these alloys, accordingly to the
phase diagram, occurs through two peritectic reactions, L
+ β → α and L + α → γ, that will hardly be complete due
to limited diffusion caused by the formation of a solid
envelope of the peritectic phase, avoiding the physical
contact between the reactants. For both peritectic reactions, Duarte et al. 192 Materials Research structures with α2 and γ lamellae; higher cooling rates are
responsible for a Widmanstätten structure; α to γ massive
transformation results from even higher cooling rates. The
γ-massive transformation nucleates at α grain boundaries
and progresses to the grain interior; for a high cooling rate
and/or coarse grain size the transformation may be incom-
plete and the grain interiors are not transformed6,7,10,11. For
Ti47.5Al and Ti48Al alloys the non transformed areas
present very fine lamellae of α2 and γ; these lamellae are
similar to the ones presented by a brine cooled totally α
structure Ti40Al after annealing in the α + γ field7,11,12. materials used were Ti and Al high purity bars. A previous
casting procedure was established in order to obtain as-cast
materials free from macro-segregation, with some ten
grams of weight and 14 mm diameter. 1. Introduction HIP was conducted in argon atmosphere at a tempera-
ture of 1250 °C during 4 h, under a pressure of about 180
MPa. Alternatively heat treatments were carried out on
as-cast aluminides, under a protective argon flux. The
samples were submitted to a set of heat treatment cycles. The pre-heating of 10 min at 800 °C was followed by a 10
°C/min heating rate until the maximum temperatures of
1150, 1250, 1350 and 1400 °C for a maximum stage of 6
h. A longer cycle was tested at 1250 °C during 36 h for the
Ti50Al alloy. All the samples were cooled inside the fur-
nace, at an average rate of 12 °C/min from the maximum
temperature to 900 °C. The fine lamellar structures are quite different from
the coarser ones resulting from slow cooling rates. These
are more stable than the fine ones and when heated to
1000 °C coarsen by fragmentation of some lamellae and
thickening at the expenses of others that dissolve and γ
grains will form at lamellar colony boundaries. If the
temperature is raised to 1200 °C, interlamellar spacing
increases and coarse α2 particles and plates are present
at non lamellar γ grains13. The as-cast aluminides chemical compositions of Ti
and Al were evaluated using 4 different techniques: energy
dispersion spectrometry (EDS), wavelength dispersion
spectrometry, inductive coupled plasma, and atomic ab-
sorption spectrometry. Similar results were achieved inde-
pendently of the technique used; the variation around
nominal compositions is ± 0.2 at. %. To measure phase
chemical compositions the quantitative EDS analysis with
standards of pure Al and Ti (FIROZ corrected) was per-
formed in the scanning electron microscope (SEM) Jeol
JSM 35 C. The accuracy of measurements is ± 0.5 at. %. 3. Results and Discussion 3. Results and Discussion 2. Materials and Procedures In this work two titanium aluminides were produced,
Ti50Al and Ti47Al (hereafter all compositions are given in
atomic percentage), by the arc melting technique under
argon atmosphere in a water cooled copper mould. The raw EM under atomic number contrast (c) Figure 1. Optical microstructures of the as-cast Ti50Al alloy (a) and (b) and SEM under atomic number contrast (c). Figure 1. Optical microstructures of the as-cast Ti50Al alloy (a) and (b) and SEM under atomic number contrast (c). As-Cast Titanium Aluminides Microstructure Vol. 2, No. 3, 1999 Vol. 2, No. 3, 1999 193 Table 1. Chemical composition of the as-cast materials, determined by EDS. Region analysed
Chemical composition (at. %)
Ti50Al
Ti47Al
Ti
Al
Ti
Al
Lamellar structure
52.0 - 53.6
46.4 - 48.0
not available
not available
Bright lamellar structure
not available
not available
53.9 - 54.9
45.1 - 46.1
Dark lamellar structure
not available
not available
50.1 - 51.2
48.8 - 49.9
γ-Phase
46.0 – 46.6
53.4 - 54.0
47.7
52.3 Table 1. Chemical composition of the as-cast materials, determined by EDS. Ref. 3. The α dendrites transform into α2+γ lamellae during
solid state cooling and the γ phase remains interdendritic. Samples not etched were SEM observed under an atomic
number contrast. The samples for optical microscopy (OM) and SEM
observation were prepared following standard metal-
lographic techniques proposed by Ref. 14. The OM studies
used differential interfferential contrast and Kroll’s solu-
tion as the etching reagent. The phase diagram shows that a decrease in Al content
of the alloy implies a progressively higher fraction of α
phase after solidification, but even for the Ti47Al alloy the
interdendritic γ remains in the as-cast structure, enhancing
the intensity of the segregation - see Fig. 2. In the lamellar
structure a higher aluminium content (see Table 1) has been
evaluated in regions near the interdendritic phase, the
darker lamellar areas in Fig. 2b. All these features support
the assumed coring hypothesis. As-cast and HIPed densities were investigated using the
Micromeritics Accupyc 1330 pycnometer with 0.001
measurement precision. 3.1. As-cast structure characterization The characteristic microstructures obtained by heat
treating Ti50Al in the α + γ field are shown in the Figs. 3
and 4. In the SEM microstructures shown in Fig. 3 the
following constituents are identified: γ-phase (dark re-
gions); α2 (thicker white regions); and lamellar structure,
remaining from dendrites. It is clear that the dendritic
fraction after 6 h at 1250 °C has decreased, showing that
solution of α2 + γ dendrites has progressed; even after an
increased stage of 36 h the α2 phase is still present - see Fig. 4a. The phase diagram states that at this temperature a
totally γ structure should be obtained, so equilibrium struc-
ture has not been reached. Increasing the temperature to
1350 °C for 6 h or 1400 °C for 1 h resulted in a dendritic
fraction smaller than the one obtained for the 6 h treatment The optical microstructure of the as-cast Ti50Al alloy
presents long dark dendrites and a white interdendritic
phase - see Fig. 1 a. Higher magnifications resolve the
lamellar nature of the dendritic areas - see Fig. 1 b. SEM
observations under atomic number contrast reveal a differ-
ent chemical composition for the lamellae, with an average
Al content of 46.4 to 48.0% - see Table 1; the interdendritic
phase has a higher Al content, from 53.4 to 54.0%. Accord-
ingly to the phase diagram15, a Ti50Al alloy should com-
plete the solidification with α dendritic structure; the
occurrence of the interdendritic phase requires coring dur-
ing α formation, with consequent Al enrichment of the
liquid, which will finally solidify as γ phase as reported by Figure 2. Optical micrographs of the as-cast Ti47Al alloy. Figure 2. Optical micrographs of the as-cast Ti47Al alloy. at 1250 °C (see Fig. 4b). Microanalysis measurements
reinforce the microstructural observations: γ phase de-
creases its Al content and lamellar structure becomes richer
(see Table 2), approaching equilibrium. Figure 5 illustrates the dendritic structure attenuation
for Ti47Al, along with the γ phase formation. The micro-
structures of samples treated in α + γ field - see Figs. 5a and
b - reveal a matrix of γ-phase and lamellar grains, duplex
194
Duarte et al. Materials Research
Figure 3. SEM micrographs of Ti50Al heat treated at 1250 °C during 6 h. Figure 4. 3.1. As-cast structure characterization Optical micrographs of heat treated Ti50Al at 1250 °C for 36 h (a) and at 1400 °C for 1 h (b). Figure 5. Optical micrographs of heat treated Ti47Al at 1250 °C for 6 h (a) and (b), and at 1400 °C for 1 h (c). 194 Materials Research Materials Research Duarte et al. Duarte et al. Figure 3. SEM micrographs of Ti50Al heat treated at 1250 °C during 6 h. Figure 4. Optical micrographs of heat treated Ti50Al at 1250 °C for 36 h (a) and at 1400 °C for 1 h (b). Figure 3. SEM micrographs of Ti50Al heat treated at 1250 °C during 6 h. Figure 4. Optical micrographs of heat treated Ti50Al at 1250 °C for 36 h (a) and at 1400 °C for 1 h (b). d t 1400 °C f
1 h (b) Figure 4. Optical micrographs of heat treated Ti50Al at 1250 °C for 36 h (a) and at 1400 °C for 1 h (b). cal micrographs of heat treated Ti50Al at 1250 °C for 36 h (a) and at 1400 °C for 1 h (b). at 1250 °C (see Fig. 4b). Microanalysis measurements
reinforce the microstructural observations: γ phase de-
creases its Al content and lamellar structure becomes richer
(see Table 2), approaching equilibrium. Figure 5 illustrates the dendritic structure attenuation
for Ti47Al, along with the γ phase formation. The micro-
structures of samples treated in α + γ field - see Figs. 5a and
b - reveal a matrix of γ-phase and lamellar grains, duplex
g
p
g p
Figure 5. Optical micrographs of heat treated Ti47Al at 1250 °C for 6 h (a) and (b), and at 1400 °C for 1 h (c). Figure 5. Optical micrographs of heat treated Ti47Al at 1250 °C for 6 h (a) and (b), and at 1400 °C for 1 h (c). Figure 5. Optical micrographs of heat treated Ti47Al at 1250 °C for 6 h (a) and (b), and at 1400 °C for 1 h (c). at 1250 °C (see Fig. 4b). Microanalysis measurements
reinforce the microstructural observations: γ phase de-
creases its Al content and lamellar structure becomes richer
(see Table 2), approaching equilibrium. Figure 5 illustrates the dendritic structure attenuation
for Ti47Al, along with the γ phase formation. The micro-
structures of samples treated in α + γ field - see Figs. 3.3. Hip structure characterization 5.Naka, S.; Thomas, M.; Sanchez, C.; Khan, T. ibid, n. 1, p. 313. HIPed microstructure of Ti50Al can not be distin-
guished from the one obtained by heat treatment, for similar
soaking conditions, in terms of both phase transformation
and chemical composition analysis. The measured rate of
densification obtained by the HIP process (0.5%) is in the
limit of the resolution of the equipment used and should be
carefully considered. 6.Kumagai, T.; Abe, E.; Nakamura, M. Metall. and Mat. Trans. A, n. 29A, p. 19, 1998. 7.Veeraraghavan, D.; Ramanath, G.; Wang, P.;
Vasudevan, V.K. Solid → Solid Phase Transforma-
tions, Jonhson, W.C., et al., eds., TMS, 273 (1994). 8.Semiatin, S.L.; Chesnutt, J.C.; Austin, C.;
Seetharaman, V. ibid, n. 1, p. 263. References likely, and a few number of α2 dispersed particles are also
observed. One α phase field treatment produced a fully-la-
mellar structure represented in the Fig. 5 c; dendrites are no
longer visible after this treatment which may be supposed
as capable of producing an equilibrium state, causing a
totally α structure after the isothermal stage. The lamellar
colony size after this treatment is very high, in agreement
with the literature9,13; the minimum colony size measure-
ment was 2 mm. likely, and a few number of α2 dispersed particles are also
observed. One α phase field treatment produced a fully-la-
mellar structure represented in the Fig. 5 c; dendrites are no
longer visible after this treatment which may be supposed
as capable of producing an equilibrium state, causing a
totally α structure after the isothermal stage. The lamellar
colony size after this treatment is very high, in agreement
with the literature9,13; the minimum colony size measure-
ment was 2 mm. 1.Kim, Y.-W.; Dimiduk, D.M. Structural Intermetallics
1997, Nathal, M.V. et al., eds., TMS, 531, 1997. 2.Froes, F.H.; Suryanarayana, C.; Eliezer, D. J. Mat. Sci., n. 27, p. 5113, 1992. 3.Shao, G.; Tsakiropoulos, P.; Miodownik, A.P. Mat. Sci. and Techn., n. 13, p. 797, 1997. 4.Muraleedharan, K.; Rishel, L.L.; De Graef, M.;
Cramb, A.W.; Pollock, T.M.; Gray III, G.T. ibid, n. 1,
p. 215. 3.1. As-cast structure characterization 5a and
b - reveal a matrix of γ-phase and lamellar grains, duplex Figure 5 illustrates the dendritic structure attenuation
for Ti47Al, along with the γ phase formation. The micro-
structures of samples treated in α + γ field - see Figs. 5a and
b - reveal a matrix of γ-phase and lamellar grains, duplex Figure 5 illustrates the dendritic structure attenuation
for Ti47Al, along with the γ phase formation. The micro-
structures of samples treated in α + γ field - see Figs. 5a and
b - reveal a matrix of γ-phase and lamellar grains, duplex at 1250 °C (see Fig. 4b). Microanalysis measurements
reinforce the microstructural observations: γ phase de-
creases its Al content and lamellar structure becomes richer
(see Table 2), approaching equilibrium. at 1250 °C (see Fig. 4b). Microanalysis measurements
reinforce the microstructural observations: γ phase de-
creases its Al content and lamellar structure becomes richer
(see Table 2), approaching equilibrium. Vol. 2, No. 3, 1999 As-Cast Titanium Aluminides Microstructure 195 Table 2. Al content of the heat treated materials, determined by EDS. Region
Ti50Al
analysed
1250 °C 6 h
1250 °C 36 h
1400 °C 1 h
γ-Phase
50.2 – 51.1
48.3 - 49.5
48.8 - 51.1
α2-phase
39.0
not available
41.1
Lamellar
structure
48.8
not available
48.6 Table 2. Al content of the heat treated materials, determined by EDS. iv) HIP microstructure modifications are close to the
ones obtained from similar heat treatment conditions. Acknowledgments This work was supported by PRAXIS XXI project with
reference 3/3.1/CTAE/1904/95. A grant BIC 3621 from
PRAXIS XXI was conceded to A. Duarte to cooperate in
this project. 4. Conclusions 9.Zhang, W.J.; Francesconi, L.; Evangelista, E. Mat. Letters, n. 27, p. 135, 1996. From this work the following conclusions are with-
drawn: 10.Ramanujan, R.V. ibid, n. 7, p. 881. i) as-cast structures of arc-melted Ti50Al and Ti47Al
present strong microsegregation, revealed by the system-
atic occurrence of interdendritic γ phase; 11.Abe, E.; Kumagai, T.; Nakamura, M. ibid, n. 1, p. 167. 12.Nakai, K.; Ono, T.; Ohtsubo, H.; Ohmori, Y. Mat. Trans., JIM, n. 37, p. 813, 1996. ii) the efficiency of the homogenizing treatment ap-
plied to the as-cast structure of Ti50Al alloy is low; even
after high temperatures and long times (1250 ºC for 36
h and 1400 ºC for 1 h) primary dendrites remain visible; 13.Ramanujan, R.V.; Maziasz, P.J.; Liu, T. Acta Mat., n. 44, p. 2611, 1996. 14.Glatz, W.; Retter, B.; Leonhard, A.; Clemens, H. Structure, n. 29, p. 3, 1996 iii) as-cast structure of Ti47Al alloy has been de-
stroyed by an α field treatment (1400 °C for 1 h), al-
though an ultra coarse fully lamellar structure resulted; 15.McCullough, C.; Valencia, J.J.; Levi, C.G.; Me-
hrabian, R. Acta Metall., n. 37, p. 1321, 1989.
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Experimental and Process Modelling Investigation of the Hydrogen Generation from Formic Acid Decomposition Using a Pd/Zn Catalyst
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Applied sciences
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cc-by
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Abstract: The use of hydrogen as a renewable fuel has attracted great attention in recent years. The
decomposition of formic acid under mild conditions was investigated using a 2%Pd6Zn4 catalyst in a
batch reactor. The results showed that the conversion of formic acid increases with reaction tempera-
ture and with the formic acid concentration. A process-simulation model was developed to predict
the decomposition of formic acid using 2%Pd6Zn4 in a batch reactor. The model demonstrated very
good validation with the experimental work. Further comparisons between the 2%Pd6Zn4 catalyst
and a commercial Pd/C catalyst were carried out. It was found that the 2%Pd6Zn4 demonstrated
significantly higher conversions when compared with the commercial catalyst. Citation: Hafeez, S.; Barlocco, I.;
Al-Salem, S.M.; Villa, A.; Chen, X.;
Delgado, J.J.; Manos, G.; Dimitratos,
N.; Constantinou, A. Experimental
and Process Modelling Investigation
of the Hydrogen Generation from
Formic Acid Decomposition Using a
Pd/Zn Catalyst. Appl. Sci. 2021, 11,
8462. https://doi.org/10.3390/
app11188462 Keywords: formic acid decomposition; H2 production; process simulation modelling; renewable
energy; green chemistry Received: 20 July 2021
Accepted: 9 September 2021
Published: 12 September 2021 Article
Experimental and Process Modelling Investigation of the
Hydrogen Generation from Formic Acid Decomposition Using
a Pd/Zn Catalyst Sanaa Hafeez 1
, Ilaria Barlocco 2,*
, Sultan M. Al-Salem 3
, Alberto Villa 2
, Xiaowei Chen 4
,
Juan J. Delgado 4, George Manos 5
, Nikolaos Dimitratos 6
and Achilleas Constantinou 7,* 1
Division of Chemical & Energy Engineering, School of Engineering, London South Bank University, 1
Division of Chemical & Energy Engineering, School of Engineering, London South Bank University,
London SE1 0AA, UK; hafeezs3@lsbu.ac.uk 2
Dipartimento di Chimica, Universitá degli Studi di Milano, Via Golgi, 20133 Milan, Italy;
alberto.villa@unimi.it 3
Environment & Life Sciences Research Centre, Kuwait Institute for Scientific Research, P.O. Box 24885,
Safat 13109, Kuwait; ssalem@kisr.edu.kw 4
Departamento de Ciencia de los Materiales, Ingeniería Metalúrgica y Química Inorgánica, Facultad de
Ciencias, Universidad de Cádiz, Campus Río San Pedro, E-11510 Puerto Real (Cádiz), Spain;
xiaowei.chen@uca.es (X.C.); juanjose.delgado@uca.es (J.J.D.) j
j
g
5
Department of Chemical Engineering, University College London, London WCIE 7JE, UK; g.manos@ucl.a 6
Dipartimento di Chimica Industriale e dei Materiali, ALMA MATER STUDIORUM Università di Bologna,
Viale Risorgimento 4, 40136 Bologna, Italy; nikolaos.dimitratos@unibo.it 7
Department of Chemical Engineering, Cyprus University of Technology, 57 Corner of Athinon and
Anexartisias, Limassol 3036, Cyprus yp
*
Correspondence: ilaria.barlocco@unimi.it (I.B.); a.konstantinou@cut.ac.cy (A.C.) applied
sciences applied
sciences applied
sciences 1. Introduction Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations. The use of conventional fossil fuels for energy production has led to serious global
consequences for the environment and climate. Renewable fuels have gained increasing
attention in recent years to combat the current climate issues. Formic acid (HCOOH) has
proven immensely popular as a favourable hydrogen storage/generation material due
to its high gravimetric and volumetric hydrogen capacity, ease of handling, non-toxicity,
stability at room temperature, and abundant supply from the conversion of biomass and
carbon dioxide (CO2) [1]. The decomposition of formic acid reaction can be conducted
in a liquid phase (at temperatures lower than 373 K) as well as in a gas phase (at excess
temperatures), resulting in the dehydrogenation or dehydration pathways [2]. The former
produces CO2 and hydrogen (H2) as products, and the latter generates carbon monoxide
(CO) and water (H2O) products. Performing the reaction at high temperatures (greater than
423 K) may lead to lower selectivity in the hydrogen production, because of the reverse
water–gas shift reaction. Therefore, selective catalysts for formic acid dehydrogenation
under these conditions are in high demand [3]. 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/). https://www.mdpi.com/journal/applsci Appl. Sci. 2021, 11, 8462. https://doi.org/10.3390/app11188462 Appl. Sci. 2021, 11, 8462 2 of 12 In recent years, heterogeneous catalysts have attracted large interest for formic acid
decomposition because of enhanced separability, reusability, and relatively low reaction
temperatures (less than 80 ◦C). The heterogeneous catalysts Pd, Au, or Ag and their
alloys have been commonly studied [4–21]. A variety of materials have additionally
been investigated as catalyst supports for the dehydrogenation of formic acid, such
as activated carbon [22–24], zeolites [16,25], amines [26–29], metal organic frameworks
(MOFs) [11,13,30,31], and macroreticular resins [32–34]. The Pd/C catalyst is the most
common and effective catalyst for H2 production from formic acid in aqueous solution [35]. Sanchez et al. investigated the performance of a commercial 5 wt.% Pd/C catalyst for
the catalytic, additive-free decomposition of formic acid at mild conditions. The catalyst
displayed an excellent 99.9% H2 selectivity and a high catalytic activity (TOF = 1136 h−1)
at 30 ◦C toward the selective dehydrogenation of formic acid to H2 and CO2 [5]. 1. Introduction y
g
Formic acid decomposition for H2 production has recently been investigated using
Pd/ZnO catalysts, which are typically used for methanol steam reforming. Bulushev et al. [3]
investigated the catalytic properties of PdZn/ZnO in formic acid decomposition. In
addition, these catalysts were compared with Pt/ZnO and Pd/Al2O3 catalysts as well
as ZnO support. The results show that the measured catalyst activity corresponds to
the following sequence: Pd/Al2O3 ≥Pd/ZnO > Pt/ZnO > ZnO. The Pd/ZnO catalyst
shows the highest selectivity to hydrogen (up to 99.3%). This is attributed to the PdZn
alloy formation during the catalyst reduction pretreatment. Increasing the pretreatment
temperature from 573 K to 773 K resulted in a significant increase in the mean size of
the PdZn (PtZn) nanoparticles. However, the activity of the catalyst remains unchanged,
though the hydrogen selectivity increases. g
y
g
y
Yao et al. [35] investigated Pd-supported catalysts for enhancing the decomposition
of formic acid. The porous carbon support was synthesised using zeolitic imidazole
frameworks (ZIFs) as a precursor. Furthermore, the porous carbon was produced by the
one-step method. By doping the Zn into the predetermined bimetallic ZnCo-ZIFs, the
operation of the catalyst was enhanced. The greatest catalytic activity of the prepared
catalyst (Pd/Co@CN-2), with a 2.6 nm average diameter of PdCo, was observed when the
Zn/Co molar ratio was 2 and demonstrated an initial turnover frequency value (TOF) as
high as 2302 h−1 at 30 ◦C. Ding et al. [36] investigated the synthesis of a hybrid material of Pd nanoparticles
encapsulated within carbon nanotubes (CNTs) (Pd-CNTs-in). The results show that the
prepared Pd-CNTs-in catalyst exhibits particularly high formic acid decomposition activity
and durability at room temperature. The TOF value was found to be 1135 h−1 in the
first 10 min, and there was no significant decrease in the continuous three-fold recovery
study. The conclusion reached is that this work can provide a promising strategy for
manufacturing cost-effective and highly active Pd-based catalysts for the dehydrogenation
of formic acid. The investigation of formic acid decomposition in batch reactors is often limited by
their ability to generate a continuous stream of H2 for fuel cell applications. Moreover,
these reactors do not permit a detailed study of the catalyst lifetime to give an indica-
tion of its long-term performance. Therefore, the study of formic acid decomposition
in continuous-flow reactors is essential to the comprehensive understanding of this re-
action. 1. Introduction The decomposition of formic acid has been explored using homogeneous and
heterogeneous catalysts in continuous stirred tank reactors (CSTR) [37–39]. Caiti et al. [40] investigated the production of hydrogen via the low-temperature
(less than 110 ◦C), additive-free decomposition of formic acid over a heterogeneous Pd/C
catalyst. The results show that, during continuous-flow operation, pore contamination and
poisoning caused by formate ions lead to catalyst deactivation. Although these factors
cause extensive deactivation in the plug-flow mode, by operating in a continuous stirred
tank reactor to reduce the steady-state formic acid concentration, encouraging results can
be obtained. Therefore, under mild conditions and without stoichiometric additives, the
system can operate continuously for more than 2500 cycles without loss of activity. Appl. Sci. 2021, 11, 8462 3 of 12 3 of 12 The decomposition of formic acid has often been investigated on an experimental
basis. The utilisation of software for conducting detailed numerical studies can facilitate an
understanding of parameter optimisation for the decomposition of formic acid reaction. Furthermore, theoretical studies are a valuable tool to assess the validity of the experimental
findings [41,42]. Hafeez and Sanchez et al. [43] investigated the liquid-phase decomposition
of formic acid using a Pd/C catalyst in a packed bed microreactor. A comprehensive
heterogeneous computational fluid dynamic (CFD) model was developed to validate the
experimental data. The results showed that the CFD model demonstrated very good
validation with the experimental work. Furthermore, the novel model could successfully
depict the deactivation of the Pd/C catalyst based on the poisoning species CO. The
model developed can be used to effectively predict the decomposition of formic acid in
microreactors for potential fuel cell applications. p
pp
The process-simulation modelling of formic acid decomposition has not been imple-
mented for the current reaction system, which enhances the uniqueness of the current work. Process-simulation or flowsheet modelling can offer a solid foundation based on mass and
energy balances, mass and heat transfer, reaction-system modelling, and phase equilibria
to simulate the decomposition of formic acid. We utilised the capabilities of Aspen Plus to
develop a simple innovative reaction system by integrating the phase-equilibrium exercises. It has been observed that both CFD and process-simulation modelling have the ability
to predict different reactions with similar results [44]. The production of H2 from the
decomposition of formic acid has been investigated using a 2%Pd6Zn4 catalyst in a batch
reactor. 2.1. Materials and Chemicals Zinc Chloride (ZnCl2, 98%), potassium tetrachloropalladate (II) (K2PdCl4, 99.99%),
sodium borohydride (NaBH4, 99.99%), and formic acid were obtained from Sigma-Aldrich
(Haverhill, MA, USA). The materials were utilised without the need for pretreatment. Deionised water was used as the reaction solvent for the reaction. The CNF PR24-HHT
(High Heat Treated carbon nanofiber) was acquired from the Applied Science Company
(Cedarville, OH, USA). 2.2. Catalyst Preparation The 2%Pd6Zn4@HHT catalyst was prepared using the galvanic replacement procedure
employed by Liao et al. [43], modified for the Pd–Zn system. This typical method consists
of a two-step synthesis. In the first part, Zn(0) was obtained using a reducing agent (NaBH4)
from ZnCl2 salt. The second step involves the spontaneous displacement reduction between
(PdCl4)2- and Zn. First, the support (HHT-CNFs) was added in milli-Q water under
sonication conditions for 40 min. Subsequently, the mixture was degassed in a vacuum
and purged in N2 for 1 h. After that, 20 mg of NaBH4 and 3.8 mg of Zn as ZnCl2 were
added. The mixture was then shaken by sonication for 5 min and placed overnight in a
vacuum under vigorous stirring. In the second part, 6.2 mg of Pd as K2PdCl4 was added. The mixture was then vigorously stirred in vacuum for 20 min. After that, the catalyst was
filtered, washed with deionised water, and dried in the oven for 48 h. 1. Introduction A comprehensive process-simulation model has been developed based on the
batch stirred reactor to validate the experimental findings. Further studies are performed
to assess the characterisation and activity of the catalyst. 2.4. Catalyst Characterisation
XPS The XPS measurements were recorded using a thermo Scientific K-alpha+ spectrome-
ter. The sample analysis was achieved using a monochromatic Al X-ray source running
at 72 W, with the signal recorded over an oval-shape area of 600 × 400 µ. The data were
recorded at 150 eV for survey scans and 40 eV for high resolution (HR) scans with 1 eV and
0.1 eV step sizes, respectively. The analysis of the data was performed using CASAXPS
(v2.3.17 PR1.1), using Scofield sensitivity factors and an energy exponent of −0.6. TEM The transmission electron microscopy (TEM) experiments were completed using a
double Cs aberration-corrected FEI Titan3 Themis 60–300 microscope. The microscope was
equipped with a monochromator, an X-FEG gun, and a high-efficiency XEDS ChemiSTEM. This comprised four windowless SDD detectors. HR-STEM imaging was completed at
200 kV, applying a high-angle annular dark-field (HAADF) detector with a camera length of
11.5 cm. The HAADF–STEM procedure is responsive to the atomic number of the elements,
in which the intensity is roughly proportional to the square of the atomic number (Z2),
and it provides the opportunity to differentiate the small nanoparticles carried on light
supports. The XEDS mappings were achieved using a beam current of 200 pA and a dwell
time per pixel of 128 µs. The elemental maps were filtered using a Gaussian blur of 0.8
(Velox software) to enhance the quality of the images. Due to the STEM–HAADF images
of the catalysts, the diameters of more than 200 randomly chosen metal particles were
measured and the resultant metal particle size distributions (PSD) were defined. As a result,
the average particle diameter (d) was computed corresponding to the following expression:
d = ∑nidi/∑ni, where ni ≥200. Equally, the total metal dispersion was evaluated according
to D = Ns/Nt, where Ns is the total number of surface-metal atoms and Nt is total number
of atoms in the metal particle. For the particle size calculations, the ImageJ software
was used. 2.3. Catalytic Tests The liquid-phase formic acid dehydrogenation was performed using a two-necked
round-bottom flask furnished with a magnetic stirrer and reflux condenser and placed
in a water bath. Usually, 10 mL of formic acid (FA) (aqueous) was loaded into the re-
actor. Different concentrations of the substrate solution were utilised: 0.5 M, 0.4 M,
0.3 M, 0.2 M, and 0.1 M. After the desired temperature had been reached, the catalyst
(FA:metal = 2000:1 mol/mol) was inserted and the mixture was agitated at 1400 rpm. Tests Appl. Sci. 2021, 11, 8462 4 of 12 to establish the stirring rate were previously shown by our group [4]. The catalyst was also
tested at various temperatures in order to study the kinetic behaviour of the catalyst, 30 ◦C,
40 ◦C, 50 ◦C, and 60 ◦C. Stability tests were performed using the procedure previously
discussed, reusing the catalyst without any further purification procedure. High-performance liquid chromatography (HPLC) was implemented to assess the
conversion of formic acid. A H+ chromatographic column (Alltech OA- 10,308, 300 mm
7.8 mm) was utilised with a UV detector established at 210 nm. The liquid samples of
200 µL were obtained after certain time intervals and subsequently diluted to 5 mL with a
solvent of H2SO4 solution (0.005 N). This isocratic effluent flow was maintained at a flow
rate of 0.5 mL/min. The volume of gases, CO and CO2, was monitored and recorded using
an online micro-gas chromatograph (Agilent 3000A) every 2 h. This equipment consisted
of a molecular sieve module, an OV-1 module (stationary phase of polydimethylsiloxilane),
and a TCD detector. The measurement of CO and CO2 was achieved from calibration
curves taken from commercial standards. 2.5. Modelling Methodology The decomposition of formic acid reaction was modelled using the software Aspen
Plus V11. The model selected to accurately predict the behaviour of the vapour–liquid
equilibrium of the reaction system was the non-random two-liquid (NRTL) model [44]. The
process-modelling studies were performed in a batch reactor with a 100 mL volume and
a temperature range of 30–60 ◦C. Characteristically, 10 mL of the required concentration
of formic acid was deposited into the batch reactor. The catalyst used was 2%Pd6Zn4, the
formic acid/metal molar ratio was 2000:1, and the stirring rate was 1400 rpm. Appl. Sci. 2021, 11, 8462 5 of 12 The most significant feature of the NRTL method [45] is its applicability to multi-
component systems, presuming that the local compositions can be depicted by a relation-
ship similar to that acquired for binary systems [46]: xji
xki
=
xj
xk
e[−
(αijgji−αikgki)
RT
]
(1) (1) where xji and xki are the mole fractions of components j and k, respectively, in the neigh-
bourhood of molecule i; gji and gki are the Gibbs energies of interaction for the j + i and k +
i pairs; and αij and αik are characteristic constants of the corresponding binary mixtures. p
;
ij
ik
p
g
y
The activity coefficients can be expressed as follows: lnγj =
∑j τjiGjixj
∑k Gkixk
+ ∑j
(
xjGij
∑k xkGkj
! ·
"
τij −
∑l xlτljGlj
∑k xkGkj
!#)
(2) (2) where where τij =
gij −gjj
RT
(3) (3) and Gij = e(−αijτij)
(4) (4) The reactor energy balance, coupled with the material balance, can determine the
heating or cooling requirements. The energy balance given for the batch stirred reactor can
be expressed as follows: dT
dt = (rAV)(∆HRX) −UA(T −Ta)
∑NiCPi
(5) (5) where T is the reaction temperature, rA is the rate of reaction, V is the volume, ∆HRX is the
constant heat of reaction, U is the overall heat transfer coefficient, A is the batch reactor
heat exchange area, Ta is the ambient temperature, Ni is the number of moles, and Cpi is
the average heat capacity of species i. The decomposition of formic acid can take place via two possible pathways dependent
upon the catalyst, reaction temperature, and reactant concentrations. The dehydrogena-
tion of formic acid is shown in Equation (1), and the dehydration reaction is shown by
Equation (2). 3. Results and Discussion
3. Results and Discussion The 2%Pd6Zn4@HHT catalyst was prepared using the galvanic replacement procedure
employed by Liao et al. [49], modified for the Pd–Zn system. High-heat-treated carbon
nanofibers (HHT-CNFs) were utilised as support for the NPs. The 2%Pd6Zn4@HHT catalyst was prepared using the galvanic replacement proce-
dure employed by Liao et al. [49], modified for the Pd–Zn system. High-heat-treated car-
bon nanofibers (HHT-CNFs) were utilised as support for the NPs. 2.5. Modelling Methodology The former reaction is slightly exothermic, while the latter reaction is slightly
endothermic. The type of catalyst used can determine which pathway is favoured for the
decomposition of formic acid [47,48]. HCOOH →CO2 + H2 ∆rH0 = 31.2 kJ/mol ∆rG0 = −33.0 kJ/mol
(6) (6) HCOOH →CO + H2O ∆rH0 = 28.4 kJ/mol ∆rG0 = −13.0 kJ/mol
(7) (7) The rate of reaction based on the dehydrogenation of formic acid can be expressed
using the Power-Law method: The rate of reaction based on the dehydrogenation of formic acid can be expressed
using the Power-Law method: r = k × Cn
(8) (8) where r is the rate of reaction, k is the kinetic rate constant, C is the concentration of formic
acid, and n is the order of reaction [5]. The estimated activation value of 22 kJ/mol was
obtained for the catalyst used in this study (Pd/Zn catalyst), and the estimation was based
on the experimental data of this study. The order of the reaction was accepted to be 0.2, the
same as for Pd/C [5], which reproduced the experimental results satisfactorily. where r is the rate of reaction, k is the kinetic rate constant, C is the concentration of formic
acid, and n is the order of reaction [5]. The estimated activation value of 22 kJ/mol was
obtained for the catalyst used in this study (Pd/Zn catalyst), and the estimation was based
on the experimental data of this study. The order of the reaction was accepted to be 0.2, the
same as for Pd/C [5], which reproduced the experimental results satisfactorily. Appl. Sci. 2021, 11, 8462 6 of 12
based
be 0.2, 3.1. Catalyst Characterisation
3.1.1. TEM Results
3.1. Catalyst Characterisation
3.1.1. TEM Results The PdZn catalyst was characterised by TEM (Figure 1) to explore the bulk properties
of the system (bulk zinc palladium atomic ratio, particle size distribution, and average
particle size). The catalysts displayed a mean particle size of 2.1 nm, with a narrow particle
size distribution (Figure 1b) and with particles well dispersed on the surface of the carbon
support. On the other hand, the Pd-monometallic counterpart (1% Pd/HHT) showed a
higher average particle size of 3.9 nm [4]. STEM–XEDS analysis confirmed the presence of
Pd–Zn bimetallic particles with an average molar ratio of 86:14 lower than the nominal one
(60:40) (Figure 1a). Figure 2 displays STEM images representing the d spacing. The PdZn catalyst was characterised by TEM (Figure 1) to explore the bulk properties
of the system (bulk zinc palladium atomic ratio, particle size distribution, and average
particle size). The catalysts displayed a mean particle size of 2.1 nm, with a narrow particle
size distribution (Figure 1b) and with particles well dispersed on the surface of the carbon
support. On the other hand, the Pd-monometallic counterpart (1% Pd/HHT) showed a
higher average particle size of 3.9 nm [4]. STEM–XEDS analysis confirmed the presence
of Pd–Zn bimetallic particles with an average molar ratio of 86:14 lower than the nominal
one (60:40) (Figure 1a). Figure 2 displays STEM images representing the d spacing. (a)
(b)
Figure 1. (a) STEM–XEDS of Pd6Zn4 catalyst (Pd–Zn atomic ratio: 86:14) and (b) particle size distri-
bution
Figure 1. (a) STEM–XEDS of Pd6Zn4 catalyst (Pd–Zn atomic ratio: 86:14) and (b) particle size
distribution. (a) (a) (b) Figure 1. (a) STEM–XEDS of Pd6Zn4 catalyst (Pd–Zn atomic ratio: 86:14) and (b) particle size distri-
Figure 1. (a) STEM–XEDS of Pd6Zn4 catalyst (Pd–Zn atomic ratio: 86:14) and (b) particle size
distribution. Appl. Sci. 2021, 11, 8462
Appl. Sci. 2021, 11, 8462 7 of 12
7 of 13 Figure 2. High Resolution STEM images (A), digital diffraction patter of a selected area (B) and
simulated diffraction pattern of Pd among the <1 0 1> axis zone. Figure 2. High Resolution STEM images (A), digital diffraction patter of a selected area (B) and
simulated diffraction pattern of Pd among the <1 0 1> axis zone. Figure 2. High Resolution STEM images (A), digital diffraction patter of a selected area (B) and
simulated diffraction pattern of Pd among the <1 0 1> axis zone. Figure 2. 3.1. Catalyst Characterisation
3.1.1. TEM Results
3.1. Catalyst Characterisation
3.1.1. TEM Results High Resolution STEM images (A), digital diffraction patter of a selected area (B) and
simulated diffraction pattern of Pd among the <1 0 1> axis zone. 3.1.2. XPS Results
3.1.2. XPS Results X-ray photoelectron spectroscopy (XPS) was performed on the material, obtaining
information on the surface properties of the PdZn catalyst. From the XPS survey spectra,
both metals were detected. The Pd/Zn atomic ratio detected by XPS is higher than the
nominal one but similar to the EDX atomic ratio (Table 1), which indicates an enrichment
of Pd on both surface and bulk. X-ray photoelectron spectroscopy (XPS) was performed on the material, obtaining
information on the surface properties of the PdZn catalyst. From the XPS survey spectra,
both metals were detected. The Pd/Zn atomic ratio detected by XPS is higher than the
nominal one but similar to the EDX atomic ratio (Table 1), which indicates an enrichment
of Pd on both surface and bulk. Table 1. Results of survey spectra. Survey
Pd 3d
Zn 2p
Pd/Znnom
Pd/ZnXPS
Pd/ZnEDX
C 1s
O 1s
Pd 3d
Zn 2p
Pd(0)
Pd(II)
Zn(II)
Pd6Zn4/HHT B.E. (eV) 284.5
532.6
337.6
1021.6
336.0
337.6
1022.1
% At. 95.6
3.6
0.6
0.1
9
91
100
1.5
6.0
6.1
In the high-resolution spectra of the Pd 3d region (Figure 3), both metallic Pd and Pd
oxide were detected with relative amounts of 9% and 91%, respectively. The novel catalyst
shown in this study possesses a high Pd(II) content with respect to the monometallic 1%
Pd/HHT, which has a Pd(0) content of 52% [4], suggesting an oxidation effect due to the
presence of Zn. By analysing the high-resolution spectra of the Zn 2p region, only ZnO
was detected. Table 1. Results of survey spectra. Survey
Pd 3d
Zn 2p
Pd/Znnom
Pd/ZnXPS
Pd/ZnEDX
C 1s
O 1s
Pd 3d
Zn 2p
Pd(0)
Pd(II)
Zn(II)
Pd6Zn4/HHT B.E. (eV)
284.5
532.6
337.6
1021.6
336.0
337.6
1022.1
% At. 95.6
3.6
0.6
0.1
9
91
100
1.5
6.0
6.1
In the high-resolution spectra of the Pd 3d region (Figure 3), both metallic Pd and Pd
oxide were detected with relative amounts of 9% and 91%, respectively. The novel catalyst
shown in this study possesses a high Pd(II) content with respect to the monometallic 1%
Pd/HHT, which has a Pd(0) content of 52% [4], suggesting an oxidation effect due to the
presence of Zn. By analysing the high-resolution spectra of the Zn 2p region, only ZnO
was detected. Table 1. Results of survey spectra. Survey
Pd 3d
Zn 2p
Pd/Znnom
Pd/ZnXPS
Pd/ZnEDX
C 1s
O 1s
Pd 3d
Zn 2p
Pd(0)
Pd(II)
Zn(II)
Pd6Zn4/HHT B.E. 3.2. Modelling Results
3.2.1. Batch Reactor Model Validation 3.2.1. Batch Reactor Model Validation
A formic acid decomposition reaction was used as a model reaction for hydrogen
production to evaluate the catalytic performance of the material. This reaction occurs in
two different pathways: dehydrogenation and dehydration. The stirring rate (1400 rpm)
was optimised in previous studies in order to ensure the chemical kinetic regime [4]. The
experimental results for the decomposition of formic acid were compared to the process-
modelling results to assess their validity. Figure 4 depicts the conversion of formic acid at
varying reaction temperatures of 30–60 °C. It can be observed that the conversion in-
creases with increasing temperatures, as expected. Furthermore, there is good validation
between the experimental and theoretical results. Usually, temperatures greater than 60
°C are not investigated due to the requirement of mild operating conditions in portable
devices, which utilise formic acid fuel cells. According to the Arrhenius expression, ݇=
ܣ݁ିಶೌ
ೃ, an apparent activation energy of approximately 22 kJ/mol was obtained for this
reaction with a 2%Pd6Zn4 catalyst for a batch reactor configuration. The catalytic deacti-
ti
i
t
t hi h
t
t
th
f
hi h
ti
t
t
A formic acid decomposition reaction was used as a model reaction for hydrogen
production to evaluate the catalytic performance of the material. This reaction occurs in
two different pathways: dehydrogenation and dehydration. The stirring rate (1400 rpm)
was optimised in previous studies in order to ensure the chemical kinetic regime [4]. The
experimental results for the decomposition of formic acid were compared to the process-
modelling results to assess their validity. Figure 4 depicts the conversion of formic acid at
varying reaction temperatures of 30–60 ◦C. It can be observed that the conversion increases
with increasing temperatures, as expected. Furthermore, there is good validation between
the experimental and theoretical results. Usually, temperatures greater than 60 ◦C are
not investigated due to the requirement of mild operating conditions in portable devices,
which utilise formic acid fuel cells. According to the Arrhenius expression, k = Ae−Ea
RT , an
apparent activation energy of approximately 22 kJ/mol was obtained for this reaction with
a 2%Pd6Zn4 catalyst for a batch reactor configuration. The catalytic deactivation is stronger
at higher temperatures; therefore, higher reaction temperatures promote the dehydration
pathway and CO formation, causing the catalyst to deactivate faster. 3.1.2. XPS Results
3.1.2. XPS Results (eV) 284.5
532.6
337.6
1021.6
336.0
337.6
1022.1
% At. 95.6
3.6
0.6
0.1
9
91
100
1.5
6.0
6.1
Table 1. Results of survey spectra. Survey
Pd 3d
Zn 2p
Pd/Znnom
Pd/ZnXPS
Pd/ZnEDX
C 1s
O 1s
Pd 3d
Zn 2p
Pd(0)
Pd(II)
Zn(II)
Pd6Zn4/HHT B.E. (eV)
284.5
532.6
337.6
1021.6
336.0
337.6
1022.1
% At. 95.6
3.6
0.6
0.1
9
91
100
1.5
6.0
6.1 Table 1 Results of survey spectra
Table 1. Results of survey spectra. In the high-resolution spectra of the Pd 3d region (Figure 3), both metallic Pd and Pd
oxide were detected with relative amounts of 9% and 91%, respectively. The novel catalyst
shown in this study possesses a high Pd(II) content with respect to the monometallic 1%
Pd/HHT, which has a Pd(0) content of 52% [4], suggesting an oxidation effect due to the
presence of Zn. By analysing the high-resolution spectra of the Zn 2p region, only ZnO
was detected. In the high-resolution spectra of the Pd 3d region (Figure 3), both metallic Pd and Pd
oxide were detected with relative amounts of 9% and 91%, respectively. The novel catalyst
shown in this study possesses a high Pd(II) content with respect to the monometallic 1%
Pd/HHT, which has a Pd(0) content of 52% [4], suggesting an oxidation effect due to the
presence of Zn. By analysing the high-resolution spectra of the Zn 2p region, only ZnO
was detected. In the high-resolution spectra of the Pd 3d region (Figure 3), both metallic Pd and Pd
oxide were detected with relative amounts of 9% and 91%, respectively. The novel catalyst
shown in this study possesses a high Pd(II) content with respect to the monometallic 1%
Pd/HHT, which has a Pd(0) content of 52% [4], suggesting an oxidation effect due to the
presence of Zn. By analysing the high-resolution spectra of the Zn 2p region, only ZnO
was detected. In the high-resolution spectra of the Pd 3d region (Figure 3), both metallic Pd and Pd
oxide were detected with relative amounts of 9% and 91%, respectively. The novel catalyst
shown in this study possesses a high Pd(II) content with respect to the monometallic 1%
Pd/HHT, which has a Pd(0) content of 52% [4], suggesting an oxidation effect due to the
presence of Zn. By analysing the high-resolution spectra of the Zn 2p region, only ZnO
was detected. 8 of 12
8 of 13 Appl. Sci. 3.1.2. XPS Results
3.1.2. XPS Results 2021, 11, 8462
Appl. Sci. 2021, 11, 8462 Figure 3. XPS (A) survey, (B) Zn 2p, and (C) Pd 3d spectra. 1200
1000
800
600
400
200
1060
1050
1040
1030
1020
1010
1000
351
348
345
342
339
336
333
330
Intensity (a.u.)
B.E. (eV)
A
Intensity (a.u.)
B.E. (eV)
B
Intensity (a.u.)
B.E. (eV)
C
Figure 3. XPS (A) survey, (B) Zn 2p, and (C) Pd 3d spectra. 1200
1000
800
600
400
200
1060
1050
1040
1030
1020
1010
1000
Intensity (a.u.)
B.E. (eV)
A
Intensity (a.u.)
B.E. (eV)
B
C Fi
3 XPS (A)
(B) Z
2
d (C) Pd 3d
t
Figure 3. XPS (A) survey, (B) Zn 2p, and (C) Pd 3d spectra. Fi
3 XPS (A)
(B) Z
2
d (C) Pd 3d
t
Figure 3. XPS (A) survey, (B) Zn 2p, and (C) Pd 3d spectra. 3.2.2. Pd/Zn vs. Pd/C Catalyst
3.2.2. Pd/Zn vs. Pd/C Catalyst Figure 5. Conversion of formic acid against inlet concentration. Reaction conditions: temperature =
30 °C, HCOOH/metal molar ratio is 2000:1, rpm = 1400, catalyst 2%Pd6Zn4. 3.2.2. Pd/Zn vs. Pd/C Catalyst
The experimental and process-modelling results obtained using the novel 2%Pd6Zn4
catalyst were compared with a commercial 5 wt% Pd/C catalyst using a batch reactor [5]. In addition, the catalytic performance of the material presented in this work was com-
pared with published data on 1% Pd/HHT [4,17]. The decomposition of formic acid under
mild conditions using a Pd/C and 1% Pd/HHT catalyst occurred under similar conditions
to those of the current work. The study was conducted using a two-necked 100 mL round-
bottom flask, which was immersed in an oil bath alongside a reflux condenser and a mag-
netic stirrer. The reaction took place at 30 °C, and 0.5 M of aqueous formic acid solution
was placed into the reactor. The substrate/metal molar ratio was 2000:1, and the stirring
rate was 1400 rpm [4 5 17]
The experimental and process-modelling results obtained using the novel 2%Pd6Zn4
catalyst were compared with a commercial 5 wt.% Pd/C catalyst using a batch reactor [5]. In addition, the catalytic performance of the material presented in this work was compared
with published data on 1% Pd/HHT [4,17]. The decomposition of formic acid under mild
conditions using a Pd/C and 1% Pd/HHT catalyst occurred under similar conditions
to those of the current work. The study was conducted using a two-necked 100 mL
round-bottom flask, which was immersed in an oil bath alongside a reflux condenser and
a magnetic stirrer. The reaction took place at 30 ◦C, and 0.5 M of aqueous formic acid
solution was placed into the reactor. The substrate/metal molar ratio was 2000:1, and the
stirring rate was 1400 rpm [4,5,17]. The experimental and process-modelling results obtained using the novel 2%Pd6Zn4
catalyst were compared with a commercial 5 wt% Pd/C catalyst using a batch reactor [5]. In addition, the catalytic performance of the material presented in this work was com-
pared with published data on 1% Pd/HHT [4,17]. The decomposition of formic acid under
mild conditions using a Pd/C and 1% Pd/HHT catalyst occurred under similar conditions
to those of the current work. The study was conducted using a two-necked 100 mL round-
bottom flask, which was immersed in an oil bath alongside a reflux condenser and a mag-
netic stirrer. 3.2. Modelling Results
3.2.1. Batch Reactor Model Validation vation is stronger at higher temperatures; therefore, higher reaction temperatures pro-
mote the dehydration pathway and CO formation, causing the catalyst to deactivate
faster. Kinetic profiles of the catalyst were obtained at different concentrations (0.2–0.5 M). Figure 5 demonstrates the effect of formic acid concentration on the conversion. The
varying concentrations of formic acid used were 0.2–0.5 M at a reaction temperature of
30 ◦C and a substrate/metal molar ratio of 2000. The kinetic regime and these optimal
reaction conditions were previously obtained by Sanchez et al. [5]. The results show that
higher concentrations lead to higher conversions. In addition, the process-modelling
studies were performed at constant reaction conditions with the experiment, and it can be
concluded that there is good agreement between the experimental and theoretical data for
the batch-reactor system. 9 of 12
9 of 13 Appl. Sci. 2021, 11, 8462
Appl. Sci. 2021, 11, 8462 Figure 4. Conversion of formic acid against reaction temperature. Reaction conditions:
HCOOH/metal molar ratio is 2000:1, inlet [HCOOH] = 0.5 M, rpm = 1400, catalyst 2%Pd6Zn4. Kinetic profiles of the catalyst were obtained at different concentrations (0.2–0.5 M). Figure 5 demonstrates the effect of formic acid concentration on the conversion. The var-
ying concentrations of formic acid used were 0.2–0.5 M at a reaction temperature of 30 °C
and a substrate/metal molar ratio of 2000. The kinetic regime and these optimal reaction
conditions were previously obtained by Sanchez et al. [5]. The results show that higher
concentrations lead to higher conversions. In addition, the process-modelling studies
were performed at constant reaction conditions with the experiment, and it can be con-
cluded that there is good agreement between the experimental and theoretical data for the
batch-reactor system. Figure 4. Conversion of formic acid against reaction temperature. Reaction conditions:
HCOOH/metal molar ratio is 2000:1, inlet [HCOOH] = 0.5 M, rpm = 1400, catalyst 2%Pd6Zn4. Figure 4. Conversion of formic acid against reaction temperature. Reaction conditions:
HCOOH/metal molar ratio is 2000:1, inlet [HCOOH] = 0.5 M, rpm = 1400, catalyst 2%Pd6Zn4. Kinetic profiles of the catalyst were obtained at different concentrations (0.2–0.5 M). Figure 5 demonstrates the effect of formic acid concentration on the conversion. The var-
ying concentrations of formic acid used were 0.2–0.5 M at a reaction temperature of 30 °C
and a substrate/metal molar ratio of 2000. The kinetic regime and these optimal reaction
conditions were previously obtained by Sanchez et al. 3.2. Modelling Results
3.2.1. Batch Reactor Model Validation [5]. The results show that higher
concentrations lead to higher conversions. In addition, the process-modelling studies
were performed at constant reaction conditions with the experiment, and it can be con-
cluded that there is good agreement between the experimental and theoretical data for the
batch-reactor system. Figure 5. Conversion of formic acid against inlet concentration. Reaction conditions: temperature =
30 °C, HCOOH/metal molar ratio is 2000:1, rpm = 1400, catalyst 2%Pd6Zn4. Figure 5. Conversion of formic acid against inlet concentration. Reaction conditions:
temperature = 30 ◦C, HCOOH/metal molar ratio is 2000:1, rpm = 1400, catalyst 2%Pd6Zn4. Figure 4. Conversion of formic acid against reaction temperature. Reaction conditions:
HCOOH/metal molar ratio is 2000:1, inlet [HCOOH] = 0.5 M, rpm = 1400, catalyst 2%Pd6Zn4. Figure 4. Conversion of formic acid against reaction temperature. Reaction conditions:
HCOOH/metal molar ratio is 2000:1, inlet [HCOOH] = 0.5 M, rpm = 1400, catalyst 2%Pd6Zn4. Figure 4. Conversion of formic acid against reaction temperature. Reaction conditions:
HCOOH/metal molar ratio is 2000:1, inlet [HCOOH] = 0.5 M, rpm = 1400, catalyst 2%Pd6Zn4. Kinetic profiles of the catalyst were obtained at different concentrations (0.2–0.5 M). Figure 5 demonstrates the effect of formic acid concentration on the conversion. The var-
ying concentrations of formic acid used were 0.2–0.5 M at a reaction temperature of 30 °C
and a substrate/metal molar ratio of 2000. The kinetic regime and these optimal reaction
conditions were previously obtained by Sanchez et al. [5]. The results show that higher
concentrations lead to higher conversions. In addition, the process-modelling studies
were performed at constant reaction conditions with the experiment, and it can be con-
cluded that there is good agreement between the experimental and theoretical data for the
batch-reactor system. Figure 4. Conversion of formic acid against reaction temperature. Reaction conditions:
HCOOH/metal molar ratio is 2000:1, inlet [HCOOH] = 0.5 M, rpm = 1400, catalyst 2%Pd6Zn4. Figure 4. Conversion of formic acid against reaction temperature. Reaction conditions:
HCOOH/metal molar ratio is 2000:1, inlet [HCOOH] = 0.5 M, rpm = 1400, catalyst 2%Pd6Zn4. Figure 4. Conversion of formic acid against reaction temperature. Reaction conditions:
HCOOH/metal molar ratio is 2000:1, inlet [HCOOH] = 0.5 M, rpm = 1400, catalyst 2%Pd6Zn4. Kinetic profiles of the catalyst were obtained at different concentrations (0.2–0.5 M). Figure 5 demonstrates the effect of formic acid concentration on the conversion. 3.2. Modelling Results
3.2.1. Batch Reactor Model Validation The kinetic regime and these optimal reaction
conditions were previously obtained by Sanchez et al. [5]. The results show that higher
concentrations lead to higher conversions. In addition, the process-modelling studies
were performed at constant reaction conditions with the experiment, and it can be con-
cluded that there is good agreement between the experimental and theoretical data for the
batch-reactor system. Figure 5. Conversion of formic acid against inlet concentration. Reaction conditions: temperature =
30 °C, HCOOH/metal molar ratio is 2000:1, rpm = 1400, catalyst 2%Pd6Zn4. Figure 5. Conversion of formic acid against inlet concentration. Reaction conditions:
temperature = 30 ◦C, HCOOH/metal molar ratio is 2000:1, rpm = 1400, catalyst 2%Pd6Zn4. Figure 5. Conversion of formic acid against inlet concentration. Reaction conditions: temperature =
30 °C, HCOOH/metal molar ratio is 2000:1, rpm = 1400, catalyst 2%Pd6Zn4. Figure 5. Conversion of formic acid against inlet concentration. Reaction conditions:
temperature = 30 ◦C, HCOOH/metal molar ratio is 2000:1, rpm = 1400, catalyst 2%Pd6Zn4. 3.2. Modelling Results
3.2.1. Batch Reactor Model Validation The var-
ying concentrations of formic acid used were 0.2–0.5 M at a reaction temperature of 30 °C
and a substrate/metal molar ratio of 2000. The kinetic regime and these optimal reaction
conditions were previously obtained by Sanchez et al. [5]. The results show that higher
concentrations lead to higher conversions. In addition, the process-modelling studies
were performed at constant reaction conditions with the experiment, and it can be con-
cluded that there is good agreement between the experimental and theoretical data for the
batch-reactor system. Figure 4. Conversion of formic acid against reaction temperature. Reactio
HCOOH/metal molar ratio is 2000:1, inlet [HCOOH] = 0.5 M, rpm = 1400, catalyst 2%
Kinetic profiles of the catalyst were obtained at different concentration
Figure 5 demonstrates the effect of formic acid concentration on the conver
ying concentrations of formic acid used were 0.2–0.5 M at a reaction temper
and a substrate/metal molar ratio of 2000. The kinetic regime and these opt
conditions were previously obtained by Sanchez et al. [5]. The results sho
concentrations lead to higher conversions. In addition, the process-mod
were performed at constant reaction conditions with the experiment and Figure 4. Conversion of formic acid against reaction temperature. Reaction conditions:
HCOOH/metal molar ratio is 2000:1, inlet [HCOOH] = 0.5 M, rpm = 1400, catalyst 2%Pd6Zn4. Figure 4. Conversion of formic acid against reaction temperature. Reaction conditions:
HCOOH/metal molar ratio is 2000:1, inlet [HCOOH] = 0.5 M, rpm = 1400, catalyst 2%Pd6Zn4. cluded that there is good agreement between the experimental and theoretical data for the
batch-reactor system. Kinetic profiles of the catalyst were obtained at different co
Figure 5 demonstrates the effect of formic acid concentration on
ying concentrations of formic acid used were 0.2–0.5 M at a react
and a substrate/metal molar ratio of 2000. The kinetic regime an
conditions were previously obtained by Sanchez et al. [5]. The
concentrations lead to higher conversions. In addition, the pr
were performed at constant reaction conditions with the experi
cluded that there is good agreement between the experimental an
batch-reactor system. Kinetic profiles of the catalyst were obtained at different concentrations (0.2–0.5 M). Figure 5 demonstrates the effect of formic acid concentration on the conversion. The var-
ying concentrations of formic acid used were 0.2–0.5 M at a reaction temperature of 30 °C
and a substrate/metal molar ratio of 2000. 3.2.2. Pd/Zn vs. Pd/C Catalyst
3.2.2. Pd/Zn vs. Pd/C Catalyst The reaction took place at 30 °C, and 0.5 M of aqueous formic acid solution
was placed into the reactor. The substrate/metal molar ratio was 2000:1, and the stirring
rate was 1400 rpm [4,5,17]. rate was 1400 rpm [4,5,17]. The process-simulation studies took place under constant reaction conditions for the
comparison between the two catalysts to assess their performance for the reaction. From
Table 2, it can be observed that, under constant reaction conditions, the performance of
the novel Pd/Zn catalyst is significantly higher than that of the commercial Pd/C catalyst
and of the monometallic counterpart, underlining the role of Zn in the catalytic process. As a result, the novel Pd/Zn catalyst could be utilised to enhance the decomposition
of formic acid reaction. Furthermore, there is good agreement (less than 5% difference)
between the process-simulation modelling and experimental data. Therefore, the theoretical
models created in this study have the ability to successfully predict the decomposition of
formic acid under mild conditions. A gas analysis was performed on the system using Appl. Sci. 2021, 11, 8462 10 of 12 10 of 12 an online micro-GC in order to calculate CO/CO2 ratio, and a selectivity of 76% for the
dehydrogenation reaction was obtained. Table 2. Comparison between 2% Pd6Zn4, 1% Pd on nanofibers and 5 wt.% Pd on carbon. Reaction
conditions: substrate/metal molar ratio: 2000:1, inlet [HCOOH] = 0.5 M, 1400 rpm 1% Pd/HHT and
Pd6Zn4, 2 h reaction time. Table 2. Comparison between 2% Pd6Zn4, 1% Pd on nanofibers and 5 wt.% Pd on carbon. Reaction
conditions: substrate/metal molar ratio: 2000:1, inlet [HCOOH] = 0.5 M, 1400 rpm 1% Pd/HHT and
Pd6Zn4, 2 h reaction time. p
6
4
conditions: substrate/metal molar ratio: 2000:1, inlet [HCOOH] = 0.5 M, 1400 rpm 1% Pd/HHT and
Pd6Zn4, 2 h reaction time. X (%)
Time (min)
2% Pd6Zn4
Model
2% Pd6Zn4
Exp
5% Pd/C
Model
5% Pd/C Exp
1% Pd Exp
0
0
0
0
0
0
5
5
5
3
5
4
15
15
17
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31 4. Conclusions 2021, 11, 8462 Conflicts of Interest: The authors declare no conflict of interest. References 1. Choi, B.-S.; Song, J.; Song, M.; Goo, B.S.; Lee, Y.W.; Kim, Y.; Yang, H.; Han, S.W. Core–shell engineering of Pd–Ag bimetallic
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Pt-Pd catalyst in the presence of formic acid. Chem. Eng. J. 2020, 398, 125541. 4. Conclusions The decomposition of formic acid has been investigated using a novel 2%Pd6Zn4
catalyst in a batch reactor. The studies were conducted experimentally, and a process-
simulation model was developed to validate the results. It was found that the conversion of
formic acid increases with temperatures in the range of 30–60 ◦C. Temperatures greater than
60 ◦C are not typically investigated due to the requirement of mild operating conditions
for fuel cell applications. Furthermore, the formic acid conversion can be enhanced by
increasing the concentration of formic acid in the batch system. For all of the parameter
studies performed, the catalyst displayed no loss of activity. p
y
p y
y
The experimental and modelling data obtained for the 2%Pd6Zn4 catalyst were com-
pared with a commercial Pd/C catalyst. It was found that the novel catalyst demonstrated
significantly higher conversions than the commercial catalyst under constant reaction
conditions. Furthermore, the theoretical models developed to predict the decomposition
of formic acid for both catalysts depicted sound validation with the experimental data,
highlighting the importance of utilising computational software to further facilitate the
design of experimental studies. The process-simulation-modelling methodology offered in
this study can potentially offer shorter computational times and requires less computing
power when compared with CFD modelling. Future research could be directed towards
implementing the catalyst in a continuous flow system to allow for constant generation of
hydrogen for fuel cell applications. Author Contributions: Conceptualization, A.C., A.V. and N.D.; methodology, I.B., S.H., A.C., A.V. and N.D.; software, S.H., A.C. and G.M.; theoretical validation, S.H., G.M., S.M.A.-S. and A.C.;
Formal analysis, I.B., A.V., N.D., X.C., J.J.D., S.H., G.M., and A.C., investigation, I.B., A.V., N.D.,
S.H. and A.C.; resources, I.B., A.V.,S.H., X.C., J.J.D. and A.C.; writing—original draft preparation,
S.H. and I.B.; writing—review and editing, S.H., I.B., A.V., N.D., X.C., J.J.D., G.M., S.M.A.-S. and
A.C.; visualization, S.H., I.B., A.C., A.V., G.M., N.D.; supervision, A.C., A.V., S.M.A.-S., G.M. project
administration, A.C. and A.V.; funding acquisition, A.V. and A.C. All authors have read and agreed
to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Exlude this statement. Data Availability Statement: Exlude this statement. Acknowledgments: The authors thank London South Bank University; School of Engineering for
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Serological evidence of single and mixed infections of Rift Valley fever virus, Brucella spp. and Coxiella burnetii in dromedary camels in Kenya
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Copyright for the publications made accessible via the Edinburgh Research Explorer is retained by the author(s)
and / or other copyright owners and it is a condition of accessing these publications that users recognise and
abide by the legal requirements associated with these rights. Link: Link:
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PLoS Neglected Tropical Diseases Edinburgh Research Explorer
Serological evidence of single and mixed infections of Rift Valley
fever virus, Brucella spp. and Coxiella burnetii in dromedary
camels in Kenya Citation for published version:
Muturi, M, Akoko, J, Nthiwa, D, Chege, B, Nyamota, R, Mutiiria, M, Maina, J, Thumbi, SM, Nyamai, M,
Kahariri, S, Sitawa, R, Kimutai, J, Kuria, W, Mwatondo, A, Bett, B & Lin, T 2021, 'Serological evidence of
single and mixed infections of Rift Valley fever virus, Brucella spp. and Coxiella burnetii in dromedary
camels in Kenya', PLoS Neglected Tropical Diseases, vol. 15, no. 3, e0009275, pp. 1-12. https://doi.org/10.1371/journal.pntd.0009275 Citation for published version:
Muturi, M, Akoko, J, Nthiwa, D, Chege, B, Nyamota, R, Mutiiria, M, Maina, J, Thumbi, SM, Nyamai, M,
Kahariri, S, Sitawa, R, Kimutai, J, Kuria, W, Mwatondo, A, Bett, B & Lin, T 2021, 'Serological evidence of
single and mixed infections of Rift Valley fever virus, Brucella spp. and Coxiella burnetii in dromedary
camels in Kenya', PLoS Neglected Tropical Diseases, vol. 15, no. 3, e0009275, pp. 1-12. https://doi.org/10.1371/journal.pntd.0009275 Citation for published version: Take down policy Take down policy
The University of Edinburgh has made every reasonable effort to ensure that Edinburgh Research Explorer
content complies with UK legislation. If you believe that the public display of this file breaches copyright please
contact openaccess@ed.ac.uk providing details, and we will remove access to the work immediately and
investigate your claim. Download date: 24. Oct. 2024 Serological evidence of single and mixed
infections of Rift Valley fever virus, Brucella spp.
and Coxiella burnetii in dromedary camels in
Kenya Mathew MuturiID1,2*, James Akoko2, Daniel Nthiwa3, Bernard Chege1,
Richard NyamotaID2, Mathew MutiiriaID1, Josphat Maina1,2, S. M. ThumbiID4,5,6,
Mutono NyamaiID4,6, Samuel KahaririID7, Rinah Sitawa8, Joshua Kimutai8, Wilson Kuria7,
Athman MwatondoID1,2, Bernard Bett2 a1111111111
a1111111111
a1111111111
a1111111111
a1111111111 a1111111111
a1111111111
a1111111111
a1111111111
a1111111111 1 Zoonotic Disease Unit Nairobi, Nairobi, Kenya, 2 International Livestock Research Institute, Nairobi,
Kenya, 3 Department of Biological Sciences, University of Embu, Embu, Kenya, 4 Center for Epidemiological
and Modelling Analysis, University of Nairobi Institute of Tropical and Infectious Diseases, Nairobi, Kenya,
5 Institute of Immunology and Infection Research, University of Edinburgh, Edinburgh, United Kingdom,
6 Paul G Allen School for Global Health, Washington State University, Pullman, Washington, United States of
America, 7 Kenya Directorate of Veterinary Services, Ministry of Agriculture, Livestock and Fisheries, Nairobi,
Kenya, 8 Food and Agriculture Organization of the United Nations, Nairobi, Kenya OPEN ACCESS Citation: Muturi M, Akoko J, Nthiwa D, Chege B,
Nyamota R, Mutiiria M, et al. (2021) Serological
evidence of single and mixed infections of Rift
Valley fever virus, Brucella spp. and Coxiella
burnetii in dromedary camels in Kenya. PLoS Negl
Trop Dis 15(3): e0009275. https://doi.org/10.1371/
journal.pntd.0009275 Abstract Camels are increasingly becoming the livestock of choice for pastoralists reeling from
effects of climate change in semi-arid and arid parts of Kenya. As the population of camels
rises, better understanding of their role in the epidemiology of zoonotic diseases in Kenya is
a public health priority. Rift Valley fever (RVF), brucellosis and Q fever are three of the top
priority diseases in the country but the involvement of camels in the transmission dynamics
of these diseases is poorly understood. We analyzed 120 camel serum samples from north-
ern Kenya to establish seropositivity rates of the three pathogens and to characterize the
infecting Brucella species using molecular assays. We found seropositivity of 24.2% (95%
confidence interval [CI]: 16.5–31.8%) for Brucella, 20.8% (95% CI: 13.6–28.1%) and 14.2%
(95% CI: 7.9–20.4%) for Coxiella burnetii and Rift valley fever virus respectively. We found
27.5% (95% CI: 19.5–35.5%) of the animals were seropositive for at least one pathogen and
13.3% (95% CI: 7.2–19.4%) were seropositive for at least two pathogens. B. melitensis was
the only Brucella spp. detected. The high sero-positivity rates are indicative of the endemic-
ity of these pathogens among camel populations and the possible role the species has in
the epidemiology of zoonotic diseases. Considering the strong association between human
infection and contact with livestock for most zoonotic infections in Kenya, there is immediate
need to conduct further research to determine the role of camels in transmission of these
zoonoses to other livestock species and humans. This information will be useful for design-
ing more effective surveillance systems and intervention measures. * muturimathew@gmail.com Editor: Tao Lin, Baylor College of Medicine,
UNITED STATES Received: November 13, 2020
Accepted: February 26, 2021
Published: March 26, 2021
Copyright: © 2021 Muturi 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. Received: November 13, 2020
Accepted: February 26, 2021
Published: March 26, 2021 Copyright: © 2021 Muturi 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. Introduction The emergence of the Middle East respiratory syndrome coronavirus (MERS-CoV) in 2012
and the subsequent detection of the virus in dromedary camels, brought into focus the role of
the species as potential reservoirs of endemic and emerging zoonotic diseases in Kenya [1–4]. As the population of camels and their contribution to food security and economy of pastoral-
ists in the country continues to rise, understanding this role is a public health priority [5–7]. The burden and transmission dynamics of zoonotic diseases in Kenyan camels are poorly
understood. This has led to the perception of low burden of camel diseases, the consequence of
which is continued neglect of the camel industry in resource allocation for disease control and
research [8, 9]. Previous studies have identified camels as reservoirs of three of the top priority zoonotic
pathogens in Kenya; Brucella spp, Rift Valley fever virus (RVFv) and Coxiella burnetii, but data
on infection patterns of these pathogens, and potential co-infections in camels is limited [2, 8,
10]. Serological studies, however, show that camels have high seroprevalences and may play an
important role in the epidemiology of the three pathogens [2, 11]. Camels have been reported
to have the highest Brucella and Coxiella burnetii infection rates among Kenyan livestock, with
one study reporting more than a two-fold increase in Brucella prevalence in livestock herds
with camels versus those with none [8, 12, 13]. Similarly, camels play an important but under-
appreciated role in the epidemiology of RVFV, one of the pathogens with the highest exposure
levels in Kenyan camels [8]. In addition to the high prevalence rates, high camel mortality
rates during RVF outbreaks in Kenya and the association of camels with confirmed human
RVF cases are indicative of role of camels in the epidemiology of the virus [14–16]. Co-exposure with multiple pathogens has been shown to alter host-pathogen interactions
(within and between host pathogen dynamics) which in turn may affect clinical consequences,
transmission dynamics and response to prevention and control measures [17–19]. Whereas,
advances in diagnostic techniques have increased interest in understanding pathogen interac-
tions, most epidemiological studies in livestock focus on single-pathogen exposures despite the
fact that most zoonotic diseases present with similar clinical symptoms and have the same risk
factors [20]. Although presence of antibodies for a given pathogen indicates exposure and not necessary
active infections, it is an integral component of disease risk assessment. Author summary Dromedary camels are well adapted to the arid and semi-arid environment that makes up
about 80% of Kenya’s landmass. As such, camels play an important role in the socio-eco-
nomic wellbeing and food security of pastoralists in the country. However, the species
remains relatively neglected in scientific research, one of the main reasons being camels
are mostly found in remote, low-income, arid regions of Africa and Asia. We carried out a
study to determine the levels of exposure of camels in northern Kenya to Brucella spp.,
Coxiella burnetii and Rift Valley fever virus, three priority zoonotic pathogens in the coun-
try. We found high levels of exposure to the three pathogens, indicating the important
role camels might play in the epidemiology of the zoonotic diseases in humans and other
livestock. Based on the study findings, we argue for the immediate need for investments
in disease surveillance and control strategies for priority zoonotic disease in camels in
Kenya. Competing interests: The authors have declared
that no competing interests exist. Competing interests: The authors have declared
that no competing interests exist. PLOS NEGLECTED TROPICAL DISEASES Rift Valley fever virus, Brucella spp. and Coxiella burnetii infection in Kenyan camels the Food and Agriculture Organization of the United
Nations. The funders had no role in study design,
data collection and analysis, decision to publish, or
preparation of the manuscript. Data Availability Statement: All relevant data are
within the manuscript and its Supporting
Information files. Data Availability Statement: All relevant data are
within the manuscript and its Supporting
Information files. Funding: This study was part of the project: Co-
infection with Rift Valley fever virus, Brucella spp
and Coxiella burnetii in humans and animals in
Kenya: Disease burden and ecological factors that
was funded by the United States Defense Threat
Reduction Agency (GRANT 12686246_R, to BB). Additional funding for field work was provided by 1 / 12 PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0009275
March 26, 2021 PLOS NEGLECTED TROPICAL DISEASES Sample collection Ten millimeters of blood was collected into plain vacutainer tubes from each animal through
jugular venipuncture. Serum was then extracted through centrifugation at the laboratories
located within county veterinary offices. After serum separation, samples were transferred into
2mls cryovials, labeled and transported in a motorized cool box at 4oc to the International
Livestock research Institute, Nairobi for laboratory analysis and testing. Study data was col-
lected electronically in Epi-Info (CDC Atlanta, Georgia, USA). Standardized and pre-tested
questionnaires were uploaded to smart phones and administered to key informants; primarily
camel owners and herders, to collect data on herd and animal level information such as age
and sex. Study area We utilized 120 camel sera samples collected from 16 herds during an outbreak investigation
of a respiratory syndrome of unknown aetiology in dromedary camels between 22nd and 28th
June 2020. Sera samples were collected from herds with history of at-least one camel present-
ing with a respiratory syndrome in the 3 months preceding the investigation. The investigation
was conducted in Isiolo and Samburu counties, both of which are semi-arid, pastoral regions
of northern Kenya (Fig 1). In Isiolo, sampling was conducted in Kinna and Burat wards, while
in Samburu camel samples were collected from Wamba West and Nyiro wards. Pastoralism,
characterized by transhumance movement is the predominant livestock production system in
both study areas. Cattle, goats, sheep and camels are the main species kept in the study area,
but goats and sheep form the bulk of livestock in the two counties. Isiolo and Samburu are also
home to several wildlife conservancies with significant population of free roaming wildlife and
human-animal-wildlife interaction. Ethics statement This study did not require ethical approvals from an institutional review board because it was
conducted as part of an outbreak investigation by the Kenya Ministry of Agriculture, Livestock
and Fisheries and Ministry of Health. However, full approval for the study was given by the
Director of Veterinary services in Kenya and local county-level Directors of Veterinary Ser-
vices in the study area. Although individual informed consent was not required for this investi-
gation, all data were handled as a de-identified set to protect farmers’ privacy and
confidentiality. Introduction In this study, we inves-
tigated the seroprevalences of RVFV, Brucella spp and Coxiella burnetii and characterized 2 / 12 PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0009275
March 26, 2021 PLOS NEGLECTED TROPICAL DISEASES Rift Valley fever virus, Brucella spp. and Coxiella burnetii infection in Kenyan camels Brucella species using molecular techniques in camels from northern Kenya counties of Isiolo
and Samburu. Laboratory procedure All serum samples were tested in duplicates for Brucella, RVFV and Coxiella burnetii antibod-
ies using ID screen Brucellosis Serum Indirect ELISA Multispecies kits (IDvet innovative diag-
nostics, France), ID screen Rift Valley Fever Competition Multispecies ELISA kits (IDvet
innovative diagnostics, France) and Q fever (Coxiella burnetii) Antibody test kit (IDEXX Labo-
ratories, Westbrook ME, USA) respectively. Samples that tested positive for Brucella on ELISA
test were further subjected to two PCR assays to detect the genus Brucella and a speciation
assay to identify Brucella species. All the testing was done according to manufacturer standard
operating procedures. 3 / 12 PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0009275
March 26, 2021 PLOS NEGLECTED TROPICAL DISEASES Rift Valley fever virus, Brucella spp. and Coxiella burnetii infection in Kenyan camels Fig 1. Map of the study area showing the sampled wards (in orange). Shape file used from: https://africaopendata.org/dataset/kenya-counties/shapefile/resource/
0b78f25e-494e-4258-96b8-a3ab2b35b121. Fig 1. Map of the study area showing the sampled wards (in orange). Shape file used from: https://africaopendata.org/dataset/kenya-counties/shapefile/resource/
0b78f25e-494e-4258-96b8-a3ab2b35b121. https://doi.org/10.1371/journal.pntd.0009275.g001 https://doi.org/10.1371/journal.pntd.0009275.g001 Based on the manufacturer’s instruction, the results were interpreted as negative if the cal-
culated S/P% was <30%, positive if the S/P was 40% and doubtful if the S/P was in between
30 and 40%. Real time PCR detection of Brucella spp. DNA extraction from serum samples. Total DNA was extracted from all the 32 Brucella
sero-positive samples using QIAamp DNA Blood Mini Kit, while following the manufacturer’s
instructions. Briefly, 20 μl of proteinase K solution, 200μl of the serum sample and lysis buffer
were added into a sterile 1.5ml Eppendorf tube, vortexed and incubated at 56˚C for 10 min-
utes. Absolute ethanol (200μl) was then added into the mixture and vortexed before applying
the lysate onto a QIAamp spin column. The columns were centrifuged at 8000 rpm for one
minute followed by a two wash steps using 500μl for each buffer AW1 and AW2 respectively. Centrifugation with buffer AW1 was done at 8000 rpm for 1 minute while AW2 was done at
maximum speed for 3 minutes. The DNA was eluted from the spin column using buffer 70μl
AE and centrifugation at 8000 rpm for one minute. Eluted DNA was spectrophotometrically PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0009275
March 26, 2021 4 / 12 PLOS NEGLECTED TROPICAL DISEASES Rift Valley fever virus, Brucella spp. and Coxiella burnetii infection in Kenyan camels checked for quality and quantified using NanoDrop 1000 UV-Vis spectrophotometer (Thermo
Scientific, Waltham, MA, USA) and stored at -20˚C until when required for analysis. Real-time PCR. Real-time PCR targeting IS711 gene was done to detect the genus Bru-
cella in the extracted DNA samples using oligonucleotide primers and probes (S1 Table). The
samples were considered PCR-positive for Brucella if they had a genus-specific amplification
with a thresh hold value (<40) in one or all the duplicate samples. The genus PCR-positive
samples were subsequently subjected to a multiplex speciation assay with B. abortus, B. meli-
tensis and B. suis species-specific oligonucleotide primers and probes. For both genus and spe-
cies-specific PCR assays, the test samples were considered positive when they had an
amplification with cycle threshold (Ct) values <40 for either or both duplicates (S1 Text). Statistical analysis. Field and laboratory data were stored in Microsoft Excel files. They
were imported to R statistical computing environment [21] for descriptive and statistical anal-
ysis. Variables that were available for analysis included (i) three dependent variables that repre-
sented exposure states of the three pathogens: RVFV, Brucella spp. and Coxiella burnetii,
recorded as either positive or negative, and two independent factors—age and sex of an ani-
mal. Herd and animal identification numbers were used for merging the data. Real time PCR detection of Brucella spp. Seroprevalence for each of the three pathogens and their 95% confidence intervals were
estimated using binom.confint function. An asymptotic method of estimating these intervals
was specified for all the tests. Chi-square or Fisher’s exact test (where values were less than 10)
were used to assess the significance of statistical associations between independent variables—
age and sex and the dependent variables. Additional dependent variables that represented co-
occurrence of two or all the target pathogens were also generated and used in similar analyses
[22]. Univariable mixed-effect logistic regression models were fitted to data with age and sex
being used as predictors in turns. Variables that were statistically significant (P<0.05) in the
univariable analyses were then used in mixed effects logistic regression model that had herd as
a random effect. Where age and sex were present and significant, their interaction terms were
included and tested for their significance using likelihood ratio tests. The Akaike’s Information
Criterion (AIC) was used to analyze the goodness of fit of the model. The data is presented in
tables and plots. We used a sjstats package to generate Intracluster Correlation Coefficient
(ICC), which measures the degree of correlation of prevalence estimates within herds after
running the mixed effects model [23]. Intracluster correlation coefficient is a measure of vari-
ability of infection between herds. Results Filling this knowledge gap in Kenya; a country
with one of the largest camel population globally, is not only key in the economic development
and growth of the camel industry, but in reducing the burden of zoonotic diseases in the coun-
try [5]. Brucellosis is one of the most important, yet understudied zoonoses in Kenya [26–28]. Our
study found more than one in every five camels had been exposed to Brucella spps., which con-
forms to reported camel brucellosis prevalence range (of up-to 40%) in the East Africa region
[26 29] Studies conducted in areas of Kenya with similar agro ecological characteristics and
Table 2. Final multivariable mixed effect model of association between select factors with exposure to one or more pathogens with random effects for herd ID. Variable and category
Targeted pathogens and their levels of co-exposure among sampled animals
Brucella spp. Coxiella burnetii
RVFV
Brucella spp. + RVFV
Brucella spp. +
Coxiella burnetii
Coxiella burnetii
+ RVFV
Odds Ratio
(95% CI)
P
value
Odds Ratio
(95% CI)
P value Odds Ratio
(95% CI)
P
value
Odds Ratio
(95% CI)
P
value
Odds Ratio
(95% CI)
P
value
Odds Ratio
(95% CI)
P
value
Fixed Effect
Animal sex
Male
1 (Ref.)
I (Ref.)
1 (Ref.)
1 (Ref.)
1 (Ref.)
1 (Ref.)
Female
0.7 (2.6–
1.9)
0.499
1.8 (0.6–
5.5)
0.278
42.6 (1.0–
1789.5)
0.049
1.3 (0.1–
22.2)
0.838
7.0 (0.5–
93.8)
0.143
2.0 (0.1–
29.0)
0.604
Age
<5 years
1 (Ref.)
1 (Ref.)
1 (Ref.)
1 (Ref.)
1 (Ref.)
1 (Ref.)
>5 years
1.6 (0.6–
4.3)
0.388
1.9 (0.7–
4.7)
0.200
13.1 (1.0–
174.3)
0.052
7.1 (0.6–
77.4)
0.108
9.4 (1.3–
69.8)
0.028
10.2 (0.2–
457.2)
0.220
Variance associated with
random effects
0.77
0.019
0.03
<0.001
14.68
0.005
73.03
0.032
1.67
0.002
5.96
0.034
The intra-herd correlation
coefficient (ICC) for herd ID
0.19
0.01
0.82
0.96
0.26
0.64
https://doi.org/10.1371/journal.pntd.0009275.t002 Table 2. Final multivariable mixed effect model of association between select factors with exposure to one or more pathogens with random effects for herd ID. Variable and category
Targeted pathogens and their levels of co-exposure among sampled animals
Brucella spp. Coxiella burnetii
RVFV
Brucella spp. + RVFV
Brucella spp. Results +
Coxiella burnetii
Coxiella burnetii
+ RVFV
Odds Ratio
(95% CI)
P
value
Odds Ratio
(95% CI)
P value Odds Ratio
(95% CI)
P
value
Odds Ratio
(95% CI)
P
value
Odds Ratio
(95% CI)
P
value
Odds Ratio
(95% CI)
P
value
Fixed Effect
Animal sex
Male
1 (Ref.)
I (Ref.)
1 (Ref.)
1 (Ref.)
1 (Ref.)
1 (Ref.)
Female
0.7 (2.6–
1.9)
0.499
1.8 (0.6–
5.5)
0.278
42.6 (1.0–
1789.5)
0.049
1.3 (0.1–
22.2)
0.838
7.0 (0.5–
93.8)
0.143
2.0 (0.1–
29.0)
0.604
Age
<5 years
1 (Ref.)
1 (Ref.)
1 (Ref.)
1 (Ref.)
1 (Ref.)
1 (Ref.)
>5 years
1.6 (0.6–
4.3)
0.388
1.9 (0.7–
4.7)
0.200
13.1 (1.0–
174.3)
0.052
7.1 (0.6–
77.4)
0.108
9.4 (1.3–
69.8)
0.028
10.2 (0.2–
457.2)
0.220
Variance associated with
random effects
0.77
0.019
0.03
<0.001
14.68
0.005
73.03
0.032
1.67
0.002
5.96
0.034
The intra-herd correlation
coefficient (ICC) for herd ID
0.19
0.01
0.82
0.96
0.26
0.64
https://doi.org/10.1371/journal.pntd.0009275.t002 sociation between select factors with exposure to one or more pathogens with random effects for herd ID. T
d
h
d h i l
l
f
l d
i
l Brucella had the highest seropositivity rate of the three pathogens at 24.2% (95% confidence
interval [CI]: 16.5–31.8%), followed by Coxiella burnetii at 20.8% (95% CI: 13.6–28.1%) and
RVFV at 14.2% (95% CI: 7.9–20.4%). Co-exposure to two or more pathogens was detected in
15.0% (95% CI: 8.6–21.4%; n = 18) of the samples. Up to 27.5% (95% CI: 19.5–35.5%) of the
animals were seropositive for at least one pathogen and 13.3% (95% CI: 7.2–19.4%) were sero-
positive for at least two pathogens. Of those with co-exposures, 92% (n = 22) had 2 co-expo-
sures detected, the rest of the samples (n = 2) had antibodies to all three pathogens. Co-
exposure with Brucella species for both RVFV and Coxiella burnetii were the most common
detections. On multivariate analysis, age was found to be significantly associated with co-expo-
sure to Brucella and Coxiella as shown in Table 2. Identification of Brucella species in camels Thirty-two Brucella (including three borderline positive samples not included in calculation of
seropositivity) sero-positive samples were run through PCR for DNA detection. Half of these
samples were positive for Brucella on PCR (n = 16). Of these, five samples (32%) were positive
for Brucella melitensis. Eleven of 16 samples positive for Brucella genus on PCR could not be
confirmed by the Brucella species PCR. Results We analyzed 120 samples collected from 16 camel herds with no history of vaccination against
brucellosis, Coxiella and RVFV. Majority of the camel samples were from females (72%,
n = 86) and camels aged above five years (49%, n = 49) as shown in Table 1. Table 1. Age and sex distribution of sampled camels. Sex
Age in years
% (n)
Female
<5
33% (39)
>5
39% (47)
Male
<5
27% (32)
>5
2% (2)
https://doi.org/10.1371/journal.pntd.0009275.t001 Table 1. Age and sex distribution of sampled camels. PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0009275
March 26, 2021 5 / 12 s://doi.org/10.1371/journal.pntd.0009275
March 26, 202 PLOS NEGLECTED TROPICAL DISEASES Rift Valley fever virus, Brucella spp. and Coxiella burnetii infection in Kenyan camels Brucella had the highest seropositivity rate of the three pathogens at 24.2% (95% confidence
interval [CI]: 16.5–31.8%), followed by Coxiella burnetii at 20.8% (95% CI: 13.6–28.1%) and
RVFV at 14.2% (95% CI: 7.9–20.4%). Co-exposure to two or more pathogens was detected in
15.0% (95% CI: 8.6–21.4%; n = 18) of the samples. Up to 27.5% (95% CI: 19.5–35.5%) of the
animals were seropositive for at least one pathogen and 13.3% (95% CI: 7.2–19.4%) were sero-
positive for at least two pathogens. Of those with co-exposures, 92% (n = 22) had 2 co-expo-
sures detected, the rest of the samples (n = 2) had antibodies to all three pathogens. Co-
exposure with Brucella species for both RVFV and Coxiella burnetii were the most common
detections. On multivariate analysis, age was found to be significantly associated with co-expo-
sure to Brucella and Coxiella as shown in Table 2. Identification of Brucella species in camels
Thirty-two Brucella (including three borderline positive samples not included in calculation of
seropositivity) sero-positive samples were run through PCR for DNA detection. Half of these
samples were positive for Brucella on PCR (n = 16). Of these, five samples (32%) were positive
for Brucella melitensis. Eleven of 16 samples positive for Brucella genus on PCR could not be
confirmed by the Brucella species PCR. Discussion
The historical neglect of camels and the perception of the species as hardy and resistant to dis-
eases has contributed to limited understanding of their role in the epidemiology of zoonotic
pathogens relative to other livestock [8, 24, 25]. Discussion However, stud-
ies in camels in Central Kenya found high Coxiella burnetii positivity rates of between 18.6% -
46%, which is in the range of our 21% prevalence finding [12, 13, 40]. The high Coxiella preva-
lence in camels is a source of public health concern because of the strong association between
Coxiella camel positivity and human infection [41, 42]. Additionally, the high exposure rates
indicate camels might play an important role as reservoirs of Coxiella in mixed livestock herds
[42–44]. Outbreaks of Rift valley fever have been frequently reported in Kenya since the first detec-
tion of the virus in 1931[45, 46]. Traditionally, cattle, sheep and goats are considered to be the
species of interest during RVF outbreaks but emerging evidence shows camels play a signifi-
cant epidemiological role during epizootics [14, 47, 48]. Our study found a positivity rate of
14% which is lower than previously reported rates of 57% and 21% from two studies conducted
in 2007 [15, 48]. Other studies in the region have found varying infection rates from 9.6% in
Sudan and 45% in Tanzania [49, 50]. The differences in infection rates can be attributed to dif-
ferences in agro-climatic zones, one of the drivers of RVF infection and different sampling
periods; epidemic versus interepidemic period [51, 52]. Our study found a strong association
between Brucella and Coxiella co-exposure exposure and camels above five years. This can be
explained by the increased likelihood of exposure to the two pathogens over time due to their
endemicity [14, 53]. Intra-herd correlation coefficient values are key in calculating sample
sizes for multistage sampling [54]. The ICC for RVFv infection was the highest of the three
pathogens. This can be inferred to mean RVFv infection within a camel herd is highly corre-
lated. This can be explained by the fact that RVF is a vector borne disease that occurs in explo-
sive outbreaks in very specific ecologies, meaning camels in such areas have much higher
exposure rates than those that are not [55]. The persistence of RVFv antibodies in camels com-
pared to the two other pathogens, a hypothesis supported by the fact that age was significantly
associated with RVFv exposure in our study, is also a plausible explanation for the high ICC
estimate within herds. Discussion The historical neglect of camels and the perception of the species as hardy and resistant to dis-
eases has contributed to limited understanding of their role in the epidemiology of zoonotic
pathogens relative to other livestock [8, 24, 25]. Filling this knowledge gap in Kenya; a country
with one of the largest camel population globally, is not only key in the economic development
and growth of the camel industry, but in reducing the burden of zoonotic diseases in the coun-
try [5]. Brucellosis is one of the most important, yet understudied zoonoses in Kenya [26–28]. Our
study found more than one in every five camels had been exposed to Brucella spps., which con-
forms to reported camel brucellosis prevalence range (of up-to 40%) in the East Africa region
[26, 29]. Studies conducted in areas of Kenya with similar agro-ecological characteristics and 6 / 12 PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0009275
March 26, 2021 PLOS NEGLECTED TROPICAL DISEASES Rift Valley fever virus, Brucella spp. and Coxiella burnetii infection in Kenyan camels animal husbandry practices in 2012 and 2014, found positivity rates of 11% (ELISA) and
6–18% (serum agglutination test) respectively [30, 31]. The high camel brucellosis prevalence
rates in northern Kenya are indicative of the high risk of human infection and interspecies
transmission [9, 32]. In literature, camels are reported to be susceptible to B. melitensis and B. abortus [33–35]. In our study however, we only detected B. melitensis after testing for B. abor-
tus, B.suis and B. melitensis. The detection of only B. melitensis is in agreement with similar
reports in literature that surmise that B. melitensis is more prevalent in African and Middle
East camels than B. abortus [26, 36]. A plausible explanation is that camels are generally herded
together with goats as opposed to cattle because of better adaptation of the former to harsher
climates; primarily less water requirement, ability to walk longer distances and well adapted
dietary preferences [36, 37]. However, our finding should not be deduced to mean that B. meli-
tensis does not affect Kenyan camels, but rather the need for comprehensive studies on the
molecular epidemiology of brucellosis in Kenyan camels [26, 38]. Data on the burden of Q fever in northern Kenyan camels is scarce [8, 39]. Discussion ICC is an important variable in the calculation of a design effect, a cor-
rection factor applied to sample sizes to correct for errors inherent from using cluster sam-
pling, however information on ICCs for most livestock diseases in developing countries are
unknown. Given our ICC estimates for exposures and co-exposures with RVFv were higher
than the 0.2 value assumed for most infectious diseases, there is need for further investigation
and to reconsider the outcome for estimation of ICC in camels [56–58]. Co-exposure with multiple pathogens is a common phenomenon in nature [19]. This is
especially true for livestock in pastoral communities in Sub-Saharan Africa with no structured
disease control programs, a consequence of which is continuous exposure to endemic patho-
gens [18, 59]. Extrinsic (environmental) and intrinsic (host-specific) factors influence burden PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0009275
March 26, 2021 7 / 12 PLOS NEGLECTED TROPICAL DISEASES Rift Valley fever virus, Brucella spp. and Coxiella burnetii infection in Kenyan camels and consequence of co-infections, but these are poorly understood for most endemic livestock
diseases in sub-Saharan Africa [59, 60]. Although the outcomes of in vivo pathogen interac-
tions are poorly understood, it is known co-infections can result in change in severity of infec-
tion, alteration of transmission dynamics and variation in physiologic response which can
change effectiveness of diagnostic techniques, control and prevention efforts [18, 53, 61, 62]. Studies on the outcomes of co-infection in livestock provide evidence of antagonistic effects of
co-infection on the host. An example is the Th1 and Th2 immunity response to Toxoplasma
gondii and nematode infection or in Trypanomosa cruzi and helminth infections where the
former activates Th1 immune response pathways and the latter the Th2 pathways [20, 63]. The
three pathogens of interest in the study can affect the immune system; Brucella species and
Coxiella burnetii can infect macrophages while RVFv is can affect monocytes [55, 64–67]. Live-
stock reared under pastoral production system are highly vulnerable to co-exposure to patho-
gens due to high population densities, widespread movement and frequent contact from
sharing pasture and watering points [42, 68]. Our study evaluated co-exposures with the three
pathogens in camels as a start to understanding burden of mixed infection and possible out-
comes. S1 Data. Database used in the investigation.
(CSV) S1 Data. Database used in the investigation. (CSV) Discussion This is important because zoonotic spillover pathways and clinical presentation of the
three infections are very similar and understanding these co-exposures can inform surveillance
strategies and integrated disease control and prevention plans in the long term. Acknowledgments The authors wish to thank the camel keeping communities of isiolo and Samburu without
whom the study would not have been possible, the County governments of Isiolo and Samburu
for the logistical support, the Kenya Directorate of Veterinary Services and the Kenya Field
Epidemiology and Laboratory Training Program for the technical support. Conclusion This study provides evidence of high exposure rates to the three priority zoonotic pathogens in
camels in northern Kenya. Considering the increasing value of camels in food nutrition for
pastoralists in Kenya, there is need for more research investment to understand the role of
camels in the epidemiology of these diseases. Because the endemicity of these diseases is
already established, further research should focus on robust studies to estimate the incidence,
transmission between livestock species and from animals to humans, and maintenance hosts
for these zoonotic infections to guide future control and elimination programs. S1 Table. Oligonucleotide primers and probes.
(DOCX) S1 Text. Supplementary material showing amplification plots for different assays run to
identify Brucella abortus, B. melitensis and B. Suis. (DOCX) Author Contributions Conceptualization: Mathew Muturi, Bernard Chege, Mathew Mutiiria, Josphat Maina, Sam-
uel Kahariri, Wilson Kuria, Bernard Bett. 8 / 12 PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0009275
March 26, 2021 PLOS NEGLECTED TROPICAL DISEASES Rift Valley fever virus, Brucella spp. and Coxiella burnetii infection in Kenyan camels Data curation: Mathew Muturi, James Akoko, Daniel Nthiwa, Richard Nyamota, S. M. Thumbi, Mutono Nyamai, Bernard Bett. Data curation: Mathew Muturi, James Akoko, Daniel Nthiwa, Richard Nyamota, S. M. Thumbi, Mutono Nyamai, Bernard Bett. Formal analysis: Mathew Muturi, James Akoko, Daniel Nthiwa, S. M. Thumbi, Mutono Nya-
mai, Bernard Bett. Funding acquisition: Mathew Muturi, Athman Mwatondo, Bernard Bett. Investigation: Mathew Muturi, James Akoko, Daniel Nthiwa, Bernard Chege, Richard Nya-
mota, Mathew Mutiiria, Josphat Maina, S. M. Thumbi, Mutono Nyamai, Samuel Kahariri,
Rinah Sitawa, Joshua Kimutai, Wilson Kuria, Bernard Bett. Methodology: Mathew Muturi, James Akoko, Daniel Nthiwa, Bernard Chege, Richard Nya-
mota, Mathew Mutiiria, Josphat Maina, S. M. Thumbi, Mutono Nyamai, Samuel Kahariri,
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Design of Interactions for Handheld Augmented Reality Devices Using Wearable Smart Textiles: Findings from a User Elicitation Study
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Received: 27 June 2019; Accepted: 31 July 2019; Published: 5 August 2019 Abstract:
Advanced developments in handheld devices’ interactive 3D graphics capabilities,
processing power, and cloud computing have provided great potential for handheld augmented
reality (HAR) applications, which allow users to access digital information anytime, anywhere. Nevertheless, existing interaction methods are still confined to the touch display, device camera,
and built-in sensors of these handheld devices, which suffer from obtrusive interactions with AR
content. Wearable fabric-based interfaces promote subtle, natural, and eyes-free interactions which
are needed when performing interactions in dynamic environments. Prior studies explored the
possibilities of using fabric-based wearable interfaces for head-mounted AR display (HMD) devices. The interface metaphors of HMD AR devices are inadequate for handheld AR devices as a typical
HAR application require users to use only one hand to perform interactions. In this paper, we aim to
investigate the use of a fabric-based wearable device as an alternative interface option for performing
interactions with HAR applications. We elicited user-preferred gestures which are socially acceptable
and comfortable to use for HAR devices. We also derived an interaction vocabulary of the wrist and
thumb-to-index touch gestures, and present broader design guidelines for fabric-based wearable
interfaces for handheld augmented reality applications. Keywords: augmented reality; handheld AR displays; mobile devices; fabric-based interface;
interaction design; user-elicitation; smart clothing applied
sciences applied
sciences Design of Interactions for Handheld Augmented
Reality Devices Using Wearable Smart Textiles:
Findings from a User Elicitation Study Vijayakumar Nanjappan 1
, Rongkai Shi 1, Hai-Ning Liang 1,*
, Haoru Xiao 1,
Kim King-Tong Lau 2 and Khalad Hasan 3 1
Department of Computer Science and Software Engineering, Xi’an Jiaotong-Liverpool Universi
Suzhou 215123, China
2
Department of Chemistry, Xi’an Jiaotong-Liverpool University, Suzhou 215123, China
3
Irving K. Barber School of Arts and Sciences, The University of British Columbia Okanagan,
Kelowna, BC V1V 1V7, Canada
*
Correspondence: HaiNing Liang@xjtlu edu cn; Tel : +86-512-8816-1516 1
Department of Computer Science and Software Engineering, Xi’an Jiaotong-Liverpool Univers
Suzhou 215123, China
2
Department of Chemistry, Xi’an Jiaotong-Liverpool University, Suzhou 215123, China
3
Irving K. Barber School of Arts and Sciences, The University of British Columbia Okanagan,
Kelowna, BC V1V 1V7, Canada
*
Correspondence: HaiNing Liang@xjtlu edu cn; Tel : +86-512-8816-1516 *
Correspondence: HaiNing.Liang@xjtlu.edu.cn; Tel.: +86-512-8816-1516
www.mdpi.com/journal/applsci 1. Introduction Augmented reality (AR) overlays computer-generated visual information—such as images,
videos, text information, and 3D virtual objects—onto the real-world [1]. Unlike virtual reality (VR),
which immerses the users inside a computer-generated environment, AR allows the users to see the
real world with virtual objects blended within the real environment and enables the users to interact the
virtual content in real-time [2]. The increasing computational capabilities, powerful graphics processors,
built-in cameras, variety of sensors, and power of cloud-supported information access have made AR
possible using handheld mobile devices, such as smartphones and tablets [3]. The evolution of AR
technology—from the heavy wearable head-mounted display (HMD) devices to smartphones and
tablets—has enabled new and rich handheld mobile AR experiences for everyday users. In handheld
augmented reality (HAR), users can see the real world overlaid with virtual information in real-time
by using the camera on their mobile devices. Current mobile devices are small in size, lightweight, Appl. Sci. 2019, 9, 3177; doi:10.3390/app9153177 www.mdpi.com/journal/applsci www.mdpi.com/journal/applsci Appl. Sci. 2019, 9, 3177 2 of 21 and portable enough to be carried wherever users go. With this ubiquitous availability, HAR allows us
to develop and design innovative applications in navigation, education, gaming, tourism, interactive
shopping, production, marketing, and others [3]. Thus, smartphones have been identified as an ideal
platform for HAR experiences in various outdoor and indoor environments [4–6]. In order to interact with the virtual world using HAR displays, a user needs to position and
orientate the device using one hand and manipulate the virtual 3D objects with the other hand. In general, the touchscreen is used as a primary interface to interact with AR content [7,8]. In addition,
the various built-in sensors in the handheld devices—such as cameras, GPS, compass, accelerometers,
and gyroscope—enable to precisely determine the position and orientation of the device in the real
world (e.g., [8–10]). Furthermore, the device’s camera is used to naturally capture the user’s mid-air
hand movements while holding the device [11,12]. Like in HMD AR, manipulations such as selecting and moving virtual 3D information are
primary interactions in HAR devices [13]. The existing HAR interaction methods, such as touch
input, offer promising solutions to manipulate virtual content (e.g., [14]). However, they still have
substantial limitations. For instance, touch input is limited by the device’s physical boundary and
usability suffers as on-screen content becomes occluded by finger (i.e., finger occlusions [15,16]). 1. Introduction Also,
2D inputs on the touch surface do not directly support manipulating the six degrees of freedom of a
virtual object in the augmented world [17]. Unlike touch-based input, device movement-based HAR
interaction methods support 3D object manipulations (e.g., [8,9]). However, orienting the device to
rotate the 3D virtual objects might force the users to lose sight of the manipulated virtual object in
the display. A mid-air hand gesture input method supports more natural and intuitive interaction
for HAR applications (e.g., [11,12]). Nevertheless, using the back-camera on the device to track the
hand and finger movements is often not feasible, due to several factors such as (1) it may not be very
accurate, (2) the device should be held at a certain distance from the user’s eye, and (3) the human arm
has a very limited region of reach. Furthermore, interactions with handheld devices also have issues,
like social acceptance, when performed in front of unfamiliar people [18]. With the recent developments in fabric-based technology and new types of electronic textiles and
smart clothing [19], we are witnessing Mark Weiser’s dream—of computers that “weave themselves
into the fabric of everyday life” [20] —has taken one step closer to reality. Integrated into users’ clothing,
fabric sensors are able to unobtrusively detect user movements [21] to convert them into input methods
to control modern devices (e.g., [22–24]). Fabric-based wearable devices are soft, natural, and flexible;
remove the need for the user to hold or carry a device; can be worn without any discomfort; and offer
more freedom of movements compared to the rigid sensors (e.g., accelerometer) (e.g., [25]). Recently,
researchers have started undertaking research on fabric-based interaction methods and attempting to
provide a more natural, unobtrusive end-user interaction experience (e.g., [24,26]). As stated by Hansson et al., an interface is considered as a “natural interface”, when it builds
upon the knowledge and experience that the user already possesses [27]. Humans are used to wear
clothes throughout their lives. In general, they do not need special training to wear or use clothes
or, in other words, to use a clothing-based wearable interface. Among the different body parts,
users’ hands enable various poses particularly important and suitable for gestural interaction for the
following reasons: (1) hands are always available, (2) support high proprioception, (3) require minimal
movement, (4) produce distinct subtle gestures, and (5) offer socially acceptable gestures. 2.1. Participatory Elicitation Studies An elicitation method involves potential users in the process of designing any new type of
interface or interaction. This approach allows designers to explore the possibilities of finding gestures
from the end-users’ perspective, which are often are not in the initial gesture set designed by the
designers. User-preferred gestures are easier to remember than expert-designed gestures [28]. To elicit
gestures from the users, an interface designer shows a result of an action for each task, known as
referents [29]. The designer then asks each participant to imagine and perform an action that would
cause each referent to occur. These user proposed actions are known as symbols [29]. The designer
compares all of the symbols for a given referent from each participant and groups them based on their
similarity. Wobbrock et al. [29] introduced a method to understand the agreement and other factors
associated with user-elicited gestures. Their method has been widely used in other elicitation studies
(e.g., [30–32]). Most recently, Vatavu and Wobbrock [33] have redefined the agreement measurement to
understand the data more precisely. This refined agreement formula and approach is applied in the
analysis of our results. In terms of user-elicited gestures for augmented reality, Piumsomboon et al. [34] presented a study
of user-defined hand gestures for head-mounted AR displays. Their participants elicited gestures
using both hands. By contrast, HAR devices are not head-worn and are often operated using only one
hand on the touchscreen. Thus, interface metaphors of HMD AR are not suitable for HAR displays. In terms of interactions based on single hand thumb-to-fingers, Chan et al. [35] presented a study of
user-elicited single hand gestures using two or more fingers. They reported that both thumb and index
fingers were highly involved in the interactions of their gesture set. Their study focused on the fingers
and did not include the wrist, which can provide richer interaction possibilities (e.g., [36,37]). The above studies do not use devices to elicit user-designed gestures. This approach enables
users to propose any gestures regardless of their feasibility of implementation and the affordance of a
device. Similarly, with their approach, the method of grouping the elicited gestures that look similar
significantly influences the magnitude of user agreement rates [38] as this process is often dependent on
the researcher’s goals for the specific domain for which gestures were elicited. 1. Introduction In this paper, we are particularly interested in exploring fabric-based interaction methods that
allow a user to use combined touch and gesture input to perform HAR interactions. To this end, we
explore the potential use of gestures performed via a hand-worn clothing-based artifact (similar to a
glove). Input based on textiles is not new (e.g., [22–25]), but their use has been directed primarily at
traditional mobile devices [24] and smartwatches [23]. To our best knowledge, there are no studies
that explored the combined use of hand and wrist gestures performed via a hand-worn device to
interact with HAR content. To fill this gap, our work investigates the use of clothing-based hand and
wrist gestures for HAR content. Our paper makes the following contributions: (1) an investigation of 3 of 21 Appl. Sci. 2019, 9, 3177 the potential design space of clothing-based gestures using the hand and wrist for HAR applications;
(2) identification of a set of user-defined gestures; and (3) a set of design guidelines for fabric-based
devices that allow users to interact with HAR content. 2.1. Participatory Elicitation Studies Therefore, by following
their method, interface designers are unable to recommend a suitable sensing technology to recognize
their user-elicited gestures. To address these issues, most recently, Nanjappan et al. [39] co-designed
a prototype with potential end-users and conducted an elicitation study for a fabric-based wearable
prototype to devise a taxonomy of wrist and touch gestures. Their non-functional prototype allowed
the users to physically feel the affordance of the interface to perform their preferred gestures that are
implementable in the functional version of the device. We adopted and extended their approach to
elicit both wrist and thumb-to-index touch gestures to perform interactions with HAR applications. 2.2. Hand-Worn Clothing-Based Interfaces The human hand is primarily used to interact and manipulate real-world objects in our everyday
life. Hence, it is not surprising that hand gestures, involving hands and fingers, are repeatedly studied
in interactive systems (e.g., [25,26,40,41]). Glove-based interfaces—the most popular systems for
capturing hand movements—started nearly thirty years ago, and are continuously attracting the
interest of a growing number of researchers. Glove-based input devices remove the requirement
of the user to carry or hold their device to perform interactions. They are implemented by directly
mounting different types of sensing technologies on the hand, mostly on each finger. The development Appl. Sci. 2019, 9, 3177 4 of 21 of fabric-based strain sensing technology allows strain sensors to be either sewed or attached to the
hand glove to detect the hand or finger movements [25,41,42]. Notably, Miller et al. [42] proposed a glove system to enter inputs using users’ thumb with two
approaches: (1) by tapping ring and little fingertips with the thumb or (2) by sliding the thumb over
the entire palmar surfaces of the index and middle fingers. Their proposed glove consists of both
conductive and non-conductive fabrics, supporting both discrete and continuous output. They used
two different approaches: (1) the palmar sides of both index and middle fingers are completely covered
with grid conductive threads woven in an over-and-under pattern and (2) pads of thumb, ring, and little
fingers are covered in conductive fabric. Typically, their glove-based input acts like a set of switches
which can be closed by making contact with the thumb. This approach allows the glove interface
to detect tap inputs on any fingertip and swipe gestures are performed on the conductive grids on
both index and middle fingers. While their approach sounds promising for single-handed eyes-free
interactions, the authors reported, however, that their sensing technology provided inaccurate results. Peshock et al. [25] presented “Argot”, a wearable one-handed keyboard glove which allows users to
type English letters, numbers, and symbols. Argot is made of multiple polyester or spandex blended
stretch knit and textile patches which provide breathability and mobility. It employs conductive thread
and textile conductive patches which improve user comfort and utilizes a simpler input language. The patches are installed on 15 different locations on finger pads (5 patches on each digit), fingernails
(4 patches except thumb), and sides of the index, middle, and ring fingers (2 patches on each finger side). 2.2. Hand-Worn Clothing-Based Interfaces In addition, it provides both tactile and auditory feedback through magnetic connection. A layering
system is developed to enclose the magnets within the conductive patches. All these materials are
insulated using fusible stitchless bonding film. However, the authors reported that the users were
always accidentally triggering the side buttons on the index, middle, and ring fingers while performing
gesture inputs. Thumb-to-finger interaction methods present a promising input mechanism that is suitable
to perform subtle, natural, and socially acceptable gestures. In particular, prior studies on hand
gestures [35,43] have found that both thumb and index fingers are preferred over the middle, ring,
and pinky fingers. As explained by Wolf et al. [43] the thumb is opposable and has the ability to
rotate and be able to touch other fingers on the same hand. Similarly, the muscles involved in moving
each finger and the biomechanics make the index finger well suited for independent movement [35]. In particular, Yoon et al. [24] proposed “Timmi”, a finger-worn piezoresistive fabric-based prototype
which supports thumb-to-finger multimodal input. Timmi comprises of elastic fabric (made of 80%
Nylon and 20% Spandex) painted with conductive elastomers and conductive threads. Diluted
conductive elastomer was used to transform a normal fabric into a piezoresistive fabric. It consists
of pressure and strain sensors made of using two different shapes and the conductive threads are
cross-stitched to control the fabric. The interface can be worn on a finger, especially the index finger;
the Spandex provides exceptional elasticity. Timmi supports the following input methods: finger bend,
touch pressure, and swipe gesture. This approach takes advantage of the digit-wise independence and
successfully uses the thumb-to-index touch input in a natural eyes-free manner. Similarly, wrist-worn interfaces are particularly suitable for performing natural, subtle,
and eyes-free interactions that can be both continuous and discrete [44] and support high
proprioception [45]. Particularly, Strohmeier et al. [46] proposed “WristFlicker”, which detects
wrist movements using a set of conductive polymer cable as strain sensors embedded in wrist-worn
clothing (e.g., a wrist warmer). They employed three sets of two counteracting strain sensors to
precisely measure the flexion/extension, ulnar/radial deviation, and pronation/supination of the human
wrist. WristFlicker supports both discrete and continuous input, and can be used in an eyes-free
manner. Recently, Ferrone et al. [47] developed a wristband equipped with stretchable polymeric strain
gauge sensors to detect wrist movements. 2.3. Summary
together. A
technology HAR is becoming promising in many different fields, such as tourism, shopping, learning,
and gaming [3]. In order to allow a more natural and intuitive way to interact with HAR content,
researchers have suggested increasing interaction methods outside of the handheld mobile device itself. Advances in fabric sensing technology allow us to combine multiple interface modalities together. A prior study on fabric-based interfaces focused on implementing the fabric sensing technology to
detect either finger (e.g., [24]) or wrist (e.g., [46]) movements, but not for HAR interactions. Thus, a
thorough investigation to understand user preferences regarding a fabric type for an interface worn
on the hand will benefit designers regarding the types of gestures to utilize in the design of HAR
interfaces that lead to a natural and intuitive interactive experience. interactions. Thus, a thorough investigation to understand user preferences regarding a fabric typ
for an interface worn on the hand will benefit designers regarding the types of gestures to utilize in
the design of HAR interfaces that lead to a natural and intuitive interactive experience. 3. Methodology
Our study explored the scenario where a fabric-based hand-worn device would be available to
users to perform interactions with HAR devices. We followed the methodology proposed by [39] to
identify user-preferred thumb-to-index touch and wrist gestures for HAR interactions using a hand
worn fabric-based prototype (see Figure 1a). 2.2. Hand-Worn Clothing-Based Interfaces The number of small filaments (0.7 mm diameter and 1
cm length) are distributed on the surface of the wristband at regular intervals to cover the muscles of
interest. These filaments are made using a mixture of thermoplastic and nano-conductive particles. 5 of 21
5 of 2 Appl. Sci. 2019, 9, 3177
Appl. Sci. 2019, 9, x F Their system presented high accuracy in sensing wrist flexion and extension. Our proposed prototype
can be implemented by combining the two approaches mentioned above: thumb-to-index touch inputs
and wrist gestures; as such, the results of this study could also be domain-independent and be useful
for designers of applications beyond augmented reality applications. HAR is becoming promising in many different fields, such as tourism, shopping, learning, an
gaming [3]. In order to allow a more natural and intuitive way to interact with HAR conten
researchers have suggested increasing interaction methods outside of the handheld mobile devic
itself. Advances in fabric sensing technology allow us to combine multiple interface modalitie 3. Methodology
In order t
based interface Our study explored the scenario where a fabric-based hand-worn device would be available to
users to perform interactions with HAR devices. We followed the methodology proposed by [39]
to identify user-preferred thumb-to-index touch and wrist gestures for HAR interactions using a
hand-worn fabric-based prototype (see Figure 1a). pp
p
q
y
g
p
p
p
p
three different gestures for each HAR task. In a typical HAR usage scenario, only one hand i
available for users to perform interactions. Therefore, we asked our participants, when given a HAR
task, to perform gestures that they thought were suitable and natural for that particular HAR task
while holding the device on their one hand. (a)
(b)
(c)
(d)
(e)
Figure 1. (a) Fabric-based prototype used in our study for righthanded participants. Three soft foam
buttons (two on the sides of the index finger on proximal and middle phalanges, and one on the
palmar side of the index proximal phalange) were glued to a fingerless hand glove design. (b)
Illustrates the finger joints on the index finger. (c–e) Sample gestures supported by in-house
developed prototype: (c) wrist only; (d) touch only; and (e) touch and wrist. Figure 1. (a) Fabric-based prototype used in our study for righthanded participants. Three soft foam
buttons (two on the sides of the index finger on proximal and middle phalanges, and one on the palmar
side of the index proximal phalange) were glued to a fingerless hand glove design. (b) Illustrates the
finger joints on the index finger. (c–e) Sample gestures supported by in-house developed prototype: (c)
wrist only; (d) touch only; and (e) touch and wrist. (b) (a) (b) (a) (e) (c) (d) (d) (c) (e) Figure 1. (a) Fabric-based prototype used in our study for righthanded participants. Three soft foam
buttons (two on the sides of the index finger on proximal and middle phalanges, and one on th
palmar side of the index proximal phalange) were glued to a fingerless hand glove design. (b
Illustrates the finger joints on the index finger. (c–e) Sample gestures supported by in-hous
developed prototype: (c) wrist only; (d) touch only; and (e) touch and wrist. Figure 1. (a) Fabric-based prototype used in our study for righthanded participants. 3. Methodology
In order t
based interface Three soft foam
buttons (two on the sides of the index finger on proximal and middle phalanges, and one on the palmar
side of the index proximal phalange) were glued to a fingerless hand glove design. (b) Illustrates the
finger joints on the index finger. (c–e) Sample gestures supported by in-house developed prototype: (c)
wrist only; (d) touch only; and (e) touch and wrist. Fabric-Based Hand-Worn Interface
Our proposed interface is a modified hand glove with a fingerless design made of Lycra and
cotton. Three soft foam buttons—two on the sides of the index finger on proximal and middl
phalanges and one on the palmar side of the index finger (see Figure 1b) —were fixed using fabri
In order to minimize the effect of users’ previous knowledge acquired by the existing touch-based
interfaces [48], we applied the production [48] technique by asking each participant to propose three
different gestures for each HAR task. In a typical HAR usage scenario, only one hand is available
for users to perform interactions. Therefore, we asked our participants, when given a HAR task, Fabric-Based Hand-Worn Interface
Our proposed interface is a modified hand glove with a fingerless design made of Lycra and
cotton. Three soft foam buttons—two on the sides of the index finger on proximal and middl
phalanges and one on the palmar side of the index finger (see Figure 1b) —were fixed using fabri
In order to minimize the effect of users’ previous knowledge acquired by the existing touch-based
interfaces [48], we applied the production [48] technique by asking each participant to propose three
different gestures for each HAR task. In a typical HAR usage scenario, only one hand is available
for users to perform interactions. Therefore, we asked our participants, when given a HAR task, Appl. Sci. 2019, 9, 3177 6 of 21 to perform gestures that they thought were suitable and natural for that particular HAR task while
holding the device on their one hand. to perform gestures that they thought were suitable and natural for that particular HAR task while
holding the device on their one hand. 4.1. Participants Thirty-three participants volunteered to participate in the study (10 female, average age 20.61,
SD = 1.13). All were university students of different educational backgrounds (mathematics,
engineering, computer science, accounting, and industrial design) and recruited using WeChat,
a widely used social media platform. All participants reported owning a handheld mobile device
(such as a smartphone or tablet). Of these, 87.9% participants (14 participants with HAR experience)
mentioned that they have some knowledge of AR. Fourteen participants reported owning at least
one wearable device and 29 participants (87.88%) expressed their interest in using wearable mid-air
gestural interfaces to perform interactions with their handheld devices. None of our participants
were lefthanded. Fabric-Based Hand-Worn Interface Our proposed interface is a modified hand glove with a fingerless design made of Lycra and cotton. Three soft foam buttons—two on the sides of the index finger on proximal and middle phalanges
and one on the palmar side of the index finger (see Figure 1b) —were fixed using fabric glue. These
three buttons all allow touch (to toggle between states) and hold (to keep one state over time) gestures. The location of these soft buttons was determined based on prior studies [24,25] and on user preferences
from our series of initial pilot studies. Our co-designed fabric-based interface is based on the human hand movements, especially
wrist [49] and finger [24] joint movements, and supports the use of both thumb-to-index touch and
wrist gesture inputs (see Figure 1c–e). The wrist joint is a flexible joint and wrist movements can take
place along different axes. Our proposed design supports both horizontal and vertical wrist movements. Flexion occurs when the palm bends downward, towards the wrist. Extension is the movement
opposite to flexion. Ulnar and radial deviation is the rightward and leftward wrist movement and
occurs when the palm is facing down. Furthermore, three soft foam buttons enable legacy-inspired
touch and hold gestures. The hand-worn fabric-based prototype used in our study supports the following wrist and
thumb-to-index touch gestures: (1) flexion and extension, ulnar and radial deviation of wrist joint
movements; (2) tap and hold gestures using thumb; and (3) combination of all these gestures. There
are only two constraints while performing a combination of wrist and thumb-to-index touch gestures. First, the two touch gestures (tap and hold) cannot be performed together and should always be
associated with any one of the wrist gestures; and, second, touch gestures must precede a wrist gesture. See Figure 1c–e for sample gestures supported in our prototype. Table 1. The selected list of 27 HAR ta
4.3. Handheld Augmented Reality Tasks categories and presented via our HAR app. User Interface (Navigation)
Object Transformation
Camera
(T1) Select next section
(T11) Move left
(T21) Switch to photo
(T2) Select previous section
(T12) Move right
(T22) Switch to front camera
(T3) Go to next target
(T13) Move closer
(T23) Switch to rear camera
(T4) Go to previous target
(T14) Move further
(T24) Take a photo
(T5) Select the target
(T15) Uniform scale up
(T25) Switch to video
(T6) Long press on the target
(T16) Uniform scale down
(T26) Start video recording
(T7) Scroll up
(T17) Yaw left
(T27) Stop video recording
(T8) Scroll down
(T18) Yaw right
(T9) Swipe left
(T19) Pitch up
We wanted to identify the list of tasks related to commonly used HAR applications. To do that,
we first looked at Piumsomboon et al.’s list of tasks list [34] for HMD AR interactions and adopted
the tasks related to the 3D object manipulations. In addition, we further surveyed the most common
operations used for HAR applications in different domains (such as IKEA’s Place app) and collected
a list of user interface (navigation) tasks. Furthermore, in HAR, the camera is the main interaction
medium and enables different kinds of HAR experience, such as creating AR-based video content and
personalized emojis (notably Apple’s Animoji). Therefore, we added the tasks related to control the
cameras. We also had to limit the tasks list of three categories after the pilot studies to reduce the
duration of the experiment to retain the participants’ commitment during the elicitation study. Table 1
presents the final set of 27 tasks which are classified in three different categories: (1) User Interface, (2)
Object Transformation, and (3) Camera. (T10) Swipe right
(T20) Pitch down
4.4. Procedure
The whole elicitation process contained four phases for each participant. All 33 participants were
video-recorded throughout the experiment, and extensive notes were taken. In addition to two
questionnaires, one of the researchers observed each session and interpreted the gestures to the
participants. The entire process lasted about 50 min for each participant. 4.4.1. Introduction
Participants were introduced to the experiment setup and a short video about HAR interactions
was played for those who were not aware of this technology. A short online questionnaire was given
to them to collect demographic and prior experience information. Each participant was given 5 min
to familiarize themselves with our HAR app
Table 1. 4.2. Experimental Setup The interaction situation was defined above a table (160 cm × 80 cm) in a dedicated experimental
laboratory space. All participants were seated in front of the table and a Nexus 5X mobile phone
running Android SDK 8.1 was used as the HAR device. Two 4K cameras mounted on tripods were
used to capture the entire experiment procedure at two different angles. A 55-inch 4K display was
used to play the video of possible gestures (see Figure 2). 7 of 21 Appl. Sci. 2019, 9, 3177 7 of 21 p
Figure 2. A participant is performing a gesture while wearing the fabric-based prototype on the right
hand and holding the handheld augmented reality (HAR) device on the left hand. Two cameras were
used the capture the entire process. Figure 2. A participant is performing a gesture while wearing the fabric-based prototype on the right
hand and holding the handheld augmented reality (HAR) device on the left hand. Two cameras were
used the capture the entire process. Figure 2. A participant is performing a gesture while wearing the fabric-based prototype on the right
hand and holding the handheld augmented reality (HAR) device on the left hand. Two cameras were
used the capture the entire process. Figure 2. A participant is performing a gesture while wearing the fabric-based prototype on the right
hand and holding the handheld augmented reality (HAR) device on the left hand. Two cameras were
used the capture the entire process. Table 1. The selected list of 27 HAR ta
4.3. Handheld Augmented Reality Tasks The selected list of 27 HAR tasks used in our study. The tasks are classified into three different
categories and presented via our HAR app. User Interface (Navigation)
Object Transformation
Camera
(T1) Select next section
(T11) Move left
(T21) Switch to photo
(T2) Select previous section
(T12) Move right
(T22) Switch to front camera
(T3) Go to next target
(T13) Move closer
(T23) Switch to rear camera
(T4) Go to previous target
(T14) Move further
(T24) Take a photo
(T5) Select the target
(T15) Uniform scale up
(T25) Switch to video
(T6) Long press on the target
(T16) Uniform scale down
(T26) Start video recording
(T7) Scroll up
(T17) Yaw left
(T27) Stop video recording
(T8) Scroll down
(T18) Yaw right
(T9) Swipe left
(T19) Pitch up
(T10) Swipe right
(T20) Pitch down (T10) Swipe right
(T20) Pitch down
Procedure
Table 1. The selected list of 27 HAR tasks used in our study. The tasks are classified into three different
categories and presented via our HAR app. 4.4.1. Introduction Participants were introduced to the experiment setup and a short video about HAR interactions
was played for those who were not aware of this technology. A short online questionnaire was given
to them to collect demographic and prior experience information. Each participant was given 5 min to
familiarize themselves with our HAR app. 4 4 2 P e Eli it
4.4. Procedure 4.4.2. Pre-Elicitation
In this second phase, participants were informed of the purpose of the study and primed [48]
with a short video to demonstrate the possible ways of using our prototype. Detailed use of the
prototype was explained to all participants, such as the possible wrist and thumb-to-index touch
gestures supported in our prototype including tap and hold gestures in the two-minute-long video
The whole elicitation process contained four phases for each participant. All 33 participants
were video-recorded throughout the experiment, and extensive notes were taken. In addition to
two questionnaires, one of the researchers observed each session and interpreted the gestures to the
participants. The entire process lasted about 50 min for each participant. Appl. Sci. 2019, 9, 3177 8 of 21 4.4.2. Pre-Elicitation In this second phase, participants were informed of the purpose of the study and primed [48]
with a short video to demonstrate the possible ways of using our prototype. Detailed use of the
prototype was explained to all participants, such as the possible wrist and thumb-to-index touch
gestures supported in our prototype, including tap and hold gestures in the two-minute-long video. The researcher clarified each participant’s questions at this stage, e.g., regarding the types of gestures
(supported by our prototype) and using a similar button, but a different gesture for multiple tasks (for
example, using button one to perform both tap and hold gestures). We informed all of our participants
that using buttons was not compulsory and they were told to use them based on their preference. After
this, participants were given a suitable fabric-based interface. We prepared three different sizes of
fabric-based wrist interfaces of black color suitable for both right and left hands, and did not constrain
participants regarding which hand they wanted to wear the interface on—i.e., they could choose to
wear it on either hand. We informed participants that all 27 tasks would be introduced, one by one, via
our HAR app, and asked them to perform three different gestures while holding the handheld device
on one hand. 4.4.3. Elicitation To elicit user-preferred gestures for HAR interactions, participants were asked to hold the AR
device (i.e., smartphone) on their preferred hand. They were encouraged to rest their arms on the table
while holding the device. The 27 HAR tasks were presented via our HAR app and also presented
via a printed A4 paper for their reference. All 33 participants were asked to follow the think-aloud
protocol [29] to think of three different gestures for each task. Each participant was given a minute to
associate their choice of three gestures for each task and perform them one by one while holding the
device on their hand (see Figure 3a,b). They were also instructed to pick their preferred gesture for
each task, and also to rate their preferred gestures in terms of “social acceptance” and “comfortable
to use” in dynamic environments. All 27 tasks were always presented in the same order for each
participant. For each task, the experimenter wrote down the gesture code of the preferred gesture and
the ratings of the preferred gesture using custom-built software. For a greater understanding of their
thought process, participants were asked to say a few words about their preferred gestures for each
task while not holding the phone. Appl. Sci. 2019, 9, 3177
understanding of t
f
d
f 9 of 21
t their (a)
(b)
Figure 3. Variety of gestures performed by our participants using one hand (right hand) while holding
the device on the other hand: (a) wrist gestures and (b) thumb-to-index touch gestures. Figure 3. Variety of gestures performed by our participants using one hand (right hand) while holding
the device on the other hand: (a) wrist gestures and (b) thumb-to-index touch gestures. (b) (a) (b) (a) (a) Figure 3. Variety of gestures performed by our participants using one hand (right hand) while holding
the device on the other hand: (a) wrist gestures and (b) thumb-to-index touch gestures. Figure 3. Variety of gestures performed by our participants using one hand (right hand) while holding
the device on the other hand: (a) wrist gestures and (b) thumb-to-index touch gestures. 5. Results We collected a total of 2673 gestures (33 participants × 27 tasks × 3 gestures) for the 27 given
HAR tasks from our 33 participants. In addition, our data collection included the video recording,
transcripts, and verbal feedback for each participant. Our results include the agreement between our
user’s wrist and thumb-to-index touch gesture proposals, a gesture taxonomy, user-preferred gesture
set, subjective feedback, and qualitative observations. 4.4.4. Semi-Structured Interview
4.4.4. Semi-Structured Interview Finally, we had a small discussion interview with each of the participants about their experience
with our fabric-based prototype, including their opinions and difficulties encountered while
performing gestures for HAR tasks. Their verbal feedback was encouraged during the experiment
and when using the wrist and thumb-to-index touch gestures for HAR interactions. Finally, we had a small discussion interview with each of the participants about their experience
with our fabric-based prototype, including their opinions and difficulties encountered while performing
gestures for HAR tasks. Their verbal feedback was encouraged during the experiment and when using
the wrist and thumb-to-index touch gestures for HAR interactions. 4 5 Measures
4.5. Measures We employed the following measures to assess and understand users’ preferences and cognition
for gestures produced with our fabric-based interface for HAR interactions: initially, we grouped and
tallied the wrist and thumb-to-index touch gestures based on the predefined unique gesture codes,
which produced a percentage score for each gesture. Then, we computed agreement rates (A-Rate)
for each of the 27 tasks, coagreement rates (CR) between pair of tasks (e.g., scroll up/scroll down),
and also between subject agreements (e.g., HAR experience) using the formulas proposed by
We employed the following measures to assess and understand users’ preferences and cognition
for gestures produced with our fabric-based interface for HAR interactions: initially, we grouped
and tallied the wrist and thumb-to-index touch gestures based on the predefined unique gesture
codes, which produced a percentage score for each gesture. Then, we computed agreement rates
(A-Rate) for each of the 27 tasks, coagreement rates (CR) between pair of tasks (e.g., scroll up/scroll
down), and also between subject agreements (e.g., HAR experience) using the formulas proposed
by Wobbrock et al. [29] and Vatavu et al. [33], respectively, and their agreement analysis application
(AGATe: Agreement Analysis Toolkit). The following equation shows their agreement rate formula: A −Rate(r) =
|P|
|P| −1
X
Pi⊆P
|Pi|
|P|
2
−
1
|P| −1
(1) (1) where, “P is the set of all proposals for referent r, |P| the size of the set, and Pi subsets of identical
proposals from P” [33]. Using this formula, we can understand how much agreement is shared between
our participants for gestures produced with our fabric-based interface for HAR interactions. Finally,
we asked participants to rate their preferred gesture in terms of social acceptance and how comfortable
they were to use for each task, on a scale of 1–7. 5.2. Consensus between the Users Table 2 shows the agreement rate for each of the 27 tasks. The participants’ agreement rate (A-Rate)
ranged between 0.053 (lowest agreement) and 0.328 (medium agreement) with the mean A-Rate of
0.144. We applied the coagreement rate (CR) formula proposed by Wobbrock et al. [29] to understand
the agreement shared between two tasks r1 and r2. For example, in most cases, users chose to perform
opposite gestures for directional pairs which have a similar meaning, such as “Swipe left/Swipe right”
and “Move closer/Move further”. In our results, both “Move closer” and “Move further” have equal
agreement rates (A-Rate for Move closer = 0.138, and A-Rate for Move down = 0.138). The CR for
“Move closer” and “Move further” was 0.095. This suggests that the opposite gestures were used to
perform these two tasks. Table 2. Agreement rate for 27 tasks in two categories: discrete and continuous with effects of
users’ prior experience (experience with HAR application and using a wearable device). The highest
agreement rates are highlighted in dark gray, while lowest are shown in light gray. Significance is
highlighted in green. Table 2. Agreement rate for 27 tasks in two categories: discrete and continuous with effects of
users’ prior experience (experience with HAR application and using a wearable device). The highest
agreement rates are highlighted in dark gray, while lowest are shown in light gray. Significance is
highlighted in green. 5.1. Classification of Gestures All elicited user-preferred gestures were organized into three categories supported by our proposed
interface: (1) wrist gestures: flexion, extension, ulnar and radial deviations; (2) touch gestures: tap Appl. Sci. 2019, 9, 3177 10 of 21 10 of 21 and hold; and (3) combination of touch and wrist gestures. These gestures were performed using the
wrist and fingers. Touch gestures were performed using the thumb by either tapping or holding any of
the three soft foam buttons on the index finger. Wrist gestures were performed by moving the wrist
joints on two different axes. As stated earlier, one of the key constraints is that the touch gesture (tap or
hold) must always precede a wrist gesture while performing a combination of thumb-to-index and
wrist gestures. 5.2. Consensus between the Users Category
Tasks
AR
HAR Experience
p
Wearable Device
p
With
Without
Used
Never
Discrete
(T1) Select next section
0.328
0.307
0.371
0.257
0.392
0.264
0.057
(T2) Select previous section
0.167
0.15
0.171
0.7
0.187
0.132
0.354
(T3) Go to next target
0.11
0.078
0.114
0.51
0.129
0.077
0.39
(T4) Go to previous target
0.11
0.085
0.114
0.588
0.123
0.077
0.44
(T5) Select the target
0.205
0.144
0.295
0.02
0.228
0.143
0.177
(T6) Long press on the target
0.21
0.124
0.343
0.003
0.234
0.154
0.201
(T21) Switch to photo
0.053
0.033
0.057
0.656
0.041
0.044
0.959
(T22) Switch to front camera
0.106
0.092
0.095
0.845
0.129
0.088
0.494
(T23) Switch to rear camera
0.106
0.065
0.114
0.375
0.123
0.088
0.553
(T24) Take a photo
0.182
0.19
0.21
0.71
0.175
0.154
0.708
(T25) Switch to video
0.064
0.059
0.038
0.701
0.058
0.044
0.801
(T26) Start recording
0.133
0.118
0.21
0.12
0.105
0.154
0.417
(T27) End recording
0.172
0.17
0.257
0.178
0.105
0.275
0.019
Continuous
(T7) Scroll up
0.11
0.131
0.067
0.258
0.105
0.132
0.65
(T8) Scroll down
0.178
0.229
0.124
0.082
0.193
0.165
0.63
(T9) Swipe left
0.189
0.19
0.162
0.616
0.24
0.154
0.175
(T10) Swipe right
0.184
0.19
0.152
0.499
0.24
0.143
0.133
(T11) Move left
0.22
0.255
0.152
0.088
0.181
0.242
0.316
(T12) Move right
0.227
0.301
0.133
0.012
0.193
0.231
0.52
(T13) Move closer
0.138
0.124
0.152
0.61
0.158
0.088
0.255
(T14) Move further
0.138
0.144
0.105
0.475
0.135
0.121
0.813
(T15) Uniform scale up
0.1
0.052
0.171
0.052
0.123
0.066
0.344
(T16) Uniform scale down
0.123
0.085
0.133
0.382
0.117
0.11
0.904
(T17) Yaw left
0.102
0.072
0.124
0.35
0.129
0.077
0.39
(T18) Yaw right
0.102
0.072
0.124
0.35
0.129
0.077
0.39
(T19) Pitch up
0.053
0.052
0.095
0.433
0.058
0.088
0.619
(T20) Pitch down
0.098
0.092
0.086
0.985
0.099
0.088
0.841 5.3. Effects of Users’ Prior Experience on Agreement Vb(2,N=33) = 177.859, p = 0.020), and “Uniform scale up” (0.171 vs. 0.052, Vb(2,N=33) = 110.071, p = 0.052),
while users with HAR experience achieved higher agreement for the tasks “Move right” (0.301 vs. 0.133, Vb(2,N=33) = 217.095, p = 0.012), “Move left” (0.255 vs. 0.152, Vb(2,N=33) = 81.504, p = 0.088),
and “Scroll down” (0.229 vs. 0.124, Vb(2,N=33) = 85.409, p = 0.082); see Table 2. Similarly, there was more
agreement for participants who do not use a wearable device for the tasks “End recording” (0.275 vs. 0.105, Vb(2,N=33) = 202.604, p = 0.019) and “Select next section” (0.392 vs. 0.2645, Vb(2,N=33) = 18.881,
p = 0.057) than those who use at least one wearable device. To further understand these differences,
we computed between-group coagreement rates for each task. For example, coagreement for the
task “Long press on the target” was CRb = 0.207, showing that only 20.7% of all pairs of users across
the two groups were in agreement regarding how to press the selected target in a HAR application,
i.e., by holding button three. The reason why the other participants disagreed was that while the
users with prior HAR experience preferred to press on the selected target by holding button three
(equivalent of performing hold gesture on the smartphone), the users with no prior HAR experience
elicited more variations, such as by tapping button one and holding button two. All of the elicited
gestures from users who had never used HAR applications before showed a clear influence from
previous experience acquired from touchscreen devices—this difference was statistically significant
(p = 0.003) for the “Long press on the target” and exposed the largest effect size (Vb = 370.806) among
all other tasks. A similar effect was observed for the “Take a photo” task as well, but this time, from
another perspective: although the agreement rates of the two groups were similar (0.190 vs. 0.210) and
the difference was not significant Vb(2,N=18) = 3.096), the coagreement rate displayed different gesture
preferences for two groups (CRb = 0.167). 5.3. Effects of Users’ Prior Experience on Agreement In our study, 33 participants were asked to perform gestures for HAR applications. We found
that there was more agreement for users who have no prior experience with HAR for the tasks “Long
press on the target“ (0.343 vs. 0.124, Vb(2,N=33) = 370.806, p = 0.003), “Select the target” (0.295 vs. 0.144, 11 of 21 Appl. Sci. 2019, 9, 3177 Vb(2,N=33) = 177.859, p = 0.020), and “Uniform scale up” (0.171 vs. 0.052, Vb(2,N=33) = 110.071, p = 0.052),
while users with HAR experience achieved higher agreement for the tasks “Move right” (0.301 vs. 0.133, Vb(2,N=33) = 217.095, p = 0.012), “Move left” (0.255 vs. 0.152, Vb(2,N=33) = 81.504, p = 0.088),
and “Scroll down” (0.229 vs. 0.124, Vb(2,N=33) = 85.409, p = 0.082); see Table 2. Similarly, there was more
agreement for participants who do not use a wearable device for the tasks “End recording” (0.275 vs. 0.105, Vb(2,N=33) = 202.604, p = 0.019) and “Select next section” (0.392 vs. 0.2645, Vb(2,N=33) = 18.881,
p = 0.057) than those who use at least one wearable device. To further understand these differences,
we computed between-group coagreement rates for each task. For example, coagreement for the
task “Long press on the target” was CRb = 0.207, showing that only 20.7% of all pairs of users across
the two groups were in agreement regarding how to press the selected target in a HAR application,
i.e., by holding button three. The reason why the other participants disagreed was that while the
users with prior HAR experience preferred to press on the selected target by holding button three
(equivalent of performing hold gesture on the smartphone), the users with no prior HAR experience
elicited more variations, such as by tapping button one and holding button two. All of the elicited
gestures from users who had never used HAR applications before showed a clear influence from
previous experience acquired from touchscreen devices—this difference was statistically significant
(p = 0.003) for the “Long press on the target” and exposed the largest effect size (Vb = 370.806) among
all other tasks. A similar effect was observed for the “Take a photo” task as well, but this time, from
another perspective: although the agreement rates of the two groups were similar (0.190 vs. 0.210) and
the difference was not significant Vb(2,N=18) = 3.096), the coagreement rate displayed different gesture
preferences for two groups (CRb = 0.167). 5.4. Taxonomy of Wrist and Thumb-to-Index Touch Gestures 5.4. Taxonomy of Wrist and Thumb-to-Index Touch Gestures To further understand wrist and thumb-to-index touch gestures used to perform HAR interactions,
we considered the following three dimensions in our analysis. We were inspired and adopted/modified
these dimensions from previous studies [35,50,51], and grouped them by the specifics of both wrist
and thumb-to-index touch gestures: 1. Complexity (see Figure 4) identifies both touch and wrist gesture as either (a) simple or (b)
complex. We define simple gestures as gestures that are performed using only one gesture
(e.g., wrist or touch gesture). For example, moving the wrist downwards toward the palm to
perform downward flexion and/or using the thumb to press any of the soft foam buttons are
identified as simple gestures. Gestures performed using two or more distinctive gestures are
identified as complex gestures (e.g., pressing a soft foam button followed by moving the wrist
downwards toward the palm). We adopted this dimension from [52]. 2. Structure (see Figure 5) represents the relative importance of the wrist and touch gestures in the
elicitation of HAR gestures, with seven categories: (a) wrist (b) touch (button one), (c) touch
(button two), (d) touch (button three), (e) touch (button one) and wrist, (f) touch (button two)
and wrist, and (g) touch (button three) and wrist. We modified this category from the taxonomy
of Vatatu and Pentiu [51]. For example, for touch only category, the tap or hold gesture was
performed using any one of the three buttons. 3. Action (see Figure 6) groups the gestures based on their actions rather than their semantic meaning
with five categories: (a) scroll, (b) swipe, (c) tap, (d) hold, and (e) compound. We adopted
and modified this classification from Chan et al. [35], who used these dimensions to define
user-designed single-hand microgestures without any specific domains. For example, downward
flexion and upward extension were grouped as scrolls, while leftward flexion and rightward
extension were grouped as swipes. 12 of 21
12
f 20 Appl. Sci. 2019, 9, 3177 Figure 4. Frequency distribution of complexity of gestures in each category. Simple gestures were
highly preferred for discrete tasks. Figure 4. Frequency distribution of complexity of gestures in each category. Simple gestures were
highly preferred for discrete tasks. Figure 4. Frequency distribution of complexity of gestures in each category. Simple gestures were
highly preferred for discrete tasks. Figure 4. Frequency distribution of complexity of gestures in each category. 5.4. Taxonomy of Wrist and Thumb-to-Index Touch Gestures 2019, 9, 3177 13 of 21 13 of 21 Figure 6. Frequency distribution of action types in the preferred gesture set for 27 tasks. Tap action
was highly preferred for the camera tasks. Figure 6. Frequency distribution of action types in the preferred gesture set for 27 tasks. Tap action
was highly preferred for the camera tasks. Figure 6. Frequency distribution of action types in the preferred gesture set for 27 tasks. Tap action
was highly preferred for the camera tasks. Figure 6. Frequency distribution of action types in the preferred gesture set for 27 tasks. Tap action
was highly preferred for the camera tasks. 5.5. Consensus Gesture Set for HAR Interactions
We isolated 891 preferred gestures (33 participants × 27 tasks) from the original 2673 gestures. 57 unique gestures were used to perform 891 preferred gestures for the 27 tasks. To create a user-
preferred gesture set, we picked the gestures which achieved the highest consensus for each task. This led us to have 13 unique gestures performed for 27 tasks, which represented the 599/891 gestures
or 67.23% of the user-preferred gestures (see Figure 7). Simple gestures were highly performed for both discrete (82.75%) and continuous tasks (49.78%). Interestingly, participants preferred to use simple touch gestures (51.05% vs. 31.70%) for discrete and
simple wrist gestures (36.58% vs. 13.2%) for continuous tasks. Similarly, complex gestures (50.22%
vs. 17.25%) were also preferred for the continuous tasks. These results were confirmed by a one-way
ANOVA test. We found a significant effect for complexity (F(1,25) = 73.274, p = 0.000) as our participants
preferred simple gestures to perform discrete HAR tasks. p
g
(
g
)
We asked our participants to rate their preferred gesture proposal with numbers from 1 (very
poor fit) to 7 (very good fit) to denote their confidence in the usability of their preferred gestures in
two categories. We compared the subjective rating for social acceptance and comfortable to use
between the user-preferred gesture set and the discarded set. We found that the average scores for
gestures that are socially acceptable for the given tasks were 6.019 (SD = 0.992) and 4.830 (SD = 1.603),
and average scores for being comfortable to use with our proposed design were 6.362 (SD = 0.366)
and 5.612 (SD = 0.844), respectively. 5.4. Taxonomy of Wrist and Thumb-to-Index Touch Gestures Independent t-tests confirmed that the user-preferred set was
Our participants preferred to use button one on the index finger to perform both simple and
complex gestures for all 27 tasks. For example, for the discrete tasks, 27.51% (button two: 23.78%;
button three: 17.01%) of gestures were performed using button one. Similarly, button one was involved
in nearly 19.91% (button two and wrist: 17.97%; button three and wrist: 12.34%) of complex gestures
for the continuous tasks. A one-way ANOVA test revealed a statistically significant effect for touch
inputs using button one for discrete tasks (F(1,25) = 14.275, p = 0.001) and button one and wrist inputs
for continuous tasks (F(1,25) = 23.773, p = 0.000). rated significantly higher than the discarded set in all two factors of social acceptance (p = 0.023) and
comfortable to use (p = 0.005). Therefore, gestures in the user-preferred set are more suitable for HAR
interactions in dynamic outdoor environments than the discarded one in terms of social acceptance
and comfortable to use. Compounds (all 27 tasks) were the most common out of the five action types, mainly used to
perform continuous tasks, particularly the object transformation tasks. Our participants mentioned
two key reasons why they preferred Compounds for object transformation tasks: (1) wrist gestures
were simple and easier to perform; and (2) buttons allowed adding additional functions to wrist
gestures to associate similar gesture for directional pairs. 5.6 Users’ Feedback
All participants were encouraged to share their opinions and suggestions during the semi-
structured discussion interview. All participants felt comfortable wearing our proposed prototype to
perform gestures for the 27 HAR tasks. Nineteen (4 females) participants expressed that our proposed
interface is most suitable for candid interaction [53] especially in a public place When asked about
Taps (20 of 27 tasks) were the next most preferred action type. Notably, Taps were mainly used for
the camera tasks. Participants mentioned that buttons are most suitable for camera tasks as (1) buttons
allowed them to perform simple interactions and (2) did not cover the visual content on the screen,
making them particularly convenient for taking photos or recording videos. Notably, Tap and Hold
actions were used to perform state toggles “Select the target” and “Long press on the target”. interface is most suitable for candid interaction [53], especially in a public place. 5.4. Taxonomy of Wrist and Thumb-to-Index Touch Gestures Simple gestures were
highly preferred for discrete tasks. Figure 4. Frequency distribution of complexity of gestures in each category. Simple gestures were
highly preferred for discrete tasks. Figure 4. Frequency distribution of complexity of gestures in each category. Simple gestures were
highly preferred for discrete tasks. Figure 5. Observed percentages of wrist and touch gestures for HAR interactions. Button one was
highly preferred for both simple and complex gestures. Simple wrist gestures were preferred for
continuous tasks and simple touch gestures for discrete tasks (shows a clear influence of prior
experience). Figure 5. Observed percentages of wrist and touch gestures for HAR interactions. Button one was
highly preferred for both simple and complex gestures. Simple wrist gestures were preferred for
continuous tasks and simple touch gestures for discrete tasks (shows a clear influence of prior
experience). Figure 5. Observed percentages of wrist and touch gestures for HAR interactions. Button one was highly
preferred for both simple and complex gestures. Simple wrist gestures were preferred for continuous
tasks and simple touch gestures for discrete tasks (shows a clear influence of prior experience). Figure 5. Observed percentages of wrist and touch gestures for HAR interactions. Button one w
Figure 5. Observed percentages of wrist and touch gestures for HAR interactions. Button one w
hi hl
f
d f
b th
i
l
d
l
t
Si
l
i t
t
f
d f
Figure 5. Observed percentages of wrist and touch gestures for HAR interactions. Button one was hig
preferred for both simple and complex gestures Simple wrist gestures were preferred for continuo Figure 5. Observed percentages of wrist and touch gestures for HAR interactions. Button one was
highly preferred for both simple and complex gestures. Simple wrist gestures were preferred for
continuous tasks and simple touch gestures for discrete tasks (shows a clear influence of prior
i
)
Figure 5. Observed percentages of wrist and touch gestures for HAR interactions. Button one was
highly preferred for both simple and complex gestures. Simple wrist gestures were preferred for
continuous tasks and simple touch gestures for discrete tasks (shows a clear influence of prior
experience)
Figure 5. Observed percentages of wrist and touch gestures for HAR interactions. Button one was highly
preferred for both simple and complex gestures. Simple wrist gestures were preferred for continuous
tasks and simple touch gestures for discrete tasks (shows a clear influence of prior experience). Appl. Sci. 5.5. Consensus Gesture Set for HAR Interactions We isolated 891 preferred gestures (33 participants × 27 tasks) from the original 2673 gestures. 57
unique gestures were used to perform 891 preferred gestures for the 27 tasks. To create a user-preferred
gesture set, we picked the gestures which achieved the highest consensus for each task. This led us to
have 13 unique gestures performed for 27 tasks, which represented the 599/891 gestures or 67.23% of
the user-preferred gestures (see Figure 7). Appl. Sci. 2019, 9, x FOR PEER REVIEW
14 of 20
glove-based single-handed interface was convenient to control the camera as it does not block the
display like in touchscreens. Five female participants particularly expressed that they would like to
have a fancy and colorful interface. Figure 7. User-preferred gesture set for HAR interactions. Simple touch gestures: (a) tap button one,
(b) tap button two, (c) tap button three, (d) hold button two, (e) hold button three; Simple wrist
gestures: (f) downward flexion, (g) upward extension, (h) leftward flexion, (i) rightward extension;
Complex gestures: (j) hold button one and downward flexion, (k) hold button two and downward
flexion, (l) hold button two and leftward flexion, (m) hold button two and rightward extension. Figure 7. User-preferred gesture set for HAR interactions. Simple touch gestures: (a) tap button one, (b)
tap button two, (c) tap button three, (d) hold button two, (e) hold button three; Simple wrist gestures:
(f) downward flexion, (g) upward extension, (h) leftward flexion, (i) rightward extension; Complex
gestures: (j) hold button one and downward flexion, (k) hold button two and downward flexion, (l)
hold button two and leftward flexion, (m) hold button two and rightward extension. Figure 7. User-preferred gesture set for HAR interactions. Simple touch gestures: (a) tap button one,
(b) tap button two, (c) tap button three, (d) hold button two, (e) hold button three; Simple wrist
gestures: (f) downward flexion, (g) upward extension, (h) leftward flexion, (i) rightward extension;
Complex gestures: (j) hold button one and downward flexion, (k) hold button two and downward
flexion, (l) hold button two and leftward flexion, (m) hold button two and rightward extension. Figure 7. User-preferred gesture set for HAR interactions. 5.4. Taxonomy of Wrist and Thumb-to-Index Touch Gestures When asked about
the positions of the buttons, all 33 participants unanimously agreed and were satisfied with the
current locations of the three soft buttons; however, only three participants (P3, P25, P27) further
recommended adding an additional button. In particular, two participants preferred to have the
fourth button on the sides of the index finger between buttons one and two, while the other
participant preferred to have it on the palmar side of the index finger. Only one user (P32) suggested
Participants associated wrist movements for the tasks which resemble symbolic actions. Scrolls
were highly preferred for tasks which resemble vertical movements (e.g., Select next section and
Select previous section) while Swipes were mostly associated for the tasks which involve in horizontal
movements (e.g., Go to next target and Go to the previous target). A one-way ANOVA test revealed a
statistically significant effect on the action (F(1,25) = 26.741, p = 0.000) and users preferred to use Tap Appl. Sci. 2019, 9, 3177 14 of 21 actions for the discrete tasks in HAR applications, which confirms the strong influence of previous
interaction experience acquired from the touchscreen devices. 5.6. Users’ Feedback All participants were encouraged to share their opinions and suggestions during the
semi-structured discussion interview. All participants felt comfortable wearing our proposed prototype
to perform gestures for the 27 HAR tasks. Nineteen (4 females) participants expressed that our proposed
interface is most suitable for candid interaction [53], especially in a public place. When asked about
the positions of the buttons, all 33 participants unanimously agreed and were satisfied with the current
locations of the three soft buttons; however, only three participants (P3, P25, P27) further recommended
adding an additional button. In particular, two participants preferred to have the fourth button on the
sides of the index finger between buttons one and two, while the other participant preferred to have
it on the palmar side of the index finger. Only one user (P32) suggested that the buttons could have
haptic feedback (e.g., vibration) while another user (P10) proposed an additional slider type of control
on the fabric-based interface. All participants mentioned that the glove-based single-handed interface
was convenient to control the camera as it does not block the display like in touchscreens. Five female
participants particularly expressed that they would like to have a fancy and colorful interface. 5.5. Consensus Gesture Set for HAR Interactions 2019, 9, 3177 15 of 21 15 of 21 6.1. Design Recommendations for Fabric-Based Interface for HAR Interactions We discuss, in this section, some of our participants’ preferred gestures in more detail as we
propose design guidelines for using wearable smart textiles for HAR interactions and recommend the
following set of suggestions to further investigate the use of fabric-based interfaces for HAR interactions. 6.1.1. Simple Gestures Were Preferred for HAR Interactions 5.5. Consensus Gesture Set for HAR Interactions Simple touch gestures: (a) tap button one, (b)
tap button two, (c) tap button three, (d) hold button two, (e) hold button three; Simple wrist gestures:
(f) downward flexion, (g) upward extension, (h) leftward flexion, (i) rightward extension; Complex
gestures: (j) hold button one and downward flexion, (k) hold button two and downward flexion, (l)
hold button two and leftward flexion, (m) hold button two and rightward extension. 6. Discussion
In this work, we adopted and extended a methodology proposed by [39] to uniquely identify
usable gestures for HAR interactions. We defined all user-preferred possible wrist and thumb-to-
index touch gestures using our prototype before the study began, and grouped the elicited gestures
using the unique gesture code. All 33 participants utilized the large degrees of the wrist and thumb-
to-index touch gestures supported by our design to elicit distinct gestures for each HAR task. We
used our HAR app to introduce all 27 tasks (most of the tasks are dichotomous pairs) in the same
order to all the participants. Though we set a one-minute time limit to think of three different gestures
for each task, all participants thought of their choice of three gestures within 45 s. Therefore, as
mentioned in the results section, our user-elicited gestures using hand-worn interface for a specific
set of HAR tasks achieved a medium agreement which is aligned with the previous elicitation study
We asked our participants to rate their preferred gesture proposal with numbers from 1 (very
poor fit) to 7 (very good fit) to denote their confidence in the usability of their preferred gestures
in two categories. We compared the subjective rating for social acceptance and comfortable to use
between the user-preferred gesture set and the discarded set. We found that the average scores for
gestures that are socially acceptable for the given tasks were 6.019 (SD = 0.992) and 4.830 (SD = 1.603),
and average scores for being comfortable to use with our proposed design were 6.362 (SD = 0.366)
and 5.612 (SD = 0.844), respectively. Independent t-tests confirmed that the user-preferred set was
rated significantly higher than the discarded set in all two factors of social acceptance (p = 0.023) and
comfortable to use (p = 0.005). Therefore, gestures in the user-preferred set are more suitable for HAR
interactions in dynamic outdoor environments than the discarded one in terms of social acceptance
and comfortable to use. Appl. Sci. 6. Discussion In this work, we adopted and extended a methodology proposed by [39] to uniquely identify
usable gestures for HAR interactions. We defined all user-preferred possible wrist and thumb-to-index
touch gestures using our prototype before the study began, and grouped the elicited gestures using the
unique gesture code. All 33 participants utilized the large degrees of the wrist and thumb-to-index
touch gestures supported by our design to elicit distinct gestures for each HAR task. We used our
HAR app to introduce all 27 tasks (most of the tasks are dichotomous pairs) in the same order to all
the participants. Though we set a one-minute time limit to think of three different gestures for each
task, all participants thought of their choice of three gestures within 45 s. Therefore, as mentioned
in the results section, our user-elicited gestures using hand-worn interface for a specific set of HAR
tasks achieved a medium agreement, which is aligned with the previous elicitation study using
non-functional fabric-based prototype [39]. All participants performed three gestures for each task while holding the mobile phone on their left
hand. Thus, they had to keep looking at the touchscreen to understand the tasks and had to associate
their preferred gesture for each task. A significant number of gestures (5 simple touch gestures; and 4
touch and wrist gestures) from the user-preferred set were touchscreen-inspired tap and hold gestures. Nevertheless, our participants were not influenced by the legacy bias as they preferred two distinct
gestures for state toggles. This finding contrasts with a prior elicitation study [35], where they did not
use any prototype. Thus, they were often forced to use the same gesture for state toggles, whereas
using our method, participants were able to perform two different gestures for state toggles. 6.1.3. Design Wrist Gestures for Continuous HAR Tasks Of all proposed gestures, 36.58% (continuous) and 31.70% (discrete) were simple wrist gestures. Participants preferred to use wrist gestures for continuous tasks even though touch gestures were
available: 13.20% (tap: 1.95%; hold: 11.26%) of touch gestures were preferred for continuous tasks. We found that participants used the flexion/extension in 86.89% of their simple wrist gestures and in
89.21% for complex gestures. Participants used in-air gestures to manipulate imaginary controls to
associate wrist gestures for the continuous tasks, e.g., pushing a virtual object down (Figure 7f) or away
in mid-air (Figure 7g), and sliding an imaginary control on both sides (Figure 7h,i). This association of
wrist gestures with imaginary objects produced more dichotomous gestures, i.e., similar gestures but
with opposite directions, as users tended to pair related tasks (Figure 7f–i). We recommend associate
wrist gestures to create an instinctive mapping between movement and action, e.g., performing
downward flexion replicates a physical action of moving an object down with control. 6.1.1. Simple Gestures Were Preferred for HAR Interactions Of all user-preferred gestures, 82.75% (discrete) and 49.78% (continuous) were “simple” gestures,
i.e., gestures performed using only one action, either a touch or wrist gesture, as mentioned in our
gesture taxonomy (see Figure 7a–i). Participants reported that simple gestures were convenient to
perform while complementing the primary task. However, they preferred to use complex gestures
for 3D object manipulation tasks (see Figure 7j–m). Also, we found that complex gestures were less
preferred for discrete (17.259%) than continuous tasks (50.22%), showing a clear preference for simple Appl. Sci. 2019, 9, 3177 16 of 21 gestures for discrete tasks. These findings align with the prior elicitation study using fabric-based
interface to perform interactions with in-vehicle systems [39]. 6.1.2. Utilize Familiar Touch-Based Input Methods for Discrete HAR Tasks 6.1.2. Utilize Familiar Touch-Based Input Methods for Discrete HAR Tasks We found that 51.05% of the gestures proposed for the discrete tasks were touch (37.53% tap
and 13.52% hold) gestures. The literature has documented the effect of users’ prior experience [48]. Interestingly, subjects preferred to use two different variations of touch gestures for discrete tasks,
as adopted from their prior experience [31]. They favored using touch gestures to toggle between states
as they wanted to perform quick actions in a fairly short time. For example, tap gesture was preferred
for switching between photo and video mode (see Figure 7a) while hold gesture was preferred to
long-press on the target (see Figure 7d), particularly influenced by the semantic expression of the task. As reported in the prior elicitation studies (e.g., [35,54]), most of our users preferred to use identical
touch gestures for state toggles and this suggests that for HAR interactions, gesture designers need to
consider the existing metaphors related to the nature of the tasks. 6.1.4. Favor Stretchable Fabric with Fingerless Design We designed a fingerless hand glove (made of cotton and Lycra) as a physical interface. Three soft
foam buttons were positioned on the index finger between metacarpophalangeal (MCP) and proximal
interphalangeal (PIP) joints. The custom-made fingerless design allowed users to have full control over
their thumb to comfortably rotate around to perform touch (Tap and Hold) gestures on the index finger. We endorse that for fabric-based interfaces to be practical and usable, they need to be thin, lightweight,
and exceptionally stretchy, with increased elasticity while enhancing comfort and breathability. 6.1.5. Consider Resistive Sensing Technique to Capture Both the Wrist and Touch Inputs Prior studies on fabric-based interfaces demonstrated the advantages of deploying resistive
sensing technology to detect unobtrusively both wrist gestures [46] and thumb-to-index inputs [24]. They used fabric-based strain sensors to capture wrist movements and pressure sensors to detect
legacy-inspired touch inputs. Our proposed design supports both wrist and thumb-to-touch input
methods. To successfully recognize the gestures from our user-defined set, we recommend using two
opposing fabric-based piezoresistive strain sensors [55] on the wrist and pressure sensors [56] on the
index finger. The strain sensors can be sewn while the pressure sensors can be glued to the hand glove
to allow detecting both wrist and thumb-to-index touch inputs. Author Contributions: Conceptualization, V.N., H.-N.L. and K.K.-T.L.; Data curation, R.S. and H.X.; Formal
analysis, V.N., R.S., H.-N.L., H.X. and K.H.; Funding acquisition, H.-N.L. and K.H.; Investigation, V.N., R.S., H.X.
and H.-N.L.; Methodology, V.N., H.-N.L. and K.H.; Project administration, H.-N.L. and K.K.-T.L.; Software, R.S.
and H.X.; Supervision, H.-N.L.; Validation, V.N. and H.-N.L.; Visualization, V.N. and R.S.; Writing – original draft,
V.N., R.S., H.-N.L., H.X., K.K.-T.L. and K.H. 6.2. Limitations We conducted our study inside a room and participants could rest their hands while holding
the AR device on the table to produce the gestures. It will be interesting to investigate if users
would produce the same interactions in other scenarios, like standing or even walking. We used a
non-functional fabric-based fingerless glove-based prototype in our study. Similarly, we excluded
pronation/supination of the forearm in our design because our participants did not prefer these
movements and mentioned difficulties associating them with the given 27 HAR tasks during the series
of pilot studies. Despite the absence of interactive functionalities and a set of distinct wrist movements,
we were still able to understand users’ behavior and responses to it as an input interface for HAR
interactions. None of our participants were lefthanded and, thus, our gesture set is only suitable for
righthanded users. 6.1.6. Design Fabric-Based Interface That Foster Socially Acceptable Gestures 6.1.6. Design Fabric-Based Interface That Foster Socially Acceptable Gestures The aim of our study is to design an unobtrusive fabric-based interface for HAR applications
so that end-users are willing to use this interface in public settings. Usability is defined by Bevan as Appl. Sci. 2019, 9, 3177 17 of 21 17 of 21 “quality in use” [57]. However, a user’s readiness to use ubiquitous devices, particularly wearable
devices, is not only limited to the quality of the device. In addition to the default usability aspect,
the decision to use a wearable device, particularly a fabric-based wearable device, is based on numerous
factors, mainly (1) wearable comfort and (2) social acceptability. Knight et al. [58] reported that factors
both internal and external to the wearers significantly influence how comfortable they feel about
wearing and using the device; as such, they influence their intention to use it. Similarly, Rice and
Brewster [59] reported that social acceptability is also influenced by various factors, such as the device’s
appearance, social status, and cultural conventions associated with it. Our proposed design allows both
the already familiar touch and mid-air gestural inputs in a subtle unobtrusive posture, and results from
the users’ subjective feedback show that the gestures in the user-preferred set are more suitable for HAR
interactions in dynamic outdoor environments in terms of social acceptance and how comfortable they
are to use. Furthermore, fabric-based wearable interfaces are useful for the places that are extremely
cold (e.g., Siberia) where touch interactions on HAR devices require users to remove their gloves. Funding: This research was funded by Xi’an Jiaotong-Liverpool University (XJTLU) Key Program Special Fund
(#KSF-A-03) and XJTLU Research Development Fund (#RDF-13-02-19). References 1. Azuma, R.T. A Survey of Augmented Reality. Presence Teleoperators Virtual Environ. 1997, 6, 355–385. [CrossRef] 2. Mekni, M.; Lemieux, A. Augmented Reality: Applications, Challenges and Future Trends. Appl. Comput. Sci. 2014, 205, 205–214. 2. Mekni, M.; Lemieux, A. Augmented Reality: Applications, Challenges and Future Trends. Appl. Comput. Sci. 2014, 205, 205–214. atzopoulos, D.; Bermejo, C.; Huang, Z.; Hui, P. Mobile Augmented Reality Survey: From Where We Are
Where We Go. IEEE Access 2017, 5, 6917–6950. [CrossRef] 4. Zhou, F.; Duh, H.B.-L.; Billinghurst, M. Trends in augmented reality tracking, interaction and display:
A review of ten years of ISMAR. In Proceedings of the 2008 7th IEEE/ACM International Symposium on
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A review of the 2nd Decade of ISMAR (2008–2017). IEEE Trans. Vis. Comput. Graph. 2018, 24, 2947–2962. [CrossRef] [PubMed] 5. Kim, K.; Billinghurst, M.; Bruder, G.; Duh, H.B.L.; Welch, G.F. Revisiting trends in augmented reality research:
A review of the 2nd Decade of ISMAR (2008–2017). IEEE Trans. Vis. Comput. Graph. 2018, 24, 2947–2962. [CrossRef] [PubMed] 6. Bekele, M.K.; Town, C.; Pierdicca, R.; Frontoni, E.; Malinverni, E.V.A.S. A Survey of Augmented, Virtual,
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and Mixed Reality for Cultural Heritage. ACM J. Comput. Cult. Herit. 2018, 11, 7. [CrossRef] 7. Polvi, J.; Taketomi, T.; Yamamoto, G.; Dey, A.; Sandor, C.; Kato, H. SlidAR: A 3D positioning method for
SLAM-based handheld augmented reality. Comput. Graph. 2016, 55, 33–43. [CrossRef] 8. Grandi, J.G.; Debarba, H.G.; Bemdt, I.; Nedel, L.; MacIel, A. Design and Assessment of a Collaborative 3D
Interaction Technique for Handheld Augmented Reality. In Proceedings of the 25th IEEE Conference Virtual
Reality 3D User Interfaces, VR 2018, Reutlingen, Germany, 18–22 March 2018; pp. 49–56. 9. Mossel, A.; Venditti, B.; Kaufmann, H. 3DTouch and HOMER-S: Intuitive Manipulation Techniques for
One-Handed Handheld Augmented Reality. 7. Conclusions In this study, we investigate the use of textile-based interface for HAR interactions. To further
explore the design space of hand-worn fabric-based interfaces as an alternative interface for HAR
applications, we designed a glove-based prototype which supports both wrist and thumb-to-index
input methods. The fingerless design allowed users to comfortably rotate their thumb to perform
touch gestures on the soft foam buttons integrated into the index finger. We recruited 33 potential
end-users to participate in the elicitation study. By following the methodology to elicit the gestures
using textile-based wearable interface, we were able to elicit gestures for HAR interactions. In addition
to reflecting the user behavior, our user-preferred gesture set has properties that make them usable
in dynamic HAR environments, such as social acceptance and how comfortable they are to use. In addition, we have presented a taxonomy of wrist and thumb-to-index tough gestures useful for
performing interactions with HAR devices. By identifying gestures in our study, we have gained insight
into the user-preferred gestures and have derived design guidelines for further exploration. Our results
suggest using fabric-based interfaces to perform HAR interactions is simple, natural, and completely
discourages the users from crossing the fingers across the screen, which could potentially cover the
content on the screen. 18 of 21 Appl. Sci. 2019, 9, 3177 Acknowledgments: We thank all the volunteers who participated in the experiment for their time. We also thank
the reviewers for their comments and suggestions that have helped improve our paper. Conflicts of Interest: The authors declare no conflicts of interest. Conflicts of Interest: The authors declare no conflicts of interest. References Nanjappan, V.; Liang, H.-N.; Lau, K.; Choi, J.; Kim, K.K. Clothing-based wearable sensors for unobtrusive
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article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).
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ECHS1, an interacting protein of LASP1, induces sphingolipid-metabolism imbalance to promote colorectal cancer progression by regulating ceramide glycosylation
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www.nature.com/cddis 1Department of Pathology, Shunde Hospital, Southern Medical University (The First People’s Hospital of Shunde), Foshan, China. 2Department of Pathology & Guangdong
Province Key Laboratory of Molecular Tumor Pathology, School of Basic Medical Sciences, Southern Medical University, Guangzhou, China. 3Department of Pathology, The
Second People’s Hospital of Longgang District, Shenzhen, China. 4Department of Pathology, Guangdong Women and Children Hospital, Guangzhou, Guangdong 511442, China.
✉email: liangsmu@foxmail.com
Edited By Alessandro Finazzi-Agrò ARTICLE
OPEN
ECHS1, an interacting protein of LASP1, induces sphingolipid-
metabolism imbalance to promote colorectal cancer
progression by regulating ceramide glycosylation © The Author(s) 2021 © The Author(s) 2021 Sphingolipid metabolic dysregulation has increasingly been considered to be a drug-resistance mechanism for a variety of tumors. In this study, through an LC–MS assay, LIM and SH3 protein 1 (LASP1) was identified as a sphingolipid-metabolism-involved protein,
and short-chain enoyl-CoA hydratase (ECHS1) was identified as a new LASP1-interacting protein through a protein assay in
colorectal cancer (CRC). Gain- and loss-of-function analyses demonstrated the stimulatory role played by ECHS1 in CRC cell
proliferation, migration, and invasion in vitro and in vivo. Mechanistic studies of the underlying tumor-supportive oncometabolism
indicate that ECHS1 enables altering ceramide (Cer) metabolism that increases glycosphingolipid synthesis (HexCer) by promoting
UDP-glucose ceramide glycosyltransferase (UGCG). Further analysis showed that ECHS1 promotes CRC progression and drug
resistance by releasing reactive oxygen species (ROS) and interfering mitochondrial membrane potential via the PI3K/Akt/mTOR-
dependent signaling pathway. Meanwhile, the phenomenon of promoting the survival and drug resistance of CRC cells caused by
ECHS1 could be reversed by Eliglustat, a specific inhibitor of UCCG, in vitro and in vivo. IHC assay showed that ECHS1 was
overexpressed in CRC tissues, which was related to the differentiation and poor prognosis of CRC patients. This study provides new
insight into the mechanism by which phospholipids promote drug resistance in CRC and identifies potential targets for future
therapies. Cell Death and Disease (2021) 12:911 ; https://doi.org/10.1038/s41419-021-04213-6 Cell Death and Disease (2021) 12:911 ; https://doi.org/10.1038/s41419-021-04213-6 Received: 17 May 2021 Revised: 14 September 2021 Accepted: 23 September 2021 Cell-line preparation p
p
The CRC cell lines RKO, HCT116, HCT15, SW620, SW480, LS174T, and
CACO2 were obtained from CBCC (Shanghai, China) and maintained as
previously described [6]. All cells were cultured in RPMI-1640 medium
(KeyGen, Keygentec, JiangSu, China) containing 10% fetal bovine serum
(Gibco-BRL, Invitrogen, Paisley, UK) in a humidity of 5% CO2 at 37 °C. Statistical analysis y
SPSS statistical software version 19.0 (SPSS; Chicago, USA) was used to
analyze the data. The t-test and one-way ANOVA were applied for RT-
PCR. Spearman rank-correlation test was used to determine the
correlation between ECHS1 and LASP1. Pearson chi-square (χ2) was
applied to determine the correlation between the expression of ECHS1
and histopathological factors. The independent t-test was used for
subcutaneous tumor detection. Kaplan–Meier chart was used to
estimate the prognostic correlation of ECHS1 in univariate analysis. The statistical significance was established at P < 0.05. All the experi-
ments were carried out in triplicate. Tumor-tissue samples The six pairs of primary colorectal cancer tissues and their paired adjacent
noncancerous tissues were obtained from the Tumor Tissue Bank of
Nanfang Hospital. All cases were from patients diagnosed with primary
CRC and undergoing surgery at Nanfang Hospital in 2017–2019. The study
was approved by the Ethics Committee of Southern Medical University,
and all aspects of the research had obtained informed consent and in
accordance with the Declaration of Helsinki. Immunohistochemistry (IHC) The CRC specimens were incubated overnight using primary antibodies
against LASP1 (Proteintech, 10515-1-AP, 1:500) and ECHS1 (Proteintech,
66117-1-Ig, 1:500) at 4 °C. Mayer’s hematoxylin was used for nuclear
counterstaining. In this study, each slide was reviewed by three blinded
pathologists. BACKGROUND
l
l Although evidence suggests a potential role of
ECHS1 involved in tumor occurrence and development, the
underlying mechanisms have not been elucidated to date. Most
importantly, in addition to abnormal FA-oxidation (FAO) synthesis,
the relevance of phospholipid metabolism associated with ECHS1
and cancer-cell function is unknown. [17] cancers. Although evidence suggests a potential role of
ECHS1 involved in tumor occurrence and development, the
underlying mechanisms have not been elucidated to date. Most
importantly, in addition to abnormal FA-oxidation (FAO) synthesis,
the relevance of phospholipid metabolism associated with ECHS1
and cancer-cell function is unknown. isopropanol to dissolve it and filter it with a membrane to prepare a
sample for LC–MS [19]. LC–MS conditions The chromatographic separation was performed in a Thermo Ultimate
3000 system that was equipped with ACQUITY UPLC® BEH C18(100 ×
2.1 mm, 1.7 µm, Waters) column and kept at 50 °C. The ESI–MSn
experiments were performed on the Thermo Q Exactive Focus mass
spectrometer, and the spray voltages of positive and negative modes were
3.5 kV and −2.5 kV, respectively. In this study, we found that LASP1 colocalizes with ECHS1 in
CRC cells and is associated with sphingomyelin metabolism to
induce apoptosis and drug resistance. The expression and
function of ECHS1 in CRC was studied, and the associated
signaling pathway was investigated. The current study helps to
elucidate the relationship between multidrug resistance and
phospholipid metabolism and provides a new insight for over-
coming clinical CRC chemotherapy resistance. Animals and tumor-growth assay g
y
The Institutional Animal Care and Use Committee of Southern Medical
University (Guangzhou, China) approved the animal experiments involved. All animal procedures were in accordance with the Helsinki Declaration. BALB/c nude mice (female, 3–5 weeks, 13–15 g) were obtained from
Southern Medical University Experimental Animal Center and kept in an
SPF animal room with free access to clean food and water and possible
adverse events were monitored. During housing, animals were monitored
twice daily for health status. For subcutaneous tumor tests for drug
sensitivity, the mice were divided into four groups (n = 20): (1) injected
with LV-ctrl-HCT116 cells (n = 5); (2) injected with LV-ctrl-HCT116 cells and
injected intraperitoneally with Oxaliplatin (1.5 mg/kg, MedChem Express,
HY-17371) every two days (n = 5); (3) injected with LV-ECHS1-HCT116 cells
and injected intraperitoneally with Oxaliplatin (1.5 mg/kg, MedChem
Express, HY-17371) every two days (n = 5); and (4) injected with LV-
ECHS1-HCT116 cells and injected intraperitoneally with Oxaliplatin (1.5 mg/
kg, MedChem Express, HY-17371) every two days and received daily
intraperitoneal injections of Eliglustat (60 mg /kg, MedChem Express, HY-
14885) [18] (n = 5). Three mutual experimenters are responsible for
grouping using a blinding and randomization method, processing, and
data collection. 6 LASP1 is associated with sphingomyelin metabolism and
regulates ECHS1 in CRC cells regulates ECHS1 in CRC cells
We first evaluated the role of LASP1 in CRC lipid metabolism. As
shown in Fig. 1A, gene-set enrichment analysis (GSEA) demonstrated
a strong enrichment of phospholipids, including ceramide (GSE4382),
sphingosine kinase/sphingosine 1-phosphate (S1P_S1P2) (GSE77955),
and S1P_META (GSE77955). Next, liquid chromatography–mass
spectrometry technology (LC–MS) was applied to detect the
lipidomics of LASP1-overexpressing CRC cells (RKO-LASP1) and
control cells. Notably, as shown in Fig. 1B, abnormal metabolism of
phospholipids, including increased ceramide and decreased sphin-
gomyelin, was found in RKO-LASP1 cells compared with the control
group. Specifically,
the
expression
of
ceramide
(d18:1_18:0)
decreased, whereas the expression of Hex2Cer (d18:1_24:1) and SM
(d36:1) increased (Fig. 1C, D). Hexose-linked glycoceramides (HexCer),
including galactosylceramide (GalCer) and glucosylceramide (GluCer). For the tumor-growth assay, 5 × 106 cells were injected into the
subcutaneous tissue of the back of the mice. After four weeks, the nude
mice were anesthetized with diethyl ether and sacrificed by cervical
dislocation before tumors reached 1500 mm3 in volume. The xenograft
tumors were harvested for subsequent histological study. The formula
(volume (mm3) = width2 (mm2) × length (mm)/2) was used to calculate the
tumor volume. g g
y
g
y
To further study the underlying mechanism of LASP1-mediated
CRC-cell malignance, 2-D DIGE assay was used to screen the
differentially expressed proteins in SW480/ SW620 cells transfected
with LASP1 siRNA or control siRNA (Fig. S1). The results demonstrated
that ECHS1, an enzyme involved in lipid metabolism, was one of the
candidates for LASP1-modulated proteins (Fig. 1E, F). RT-PCR (Fig. 1G)
and Western blotting (Fig. 1H) results showed that LASP1 regulates
ECHS1 at the protein, rather than the RNA level. Furthermore, as
shown in Fig. 1I, the results of the Transwell assay demonstrated that
the ability of LASP1 to promote CRC-cell migration and invasion was
related to its regulation of ECHS1. BACKGROUND
l
l metabolism is found to interact with LASP1 to promote aggressive
phenotype of CRC cells. Moreover, microRNAs, including miR-1 [9],
miR-133a [10], and miR-145 [11], suppress colon-cancer cell
invasion and metastasis by targeting LASP1. Although there have
been many researches on the molecular mechanism of LASP1
promoting the progression of CRC, it promotes cell metabolism,
especially regarding sphingolipid metabolism, which has not been
elucidated in detail. However, the molecular mechanism under-
lying this interaction, especially regarding sphingolipid metabo-
lism, has not been elucidated in detail. Colorectal cancer (CRC) is one of the most common malignant
tumors threatening human health worldwide [1]. Chemotherapy is
the most commonly used adjuvant and conversion therapy for
patients with advanced CRC. Despite significant progress in
chemotherapy, inherent or acquired chemoresistance, especially
multidrug
resistance
(MDR),
is
the
main
obstacle
to
this
development,
leading
to
inefficient
cancer-cell
killing
and
subsequent patient relapse [2]. Therefore, it is essential to
understand the biological mechanisms underlying MDR more
comprehensively to benefit patients with CRC or other tumors. Short-chain enoyl-CoA hydratase (ECHS1) is responsible for the
second step of hydration of fatty-acid β oxidation(FAO) and its
activity was first observed by Del Campillo and Stern in cow hearts
and livers, and the human ECHS1 cDNA clones were first isolated
in 1993 [12]. The ECHS1 gene mapped to human chromosome
10q26.2–q26.3, which encodes eight exons, including 50 and 30
untranslated regions containing exons I and VIII, respectively [13]. The expression of ECHS1 has been identified in numerous types of
cancer cells or patient tissues by gene or proteomic expression
profiling. It was reported that the expression of ECHS1 was
significantly increased in colorectal [14, 15], liver [16], and gastric LIM and SH3 protein 1 (LASP1) was identified as a tumor-
promotion-related gene and its abnormal expression has been
reported in several tumor types, including breast cancer [3],
ovarian cancer [4], hepatocellular carcinoma [5], and CRC [6]. On
the basis of our previous study, we found that LASP1 was highly
overexpressed in CRC tissues which is positively associated with
lymph node and distant metastasis [7]. By using comparative
proteomics methods to identify LASP1-related proteins involved in
regulating tumor progression, a series of proteins related to
cytoskeleton [8], epithelial–mesenchymal transition (EMT), and Official journal of CDDpress Official journal of CDDpress R. Li et al. 2 [17] cancers. Western blot analysis Western blot analysis was performed to evaluate protein expression
(30–60 mg) in the presence of antibodies to ECHS1 (Proteintech, 66117-1-
Ig, 1:1000), LASP1 (Proteintech, 10515-1-AP, 1:1000), Phospho-Akt (CST,
4060, 1:1000), PI3K (CST, 4249, 1:1000), Phospho-mTOR (CST,5536, 1:500),
Beclin1 (CST, 3495, 1:1000), ATG5 (CST, 12994 S, 1:500), LC3I/II (Novus,
NB600-1384, 1:500), Caspase 3 (Proteintech, 66470-2-Ig, 1:500), UGCG (SAB,
35753, 1:300), BCL2 (Proteintech, 12789-1-AP, 1:500), HA-Tag (CST, 3724 S,
1:1000), Flag-Tag (CST,8146 S, 1:1000), Tubulin (Zsbio, TA-10, 1:1000), and
GAPDH (Zsbio,TA-08, 1:1000). RESULTS LASP1 is associated with sphingomyelin metabolism and
regulates ECHS1 in CRC cells
We first evaluated the role of LASP1 in CRC lipid metabolism. As
shown in Fig. 1A, gene-set enrichment analysis (GSEA) demonstrated
a strong enrichment of phospholipids, including ceramide (GSE4382),
sphingosine kinase/sphingosine 1-phosphate (S1P_S1P2) (GSE77955),
and S1P_META (GSE77955). Next, liquid chromatography–mass
spectrometry technology (LC–MS) was applied to detect the
lipidomics of LASP1-overexpressing CRC cells (RKO-LASP1) and
control cells. Notably, as shown in Fig. 1B, abnormal metabolism of
phospholipids, including increased ceramide and decreased sphin-
gomyelin, was found in RKO-LASP1 cells compared with the control
group. Specifically,
the
expression
of
ceramide
(d18:1_18:0)
decreased, whereas the expression of Hex2Cer (d18:1_24:1) and SM
(d36:1) increased (Fig. 1C, D). Hexose-linked glycoceramides (HexCer),
including galactosylceramide (GalCer) and glucosylceramide (GluCer). To further study the underlying mechanism of LASP1-mediated
CRC-cell malignance, 2-D DIGE assay was used to screen the
differentially expressed proteins in SW480/ SW620 cells transfected
with LASP1 siRNA or control siRNA (Fig. S1). The results demonstrated
that ECHS1, an enzyme involved in lipid metabolism, was one of the
candidates for LASP1-modulated proteins (Fig. 1E, F). RT-PCR (Fig. 1G)
and Western blotting (Fig. 1H) results showed that LASP1 regulates
ECHS1 at the protein, rather than the RNA level. Furthermore, as
shown in Fig. 1I, the results of the Transwell assay demonstrated that
the ability of LASP1 to promote CRC-cell migration and invasion was
related to its regulation of ECHS1. Lipid extraction
F
l For sample preparation, 1500 μL of chloroform methanol mixed solution
(2:1) (precooled at −20 °C) and 100-mg glass beads were added into the
sample. The samples were frozen in liquid nitrogen and then lysed
ultrasonically at a frequency of 50 Hz. After adding ddH2O and standing on
ice for 10 min, centrifuge at 12,000 rpm for 5 min at room temperature. Take the lower layer and add 1000 μL of chloroform methanol mixed
solution (2:1) (precooled at −20 °C), then centrifuge at 12,000 rpm at room
temperature for 5 min. Remove the lower layer of liquid and add 200 μL of Cell Death and Disease (2021) 12:911 ECHS1 resists autophagy and apoptosis of CRC cells through
the PI3K-Akt-mTOR pathway mediated by ceramide One of the important mechanisms by which ceramide promotes
apoptosis is to promote lethal autophagy by inhibiting the PI3K/
Akt/mTOR pathway [21]. We doubt whether ECHS1 is related to
this mechanism. As shown in Fig. 4A, Western blot assays showed
that the PI3K/Akt/mTOR pathway in HCT116-LV-ECHS1 cells was
reduced compared to that in the control group and activated
when ECHS1 was knocked out in SW480 cells, and the opposite
results were achieved. In the recovery experiment, whether the
application of eliglustat, the inhibitor of UGCG, in ECHS1-
overexpressing cells or the addition of C2 ceramide to ECHS1-
knockout cells, the activation or inhibition of the PI3K/Akt/mTOR
pathway could be reversed accordingly (Fig. 4B). p
g
To verify whether LASP1 regulates phospholipid metabolism in
CRC through ECHS1, we knocked down ECHS1 in LASP1-
overexpressing cells and detected its lipidomics by LC–MS. As
shown in Fig. 2-G, after knocking down ECHS1, the imbalance of
phospholipid metabolism caused by LASP1 overexpression was
restored. Meanwhile, as shown in Fig. 2-H, single-fatty-acid
analysis showed that ceramide (d18:1_18:0) was increased and
Hex2Cer (d18:1_24:1) and SM (d44:2) were decreased when ECHS1
was knocked down in LASP1-overexpressing cells. Generally,
ECHS1
interacted
with
LASP1
and
regulated
ceramide
to
glycosylated ceramide in CRC. p
y
g y ( g
)
We also detected the expression of lethal autophagy-related
proteins, such as ATG5, Beclin1, and LC3. As shown in Fig. 4C, the
expression of ATG5 and Beclin1, autophagy-initiation-related
proteins, was reduced, and the LC3II/LC3I ratio was increased. Furthermore, after the application of Eliglustat to inhibit the
conversion of ceramide to glycosylated ceramide, the expression
of the lethal autophagy-related proteins BCL-2 and Beclin1 and
the apoptosis-related protein Caspase3 was restored (Fig. 4D). The
release of reactive oxygen species (ROS) and the changes in
mitochondrial membrane potential are usually thought to be
important signatures of mitochondrial dysfunction-induced apop-
tosis [22]. Therefore, flow cytometry and immunofluorescence
have been used to detect ROS and mitochondrial membrane
potential. As shown in Fig. 4E and F, ROS production (Fig. 4E)
increased
and
mitochondrial
membrane
potential
(Fig. 4F)
decreased when exogenous ECHS1 was overexpressed, which
could also be reversed by Eliglustat. Furthermore, we observed the
morphological changes of SW480sgECHS1 and the control group
by electron microscopy. The results showed that after knocking
out ECHS1, the mitochondrial double-membrane structure was
lost, and the dorsal membrane was blurred. The number of
autophagosomes near lysosomes also increased (Fig. 4G). ECHS1 resists autophagy and apoptosis of CRC cells through
the PI3K-Akt-mTOR pathway mediated by ceramide Finally,
we observed the effect of ECHS1 on drug-induced apoptosis of
CRC cells. As shown in Fig. 4H, endogenous overexpression of
ECHS1 reduced the drug-induced apoptosis of SW480 and HCT116
cells, and Eliglustat reversed this change. 2D),
therefore, we constructed full-length Flag-LASP1 (1–261a), Flag-
LASP1 (1–131 aa), Flag-LASP1 (60–198 aa), Flag-LASP1 (131–261
aa),
and
HA-ECHS1-overexpression
vectors
and
transfected
293 T cells. As shown in Fig. 2E, ECHS1 was detected by Co-IP
assay only after Flag-LASP1 (132–261aa) and full-length Flag-
LASP1 (1–261aa) were transfected into 293 T cells, which indicated
that ECHS1 might directly bind to the SH3 domain of LASP1. Meanwhile, as shown in Fig. 2F, western blot assays showed that
LASP1 prevented ECHS1 degradation induced by CHX only after
transfection with plasmids containing LASP1–SH3 domain. LASP1 evidently prevented ECHS1 degradation, thus prolonging
its
half-life. Furthermore,
western
blot
assay
showed
that
chloroquine can reverse the degradation of ECHS1 protein
prevented by LASP1 instead of MG132, indicating that LASP1
inhibits the degradation of ECHS1 by inhibiting its hydrolysis in
the
proteasome
(Fig. 2C). Given
that
LASP1 contains
the
N-terminal LIM domain and the C-terminal SH3 domain (Fig. 2D),
therefore, we constructed full-length Flag-LASP1 (1–261a), Flag-
LASP1 (1–131 aa), Flag-LASP1 (60–198 aa), Flag-LASP1 (131–261
aa),
and
HA-ECHS1-overexpression
vectors
and
transfected
293 T cells. As shown in Fig. 2E, ECHS1 was detected by Co-IP
assay only after Flag-LASP1 (132–261aa) and full-length Flag-
LASP1 (1–261aa) were transfected into 293 T cells, which indicated
that ECHS1 might directly bind to the SH3 domain of LASP1. Meanwhile, as shown in Fig. 2F, western blot assays showed that
LASP1 prevented ECHS1 degradation induced by CHX only after
transfection with plasmids containing LASP1–SH3 domain. a critical step to regulate the modulation of cellular activities by
regulating the metabolism of ceramide and glycosphingolipids
(GSLs) [20] (Fig. 3G). We then detected the expression of UGCG
while endogenous ECHS1 changed. As shown in Fig. 3H, western
blotting showed that the expression of UGCG and ECHS1 was
synchronous, and ELISA indicated that the ceramide decreased
when ECHS1 was knocked down (Fig. 3I), and it could be reversed
when Eliglustat, a specific inhibitor of UGCG, was applied, which
suggested that ECHS1 regulates UGCG to promote the ceramide
to glycosylated ceramide. B Heatmap of the LC–MS assay for RKO-LASP1 and control
cells. C Heat map depicting the differentially expressed lipomics for RKO-LASP1 and control cells. D Histogram of c. The asterisk (*) indicates P
< 0.05. The double asterisk (**) indicates P < 0.01. The quadra asterisk (****) indicates P < 0.0001. E Fluorescence images show the two
differentially expressed protein spots in DIGE analysis. The interacting protein spots are indicated (white arrows). F MS of in-gel trypsin digests
of the protein and analysis of the depicted peptide spectrum resulted in the identification of ECHS1. G, H RT-PCR (G) and Western blotting
(H) were performed to detect the expression of ECHS1 and LASP1 in LASP1-overexpressing and LASP1-knockdown cells. I Representative
figures and data of the Transwell assay for LV-control-, LV-LASP1-, and LV-LASP1 plus siRNA-ECHS1-transfected RKO cells. Bars in the right
panel represent the number of migrated and invaded cells. Fig. 1
LASP1 is associated with sphingomyelin metabolism and regulates ECHS1 in CRC cells. A GSEA of the top 100 upregulated LASP1-
related pathways, including biocarta ceramide, PID_S1P_S1P2 and PID_S1P_META. B Heatmap of the LC–MS assay for RKO-LASP1 and control
cells. C Heat map depicting the differentially expressed lipomics for RKO-LASP1 and control cells. D Histogram of c. The asterisk (*) indicates P
< 0.05. The double asterisk (**) indicates P < 0.01. The quadra asterisk (****) indicates P < 0.0001. E Fluorescence images show the two
differentially expressed protein spots in DIGE analysis. The interacting protein spots are indicated (white arrows). F MS of in-gel trypsin digests
of the protein and analysis of the depicted peptide spectrum resulted in the identification of ECHS1. G, H RT-PCR (G) and Western blotting
(H) were performed to detect the expression of ECHS1 and LASP1 in LASP1-overexpressing and LASP1-knockdown cells. I Representative
figures and data of the Transwell assay for LV-control-, LV-LASP1-, and LV-LASP1 plus siRNA-ECHS1-transfected RKO cells. Bars in the right
panel represent the number of migrated and invaded cells. LASP1 evidently prevented ECHS1 degradation, thus prolonging
its
half-life. Furthermore,
western
blot
assay
showed
that
chloroquine can reverse the degradation of ECHS1 protein
prevented by LASP1 instead of MG132, indicating that LASP1
inhibits the degradation of ECHS1 by inhibiting its hydrolysis in
the
proteasome
(Fig. 2C). Given
that
LASP1 contains
the
N-terminal LIM domain and the C-terminal SH3 domain (Fig. R. Li et al. 3 is essential for LASP1-mediated sphingomyelin-
bolism imbalance by interacting with SH3 domain of
in CRC
teraction between LASP1 and ECHS1 was further verified by
noprecipitation
(Co-IP)
assays
(Fig. 2A)
using
protein
extraction of SW480 cells. As shown in Fig. 2B, when
synthesis is blocked by CHX, ECHS1 gradually degrades w
CRC cells were transfected with plasmid-encoding L
control plasmid, and the degradation of ECHS1 pro
monitored after CHX treatment. Compared with the contr
P=0.0096
P=0.00
P=0.0041
ath and Disease (2021) 12:911 P=0.0041 P=0.00 P=0.0096 P=0.0096
P=0.00 P=0.0096
P=0.00
P=0.0041 extraction of SW480 cells. As shown in Fig. 2B, when protein
synthesis is blocked by CHX, ECHS1 gradually degrades with time. CRC cells were transfected with plasmid-encoding LASP1 or
control plasmid, and the degradation of ECHS1 protein was
monitored after CHX treatment. Compared with the control group, ECHS1 is essential for LASP1-mediated sphingomyelin-
metabolism imbalance by interacting with SH3 domain of
LASP1 in CRC The interaction between LASP1 and ECHS1 was further verified by
immunoprecipitation
(Co-IP)
assays
(Fig. 2A)
using
protein Cell Death and Disease (2021) 12:911 Fig. 1
LASP1 is associated with sphingomyelin metabolism and regulates ECHS1 in CRC cells. A GSEA of the top 100 upregulated LASP1-
related pathways, including biocarta ceramide, PID_S1P_S1P2 and PID_S1P_META. B Heatmap of the LC–MS assay for RKO-LASP1 and control
cells. C Heat map depicting the differentially expressed lipomics for RKO-LASP1 and control cells. D Histogram of c. The asterisk (*) indicates P
< 0.05. The double asterisk (**) indicates P < 0.01. The quadra asterisk (****) indicates P < 0.0001. E Fluorescence images show the two
differentially expressed protein spots in DIGE analysis. The interacting protein spots are indicated (white arrows). F MS of in-gel trypsin digests
of the protein and analysis of the depicted peptide spectrum resulted in the identification of ECHS1. G, H RT-PCR (G) and Western blotting
(H) were performed to detect the expression of ECHS1 and LASP1 in LASP1-overexpressing and LASP1-knockdown cells. I Representative
figures and data of the Transwell assay for LV-control-, LV-LASP1-, and LV-LASP1 plus siRNA-ECHS1-transfected RKO cells. Bars in the right
panel represent the number of migrated and invaded cells. R. Li et al. R. Li et al. 4 Fig. 1
LASP1 is associated with sphingomyelin metabolism and regulates ECHS1 in CRC cells. A GSEA of the top 100 upregulated LASP1-
related pathways, including biocarta ceramide, PID_S1P_S1P2 and PID_S1P_META. ECHS1 promotes the conversion of ceramide to glycosylated
ceramide by regulating UGCG y
g
g
ECHS1 is generally considered to be involved in the hydration step of
fatty-acid oxidative phosphorylation, but its expression and role in
CRC are still controversial. As shown in Fig. 3A and B, ECHS1 was
highly overexpressed in CRC cells either in mRNA or protein level
compared
with
nonneoplastic
cell
line
NCM460. To
further
investigate the biological behaviors of ECHS1, CRISPR–Cas9 was
used to construct ECHS1-knockout cells (SW480-sgECHS1), and
lentivirus was used to build ECHS1-overexpressing cells (HCT116-
LV-ECHS1) (Fig. 3C). Meanwhile, as shown in Fig. 3E, Transwell assays
showed that overexpression of ECHS1 dramatically enhanced the
migration and invasion of HCT116 cells, whereas knocking out ECHS1
inhibited the migration and invasion of SW480 cells. g
Then, we detected lipid changes in CRC cells by changing the
endogenous expression of ECHS1 only. As shown in Fig. 3D, after
overexpression of ECHS1, the number of HexCer in HCT116 cells
increased, whereas ceramide decreased. Consistent results were
obtained in SW480 cells with ECHS1 knocked out. Further analysis
focused on single-lipid metabolism. By taking the intersection of
the increased phospholipid of the overexpression group and the
decreased of the knockout group of ECHS1, we observed six kinds
of lipids with positive correlation with ECHS1, including 2
BisMePA, 2 HexCer, 1 SM, and 1 Cer (Fig. 3F). Combined with
the previous lipidomics results, we suspected that the dysregula-
tion of ECHS1 regulated by LASP1 was related to ceramide
glycosylation. UDP-glucose ceramide glycosyltransferase (UGCG) is ECHS1 contributes to aggressive phenotypes and drug
resistance of CRC cells both in vitro and in vivo
As shown in Fig. 5A and B, exogenous overexpression of ECHS1
dramatically enhanced the proliferation ability of HCT116 cells in ECHS1 contributes to aggressive phenotypes and drug
resistance of CRC cells both in vitro and in vivo ECHS1 contributes to aggressive phenotypes and drug
resistance of CRC cells both in vitro and in vivo As shown in Fig. 5A and B, exogenous overexpression of ECHS1
dramatically enhanced the proliferation ability of HCT116 cells in Cell Death and Disease (2021) 12:911 R. Li et al. g. 2
ECHS1 is essential for LASP1-mediated sphingomyelin-metabolism imbalance by interacting with SH3 domain of LASP1 in CR
Immunoprecipitation-assay analysis of endogenous interaction between ECHS1 and LASP1 in SW480 cells. B Western blot assay of LAS
nd ECHS1 protein and quantification of ECHS1 relative level at the indicated time in HCT116 cells transfected with siLASP1 or siNC (upp
gure) or RKO cells transfected with LV-LASP1 or LV-Ctrl (lower figure) after CHX treatment to block protein synthesis. C Western blot assay
e level of LASP1 and ECHS1 protein with application of chloroquine (10uM) or MG132 (10uM) after 8 h. D Schematic presentation
ASP1 structure features. The structures of truncated LASP1 are listed behind those of the full-length proteins. E Western blotting resu
dicate the binding domain of ECHS1 and LASP1 in 293 T cells. F Western blot assay of HA and ECHS1 protein in 293 T cells transfected w
asmid HA-LASP1 full length (1–261aa), HA-LASP1 (60–199aa), or HA-LASP1(131–261aa) after CHX treatment to block protein synthes
Heatmap of the LC–MS assay for RKO-LASP1 and RKO-LASP1 transfected with siECHS1 cells. H Heatmap and histogram of depicting t
fferentially expressed lipomics for RKO-LASP1 and RKO-LASP1 plus siRNA-ECHS1 cells. R. Li et al. 5 Fig. 2
ECHS1 is essential for LASP1-mediated sphingomyelin-metabolism imbalance by interacting with SH3 domain of LASP1 in CRC. A Immunoprecipitation-assay analysis of endogenous interaction between ECHS1 and LASP1 in SW480 cells. B Western blot assay of LASP1
and ECHS1 protein and quantification of ECHS1 relative level at the indicated time in HCT116 cells transfected with siLASP1 or siNC (upper
figure) or RKO cells transfected with LV-LASP1 or LV-Ctrl (lower figure) after CHX treatment to block protein synthesis. C Western blot assay of
the level of LASP1 and ECHS1 protein with application of chloroquine (10uM) or MG132 (10uM) after 8 h. D Schematic presentation of
LASP1 structure features. The structures of truncated LASP1 are listed behind those of the full-length proteins. E Western blotting results
indicate the binding domain of ECHS1 and LASP1 in 293 T cells. F Western blot assay of HA and ECHS1 protein in 293 T cells transfected with
plasmid HA-LASP1 full length (1–261aa), HA-LASP1 (60–199aa), or HA-LASP1(131–261aa) after CHX treatment to block protein synthesis. ECHS1 contributes to aggressive phenotypes and drug
resistance of CRC cells both in vitro and in vivo G Heatmap of the LC–MS assay for RKO-LASP1 and RKO-LASP1 transfected with siECHS1 cells. H Heatmap and histogram of depicting the
differentially expressed lipomics for RKO-LASP1 and RKO-LASP1 plus siRNA-ECHS1 cells. Cell Death and Disease (2021) 12:911 R. Li et al. 8 and clone-formation assays. Meanwhile, knockout of ECHS1
bited SW480 cells. In addition, knocking down endogenous
S1 by siRNA restored the proliferation of CRC cells caused by
P1 (Fig. S2, Fig. 5C). Additionally, in vivo subcutaneous tumor
ation in BALB/C nude mice supported a stimulatory effect of
ECHS1 on CRC cell proliferation (0.1 ± 0.05 g vs. 0.4 ± 0.3 g
0.01; Fig. 5D), while knocking out ECHS1 dramatically inhibite
growth of subcutaneous tumors (0.2 ± 0.05 g vs. 0.08 ± 0.05 g
0.01; Fig. 5E). We observed that endogenous overexpressio
ECHS1 could reduce Oxaliplatin-induced apoptosis of CRC 6 ECHS1 on CRC cell proliferation (0.1 ± 0.05 g vs. 0.4 ± 0.3 g, P <
0.01; Fig. 5D), while knocking out ECHS1 dramatically inhibited the
growth of subcutaneous tumors (0.2 ± 0.05 g vs. 0.08 ± 0.05 g, P <
0.01; Fig. 5E). We observed that endogenous overexpression of
ECHS1 could reduce Oxaliplatin-induced apoptosis of CRC cells, CCK-8 and clone-formation assays. Meanwhile, knockout of ECHS1
inhibited SW480 cells. In addition, knocking down endogenous
ECHS1 by siRNA restored the proliferation of CRC cells caused by
LASP1 (Fig. S2, Fig. 5C). Additionally, in vivo subcutaneous tumor
formation in BALB/C nude mice supported a stimulatory effect of Cell Death and Disease (2021) 12:911 Furthermore,
in vivo subcutaneous tumor formation in BALB/C nude mice was
significantly
increased
in
the
ECHS1-overexpressing
groups
compared with the control groups after the injection of Oxaliplatin
(1.5 mg/kg). Administration of Eliglustat (60 mg/kg) reversed
ECHS1-induced drug resistance and subcutaneous tumor growth
(Fig. 5G). Meanwhile, as shown in Fig. 5H, immunohistochemical
staining demonstrated that the expression of p-mTOR and the
mitochondrial membrane stability marker BCL2 was increased
when the autophagy-related indicator Beclin1 was decreased in
the HCT116-LV-ECHS1 group compared with the HCT116-Ctrl
group. Meanwhile, along with the increase in ECHS1 expression,
the number of Ki-67-positive tumor cells significantly increased in
tumors. More importantly, application of Eliglustat (60 mg/kg)
reversed the expression of pathway proteins activated by ECHS1. ECHS1 is a key enzyme that catalyzes the second step of the
β-oxidation pathway in fatty-acid metabolism. Apart from its
critical roles in regulating fatty acid metabolism, numerous
literatures have indicated that ECHS1 might be involved in the
development of tumor, including colon [14, 15], liver [27],
gastric [17], and renal [28] cancer. In CRC cell lines, it has been
reported that ECHS1 is able to suppress proliferation and
migration through PI3K–Akt–GSKβ signaling pathways [15], and
the knockdown of ECHS1 attenuates HCC proliferation by
impairing cell metabolism and inducing cell apoptosis and
autophagy by activating the AMP protein kinase (AMPK)
pathway [16]. Additionally, in the human gastric cancer cell
lines, the protein levels of ECHS1 were significantly higher than
those in nonneoplastic gastric epithelial mucosa cells [17]. Constitutive knockdown of ECHS1 significantly inhibited cell
proliferation and migration through the PI3K–PKB and GSK3β
signaling pathways. Although several studies have shown that
ECHS1 expression is absent in clear-cell renal-cell carcinoma
(ccRCC) [28] or CRC [29], Western blot and IHC assays clearly
showed that the expression of ECHS1 is highly expressed in
colon-cancer cells and tissues and associated with advanced
progression and poor prognosis based on our study. Over-
expression
of
ECHS1
could
promote
tumor-cell
invasion,
migration, and proliferation of CRC cells in vitro and in vivo. Upon further detection of cell-lipid metabolism by LC–MS,
except for the well-known function of ECHS1 in regulating FAO,
we found that after overexpression of ECHS1, the content of
ceramide decreased, while glycosylated ceramide increased. The results of the Western blot assay showed that ECHS1 could
regulate UGCG, which catalyzes the first step of ceramide
glycosylation to convert ceramide to glucosylceramide in
sphingolipid metabolism. R. Li et al. 7 Fig. 3
ECHS1 promotes the conversion of ceramide to glycosylated ceramide by regulating UGCG. A, B RT-PCR (A) and Western blot
analysis (B) were used to detect the expression of ECHS1 in CRC cell lines. C Western blot analysis showed that ECHS1-knockout and
-overexpression cells were constructed. D Left panel: heatmap of the LC–MS assay for SW480sgECHS1 and SW480sgNC cells. Right panel:
heatmap of the LC–MS assay for HCT116-LV-ECHS1 and HCT116-LV-Ctrl cells. E Representative figures and data of the Transwell assay for
SW480sgECHS1, SW480sgNC, and HCT116-LV-ECHS1 and HCT116-LV-Ctrl cells. Bars in the right panel represent the number of migrated and
invaded cells. The triple asterisk (***) indicates P < 0.001. F Upper panel: pi chart of lipids associated with the elevation of endogenous ECHS1. Bars in the right panel represent the lipids associated with phospholipid metabolism. Lower panel: pi chart of lipids associated with the
reduction in endogenous ECHS1. Bars in the right panel represent the lipids associated with phospholipid metabolism. G Schematic diagram
of UGCG regulating ceramide glycosylation. H Western blot analysis showed the expression of UGCG when endogenous ECHS1 changed in
CRC cells. I Histogram of ELISA results showing the expression of endogenous ceramide when ECHS1 was overexpressed or knocked out in
CRC cells of LASP1 and HSPA1A had been reported in neck squamous cell
carcinoma [23]. HSPA1A, which is also known as HSP70, was
widely found in the surface of the lysosome membrane, and
protected its chaperone protein from the ubiquitin–lysosomal-
degradation pathway by inhibiting lysosomal permeability [24]. Moreover, many studies had shown that HSP70 was not only
involved in the ubiquitination and subsequent degradation by
dissociating 26 S proteasome complexes (into free 20 S protea-
somes and bound 19 S regulators), preserving 19 S regulators, and
reconstituting 26 S proteasomes [25], but also could be cleaved
independent of ubiquitination by the 20 S proteasome since
HSP70 itself was a substrate of 20 S proteasomes [26]. Therefore,
LASP1 may regulate the proteasome-degradation pathway via its
chaperone protein. and then we wanted to explore whether endogenous ECHS1
affected the drug sensitivity of cells. As shown in Fig. 5F,
regardless of whether Oxaliplatin or 5-Fluorouracil was applied,
the
survival
rate
was
significantly
increased
in
ECHS1-
overexpressing cells and decreased in ECHS1-knockout cells
compared with the corresponding control cells. Thus, we suspect that ECHS1 affects
the phospholipid metabolism–glycosylation of ceramide in CRC
cells by regulating UGCG in CRC cells. Although numerous
studies have demonstrated abnormal metabolism of phospho-
lipids in CRC progression, the underlying mechanism remains
unclear. Overexpression of ECHS1 is associated with tumor
progression and poor prognosis of CRC p
g
p
p
g
As shown in Fig.6A, the immunohistochemical results showed that
relatively high expression of ECHS1 was often observed in CRC
samples overexpressing LASP1 (R = 0.62, P < 0.0001). Next, by
detecting the expression of ECHS1 in six CRC tissues, the western
blot assay results showed that the expression of ECHS1 was
overexpressed in CRC tissues compared with matched nontumor
tissues (Fig. 6B). Immunohistochemistry further demonstrated that
the overexpression of ECHS1 was significantly upregulated in 64%
(25/39) of the CRC tissues compared with 30% (12/39) of the
adjacent normal tissues (Fig. 6C, P < 0.001). IHC also demonstrated
that ECHS1 overexpressed at a much higher rate in CRC tissues
with distant lymph-node metastasis (N1 + N2) (Fig. 6C, P < 0.05). Kaplan–Meier survival analysis of a previously published CRC
dataset (TCGA-READ, n = 79) revealed that ECHS1 expression is
closely correlated with patient overall survival (OR) and disease-
free survival (DSS). Patients with low ECHS1 expression had a
significantly better prognosis (Fig. 6D). DISCUSSION A Western blot
analysis of the expression of PI3K/Akt/mTOR pathway members (PI3K, Akt, phosphorylated AKT at Ser473, and phosphorylated mTOR) in
ECHS1-overexpressing or ECHS1-knockout CRC cells. B Western blot analysis of the expression of PI3K/Akt/mTOR pathway members in ECHS1-
overexpressing cells with the application of the inhibitor of UGCG eliglustat (left panel) or in ECHS1-knockout cells with the addition of C2
ceramide (right panel). C Western blot analysis of the expression of autophagy-related proteins (ATG5, Beclin1, and LC3II/LC3I). D Western blot
analysis of phosphorylated mTOR, Beclin1, BCL2, and caspase3 in CRC cells with or without ECHS1 overexpression and Eliglustat (100 nmol). E Flow cytometry analysis of intracellular ROS content. Bars in the right panel represent the average fluorescence intensity. F IF assay analysis
of JC-10 aggregates (red) and monomers (green) in the mitochondrial matrix with or without starvation (2 h) and Eliglustat (100 nmol). Bars of
the right panel represent the ratio of the average fluorescence intensity of monomers (green) and aggregates (red). G Electron microscopy
results show mitochondrial morphology (yellow arrows) and autophagosomes (red arrows) in ECHS1-knockout SW480 cells. H Histogram of
flow cytometry assay showing the ratio of the average fluorescence intensity of apoptotic cells with or without Oxaliplatin (20 µg/µl) and
Eliglustat (100 nmol). Fig. 4
ECHS1 resists autophagy and apoptosis of CRC cells through the PI3K-Akt-mTOR pathway mediated by ceramide. A Western blot
analysis of the expression of PI3K/Akt/mTOR pathway members (PI3K, Akt, phosphorylated AKT at Ser473, and phosphorylated mTOR) in
ECHS1-overexpressing or ECHS1-knockout CRC cells. B Western blot analysis of the expression of PI3K/Akt/mTOR pathway members in ECHS1-
overexpressing cells with the application of the inhibitor of UGCG eliglustat (left panel) or in ECHS1-knockout cells with the addition of C2
ceramide (right panel). C Western blot analysis of the expression of autophagy-related proteins (ATG5, Beclin1, and LC3II/LC3I). D Western blot
analysis of phosphorylated mTOR, Beclin1, BCL2, and caspase3 in CRC cells with or without ECHS1 overexpression and Eliglustat (100 nmol). E Flow cytometry analysis of intracellular ROS content. Bars in the right panel represent the average fluorescence intensity. F IF assay analysis
of JC-10 aggregates (red) and monomers (green) in the mitochondrial matrix with or without starvation (2 h) and Eliglustat (100 nmol). Bars of
the right panel represent the ratio of the average fluorescence intensity of monomers (green) and aggregates (red). DISCUSSION LIM and SH3 protein 1 (LASP1) has been found overexpressed in
the amount of tumors including breast cancer [3] and ovarian
cancer [4]. According to our previous study, LASP1 was highly
overexpressed in CRC tissues, which is closely associated with
lymph nodes and distant metastases and the poor prognosis of
patients [6]. In this study, we first found that LASP1 is related to
the metabolism of phospholipids, especially ceramides, in tumor
cells, and regulates the lipid-metabolism enzyme ECHS1. Simulta-
neously, ECHS1 tends to interact with the SH3 domain at the C
terminus of LASP1 to stimulate ceramide metabolism and cancer
aggressiveness. As for the mechanism behind LASP1-driven
prevention of ECHS1 degradation in the proteasome, interaction Ceramide (Cer) is a tumor suppressor that can enhance the signal-
initiating apoptosis, autophagy, and cell-cycle arrest, and its
reduction helps prolong the survival time of cancer cells [30]. Mechanistic research clarified that ceramide triggers autophagy by Cell Death and Disease (2021) 12:911 Fig. 4
ECHS1 resists autophagy and apoptosis of CRC cells through the PI3K-Akt-mTOR pathway mediated by ceramide. A Western
analysis of the expression of PI3K/Akt/mTOR pathway members (PI3K, Akt, phosphorylated AKT at Ser473, and phosphorylated mTO
ECHS1-overexpressing or ECHS1-knockout CRC cells. B Western blot analysis of the expression of PI3K/Akt/mTOR pathway members in EC
overexpressing cells with the application of the inhibitor of UGCG eliglustat (left panel) or in ECHS1-knockout cells with the addition
ceramide (right panel). C Western blot analysis of the expression of autophagy-related proteins (ATG5, Beclin1, and LC3II/LC3I). D Western
analysis of phosphorylated mTOR, Beclin1, BCL2, and caspase3 in CRC cells with or without ECHS1 overexpression and Eliglustat (100 n
E Flow cytometry analysis of intracellular ROS content. Bars in the right panel represent the average fluorescence intensity. F IF assay an
of JC-10 aggregates (red) and monomers (green) in the mitochondrial matrix with or without starvation (2 h) and Eliglustat (100 nmol). Ba
the right panel represent the ratio of the average fluorescence intensity of monomers (green) and aggregates (red). G Electron micros
results show mitochondrial morphology (yellow arrows) and autophagosomes (red arrows) in ECHS1-knockout SW480 cells. H Histogra
flow cytometry assay showing the ratio of the average fluorescence intensity of apoptotic cells with or without Oxaliplatin (20 µg/µl
Eliglustat (100 nmol). R. Li et al. R. Li et al. 8 Fig. 4
ECHS1 resists autophagy and apoptosis of CRC cells through the PI3K-Akt-mTOR pathway mediated by ceramide. DISCUSSION G Electron microscopy
results show mitochondrial morphology (yellow arrows) and autophagosomes (red arrows) in ECHS1-knockout SW480 cells. H Histogram of
flow cytometry assay showing the ratio of the average fluorescence intensity of apoptotic cells with or without Oxaliplatin (20 µg/µl) and
Eliglustat (100 nmol). Cell Death and Disease (2021) 12:911 R. Li et al. 9 Fig. 5
ECHS1 contributes to aggressive phenotypes and drug resistance of CRC cells, both in vitro and in vivo. A Representative figures o
the clone-formation assay for the indicated cells. Bars on the right represent the number of formed clones. B CCK-8 assay for ECHS1 loss- an
gain-of-function analysis of SW480 cells and HCT116 cells on CRC cell proliferation. C CCK-8 assay for LV-Ctrl-, LV-LASP1-, and LV-LASP1 plu
siRNA-ECHS1-transfected RKO cells. D, E LV-Ctrl and LV-ECHS1 HCT116 cells (D) and sgNC and sgECHS1 SW480 cells (E) were injecte
subcutaneously into the backs of nude mice to access tumor growth. The representative figures of the tumors are shown. The right ba
represents the weight of the tumors. The double asterisk (**) indicates P < 0.01. F CCK-8 assay analysis of cell viability with gradient applicatio
of 5-Fluorouracil and Oxaliplatin. G LV-Ctrl and LV-ECHS1 HCT116 cells were injected subcutaneously into the backs of nude mice with o
without Oxaliplatin (1.5 mg/kg) and Eliglustat (60 mg/kg) to evaluate tumor growth. A representative figure of the tumors is shown. The righ
bar represents the weight of the tumors. H Subcellular localization of Ki-67, p-mTOR, Beclin1, and BCL2 in the indicated cells was assesse
through immunohistochemical staining. Fig. 5
ECHS1 contributes to aggressive phenotypes and drug resistance of CRC cells, both in vitro and in vivo. A Representative figures of
the clone-formation assay for the indicated cells. Bars on the right represent the number of formed clones. B CCK-8 assay for ECHS1 loss- and
gain-of-function analysis of SW480 cells and HCT116 cells on CRC cell proliferation. C CCK-8 assay for LV-Ctrl-, LV-LASP1-, and LV-LASP1 plus
siRNA-ECHS1-transfected RKO cells. D, E LV-Ctrl and LV-ECHS1 HCT116 cells (D) and sgNC and sgECHS1 SW480 cells (E) were injected
subcutaneously into the backs of nude mice to access tumor growth. The representative figures of the tumors are shown. The right bar
represents the weight of the tumors. The double asterisk (**) indicates P < 0.01. F CCK-8 assay analysis of cell viability with gradient application
of 5-Fluorouracil and Oxaliplatin. DISCUSSION G LV-Ctrl and LV-ECHS1 HCT116 cells were injected subcutaneously into the backs of nude mice with or
without Oxaliplatin (1.5 mg/kg) and Eliglustat (60 mg/kg) to evaluate tumor growth. A representative figure of the tumors is shown. The right
bar represents the weight of the tumors. H Subcellular localization of Ki-67, p-mTOR, Beclin1, and BCL2 in the indicated cells was assessed
through immunohistochemical staining. Cell Death and Disease (2021) 12:911 regulating the mTOR signaling pathway and interfering with Beclin 1:
BCL-2 complex in a phosphorylation-dependent manner mediated
important mechanism of the mitochondrial pathway is leading to ce
apoptosis In summary ECHS1 resists apoptosis and autophagy o
Fig. 6
Overexpression of ECHS1 is associated with tumor progression and poor prognosis of CRC. A Representative images of IHC stainin
analyses of LASP1 and ECHS1 in CRC tissues. B Western blot analysis of ECHS1 in CRC tissues (T) and adjacent nontumor tissues (N). Th
scatterplot on the right shows the relative expression of ECHS1 in normal and CRC tissues (normalized to GAPDH, P < 0.05). C Left pane
immunohistochemistry (IHC) analysis of ECHS1 in CRC tissues and adjacent nontumor tissues. Right panel: histogram representing IHC result
in CRC tissues (T) and adjacent nontumor tissues (N) and with or without lymph-node metastasis. D Kaplan–Meier survival curves an
univariate analyses (log rank) for CRC patients with distinct expression levels of ECHS1. E Sketch map of the mechanism of ECHS1-mediate
CRC proliferation and drug resistance. R. Li et al. 0 10 Fig. 6
Overexpression of ECHS1 is associated with tumor progression and poor prognosis of CRC. A Representative images of IHC stain
analyses of LASP1 and ECHS1 in CRC tissues. B Western blot analysis of ECHS1 in CRC tissues (T) and adjacent nontumor tissues (N). T
scatterplot on the right shows the relative expression of ECHS1 in normal and CRC tissues (normalized to GAPDH, P < 0.05). C Left pa
immunohistochemistry (IHC) analysis of ECHS1 in CRC tissues and adjacent nontumor tissues. Right panel: histogram representing IHC res
in CRC tissues (T) and adjacent nontumor tissues (N) and with or without lymph-node metastasis. D Kaplan–Meier survival curves a
univariate analyses (log rank) for CRC patients with distinct expression levels of ECHS1. E Sketch map of the mechanism of ECHS1-media
CRC proliferation and drug resistance. Fig. 6
Overexpression of ECHS1 is associated with tumor progression and poor prognosis of CRC. DISCUSSION 11 cell lines [33]. Different from Cer and SM, glycosphingolipids,
including LacCers and HexCers (GalCers and GluCers), are generally
regarded as an important biomarker for maintaining tumor growth
and promoting chemotherapy resistance [34]. Glucosylceramide
synthase has been observed upregulated in a series of tumors after
drug treatment, which suggests that glycolipids may be involved in
promoting tumor-drug resistance [35]. In CRC cells, GalCer and
LacCer had been reported that characteristically increased in
Oxaliplatin-resistant cells and the overexpression of UGCG gene is
significantly associated with the decrease in disease-free survival of
patients with colorectal cancer, which suggests that glycosylation
plays a pivotal role in Oxaliplatin chemosensitivity [36]. cell lines [33]. Different from Cer and SM, glycosphingolipids,
including LacCers and HexCers (GalCers and GluCers), are generally
regarded as an important biomarker for maintaining tumor growth
and promoting chemotherapy resistance [34]. Glucosylceramide
synthase has been observed upregulated in a series of tumors after
drug treatment, which suggests that glycolipids may be involved in
promoting tumor-drug resistance [35]. In CRC cells, GalCer and
LacCer had been reported that characteristically increased in
Oxaliplatin-resistant cells and the overexpression of UGCG gene is
significantly associated with the decrease in disease-free survival of
patients with colorectal cancer, which suggests that glycosylation
plays a pivotal role in Oxaliplatin chemosensitivity [36]. 11. Wang W, Ji G, Xiao X, Chen X, Qin WW, Yang F, et al. Epigenetically regulated miR-
145 suppresses colon cancer invasion and metastasis by targeting LASP1. Oncotarget. 2016;7:68674–87. 12. Kanazawa M, Ohtake A, Abe H, Yamamoto S, Satoh Y, Takayanagi M, et al. Molecular cloning and sequence analysis of the cDNA for human mitochondrial
short-chain enoyl-CoA hydratase. Enzyme Protein. 1993;47:9–13. 13. Sharpe AJ, McKenzie M. Mitochondrial fatty acid oxidation disorders associated
with short-chain Enoyl-CoA hydratase (ECHS1) deficiency. Cells. 2018; 7: 46. 14. Xie JP, Zhu XS, Dai YC, Yu C, Xie T, Chen ZX. Expression of enoyl coenzyme A
hydratase, short chain, 1, in colorectal cancer and its association with clin-
icopathological characteristics. Mol Clin Oncol. 2014;2:1081–4. 15. Zhao QM, Kuang F, Wu H, Zhang YH. Attenuation of enoyl coenzyme A hydratase
1 expression in colorectal cancer cells using small interfering RNA inhibits cell
proliferation and migration. Mol Med Rep. 2015;12:470–4. In this study, we found that both in vivo and in vitro ECHS1
promoted UGCG-mediated ceramide glycosylation, which led to
drug resistance, including 5-Fluorouracil and Oxaliplatin, in CRC
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I 19. Dunn WB, Broadhurst D, Begley P, Zelena E, Francis-McIntyre S, Anderson N, et al. Procedures for large-scale metabolic profiling of serum and plasma using gas
chromatography and liquid chromatography coupled to mass spectrometry. Nat
Protoc. 2011;6:1060–83. In summary, we found that LASP1 is associated with sphingo-
myelin metabolism in CRC cells and identified ECHS1 as a new
LASP1-interacting protein. ECHS1 regulates UGCG-mediated cer-
amide glycosylation, which further stimulates CRC progression
and resists apoptosis and autophagy through the PI3K/Akt/mTOR
pathway. Furthermore, the application of Eliglustat could restore
chemotherapeutic drug resistance caused by ECHS1-mediated
phospholipid metabolism disorders (Fig. 6E). The current research
illustrates ECHS1 as a novel predictive biomarker and provides a
new insight for clinical CRC chemotherapy resistance. 20. Ichikawa S, Sakiyama H, Suzuki G, Hidari KI, Hirabayashi Y. Expression cloning of a
cDNA for human ceramide glucosyltransferase that catalyzes the first glycosyla-
tion step of glycosphingolipid synthesis. Proc Natl Acad Sci USA. 1996;93:12654. 21. Jęśko H, Stępień A, Lukiw WJ, Strosznajder RP. The cross-talk between sphingo-
lipids and insulin-like growth factor signaling: significance for aging and neuro-
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metastasis, invasion in head and neck squamous cell carcinoma and through
direct interaction with HSPA1A. J Cell Mol Med. 2020;24:1626–39. DISCUSSION More importantly, the application of Eliglustat, an oral UGCG
inhibitor, could reverse the drug resistance caused by ECHS1,
which provides a new target for chemotherapy of CRC. 16. Zhu XS, Dai YC, Chen ZX, Xie JP, Zeng W, Lin YY, et al. Knockdown of ECHS1
protein expression inhibits hepatocellular carcinoma cell proliferation via sup-
pression of Akt activity. Crit Rev Eukaryot gene Expr. 2013;23:275–82. 17. Zhu XS, Gao P, Dai YC, Xie JP, Zeng W, Lian QN. Attenuation of enoyl coenzyme A
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and migration in vitro. Cell Mol Biol Lett. 2014;19:576–89. 18. Vykoukal J, Fahrmann JF, Gregg JR, Tang Z, Basourakos S, Irajizad E, et al. Caveolin-1-mediated sphingolipid oncometabolism underlies a metabolic vul-
nerability of prostate cancer. Nat Commun. 2020, 11: 4279. DISCUSSION A Representative images of IHC staining
analyses of LASP1 and ECHS1 in CRC tissues. B Western blot analysis of ECHS1 in CRC tissues (T) and adjacent nontumor tissues (N). The
scatterplot on the right shows the relative expression of ECHS1 in normal and CRC tissues (normalized to GAPDH, P < 0.05). C Left panel:
immunohistochemistry (IHC) analysis of ECHS1 in CRC tissues and adjacent nontumor tissues. Right panel: histogram representing IHC results
in CRC tissues (T) and adjacent nontumor tissues (N) and with or without lymph-node metastasis. D Kaplan–Meier survival curves and
univariate analyses (log rank) for CRC patients with distinct expression levels of ECHS1. E Sketch map of the mechanism of ECHS1-mediated
CRC proliferation and drug resistance. important mechanism of the mitochondrial pathway is leading to cell
apoptosis. In summary, ECHS1 resists apoptosis and autophagy of
CRC cells through the ceramide-mediated PI3K–Akt–mTOR pathway. Evidence suggests that the sphingolipidome plays an important
role in cancer-drug resistance. It has been reported that in the
human ovarian carcinoma-cell lines, application of drugs such as
Taxol could change the phospholipid metabolism of tumor cells to
activate SMase to generate Cer [32]. Similarly, under the induction of
drugs, lung-cancer cell A549 produces more dihydroceramide and
ceramide through de novo synthesis than matched drug-resistant important mechanism of the mitochondrial pathway is leading to cell
apoptosis. In summary, ECHS1 resists apoptosis and autophagy of
CRC cells through the ceramide-mediated PI3K–Akt–mTOR pathway. regulating the mTOR signaling pathway and interfering with Beclin 1:
BCL-2 complex in a phosphorylation-dependent manner mediated
by c-Jun N-terminal kinase 1 (JNK1) [31]. Western blot results showed
that
the
PI3K/Akt/mTOR
pathway
was
suppressed
and
that
autophagy-related proteins, such as ATG5, Beclin1, and LC3II/LC3I,
were activated when ECHS1 was knocked out in CRC cells, which
could be resorted by Eligustat, an inhibitor of UGCG. Thus, flow
cytometry and immunofluorescence assays showed that ROS
production
increased
and
mitochondrial
membrane
potential
decreased when exogenous ECHS1 was knocked out, in which an Evidence suggests that the sphingolipidome plays an important
role in cancer-drug resistance. It has been reported that in the
human ovarian carcinoma-cell lines, application of drugs such as
Taxol could change the phospholipid metabolism of tumor cells to
activate SMase to generate Cer [32]. Similarly, under the induction of
drugs, lung-cancer cell A549 produces more dihydroceramide and
ceramide through de novo synthesis than matched drug-resistant Cell Death and Disease (2021) 12:911 R. Li et al. Reprints and permission information is available at http://www.nature.com/
reprints Reprints and permission information is available at http://www.nature.com/
reprints Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims
in published maps and institutional affiliations. REFERENCES Wang H, Shi J, Luo Y, Liao Q, Niu Y, Zhang F, et al. LIM and SH3 protein 1 induces
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synthase and cancer drug resistance. Adv Cancer Res. 2013;117:59–89. 9. Xu L, Zhang Y, Wang H, Zhang G, Ding Y, Zhao L. Tumor suppressor
miR-1
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colorectal carcinoma via the MAPK and PI3K/AKT pathway. J Transl Med. 2014;12:244. 35. Kopecka J, Trouillas P, Gašparović A, Gazzano E, Assaraf YG, Riganti C. Phos-
pholipids and cholesterol: Inducers of cancer multidrug resistance and ther-
apeutic targets. Drug Resist Updat. 2020;49:100670. 10. Wang H, An H, Wang B, Liao Q, Li W, Jin X. et al. miR-133a represses
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Cancer. 2013;49:3924–35. 36. Madigan JP, Robey RW, Poprawski JE, Huang H, Clarke CJ, Gottesman MM, et al. A
role for ceramide glycosylation in resistance to oxaliplatin in colorectal cancer. Exp Cell Res. 2020;388:111860. Cell Death and Disease (2021) 12:911 R. Li et al. 12 Correspondence and requests for materials should be addressed to Liang Zhao. AUTHOR CONTRIBUTIONS Correspondence and requests for materials should be addressed to Liang Zhao. LZ led the study design and prepared the manuscript. RL, Y-YH and Q-HW carried out
the experiments. YM and K-HW performed statistical analysis; C-QL and L-JX assisted
in tissue sample collection. Z-GL performed data analysis and interpretation. All the
authors have approved the final version of the paper. FUNDING INFORMATION This work was supported by the National Natural Science Foundation of China (Nos. 81972813, 81902946), China Postdoctoral Science Foundation (F119280111), and
Guangdong Basic and Applied Basic Research Foundation (2019A1515010974). Open Access This article is licensed under a Creative Commons
Attribution 4.0 International License, which permits use, sharing,
adaptation, distribution and reproduction in any medium or format, as long as you give
appropriate credit to the original author(s) and the source, provide a link to the Creative
Commons license, and indicate if changes were made. The images or other third party
material in this article are included in the article’s Creative Commons license, unless
indicated otherwise in a credit line to the material. If material is not included in the
article’s Creative Commons license and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly
from the copyright holder. To view a copy of this license, visit http://creativecommons. org/licenses/by/4.0/. © The Author(s) 2021 ETHICS APPROVAL AND CONSENT TO PARTICIPATE All experiments involving patients are endorsed by the Ethics Committee of Southern
Medical University and complied with the Declaration of Helsinki. No informed
consent was required because data were going to be analyzed anonymously. All
animal experiments involved ethical and humane treatment under a license from the
Guangdong Provincial Bureau of Science. © The Author(s) 2021 ADDITIONAL INFORMATION Supplementary information The online version contains supplementary material
available at https://doi.org/10.1038/s41419-021-04213-6. Supplementary information The online version contains supplementary material
available at https://doi.org/10.1038/s41419-021-04213-6. Cell Death and Disease (2021) 12:911
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Digitalisation in accounting
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Вестник университета № 2, 2022 Вестник университета № 2, 2022 УДК 330.101.22 JEL M41
Гилева Дарья Валерьевна
студент магистратуры, ЧОУ ВО ЮУ
«Южный университет (Институт
управления бизнеса и права)»,
г. Ростов-на-Дону, Российская
Федерация
ORCID: 0000-0002-5220-860Х
e-mail: d.giliova@yandex.ru Darya V. Gileva
Graduate Student, Southern University
(Institute of Business Management and
Law), Rostov-on-Don, Russia
ORCID: 0000-0002-5220-860Х
e-mail: d.giliova@yandex.ru DOI 10.26425/1816-4277-2022-2-108-113
ЦИФРОВИЗАЦИЯ В БУХГАЛТЕРСКОМ УЧЕТЕ Аннотация. Целью статьи является выявление проблем, возникающих в процессе
цифровизации в бухгалтерском учете, рассмотрение способов внедрения цифровой
трансформации, и предложения по повышению качества и эффективности работы
бухгалтерского аппарата предприятия. В соответствии с поставленной целью, определены
следующие задачи исследования: обосновать необходимость внедрения цифровизации в сферу
бухгалтерского учета, определить ключевые цифровые технологии, выявить проблемы
и преимущества трансформации. В ходе исследования предложены способы решения
актуальных проблем, таких как планирование цифровой трансформации и отсутствие
квалифицированных сотрудников в данной сфере. Научная новизна данной статьи
заключается в предпосылках внедрения цифровых технологий в бухгалтерию предприятия. В качестве результатов исследования выявлена необходимость составления компетенций
по цифровым технологиям и внедрения цифровизации в бухгалтерию. Основным выводом
статьи является необходимость цифровизации аспекта бухгалтерского учета с целью
формирования достоверных данных о финансовом положении компании. ORCID: 0000-0002-5220-860Х
e-mail: d.giliova@yandex.ru Ключевые слова: цифровая экономика, цифровые технологии, цифровая трансформация,
цифровизация, диджитализация, бухгалтерский учет, цифровые компетенции, дорожная карта Ключевые слова: цифровая экономика, цифровые технологии, цифровая трансформация,
цифровизация, диджитализация, бухгалтерский учет, цифровые компетенции, дорожная карта Для цитирования: Гилева Д.В. Цифровизация в бухгалтерском учете//Вестник университета. 2022. № 2. С. 108–113. DIGITALISATION IN ACCOUNTING Abstract. The aim of the article is to identify the problems encountered in the digitalisation pro-
cess in accounting, consider ways of implementing digital transformation, and proposals to im-
prove the enterprise’s accounting apparatus quality and efficiency. In accordance with this aim,
the following research objectives have been defined: to justify the need to introduce digitalisa-
tion in the accounting field, determine key digital technologies, identify the transformation prob-
lems and advantages. In the course of the study, ways to solve urgent problems have been pro-
posed, such as planning for digital transformation and the qualified employees lack in this field. The scientific novelty of this article lies in the prerequisites for the digital technologies intro-
duction into the enterprise accounting department. As the results of the study, the need to com-
pile competencies in digital technologies and introduce digitalisation into accounting has been
identified. The main conclusion of the article is the necessity to digitalise the accounting aspect
in order to generate reliable data on a company’s financial position. Keywords: digital economy, digital technologies, digital transformation, digitalisation, digitalisation,
accounting, digital competencies, roadmap For citation: Gileva D.V. (2022) Digitalisation in accounting. Vestnik universiteta, no. 2, pp. 108–113. DOI: 10.26425/1816-4277-2022-2-108-113 © Гилева Д.В., 2022.
Статья доступна по лицензии Creative Commons «Attribution» («Атрибуция») 4.0. всемирная (http://creativecommons.org/licenses/by/4.0/).
© Gileva D.V., 2022.
This is an open access article under the CC BY 4.0 license (http://creativecommons.org/licenses/by/4.0/). This is an open access article under the CC BY 4.0 license (http://creativecommons.org/licenses/by/4.0/). 22.
по лицензии Creative Commons «Attribution» («Атрибуция») 4.0. всемирная (http://creativecommons.org/licenses/by/4.0/).
22 УДК 330.101.22 JEL M41
Гилева Дарья Валерьевна
студент магистратуры, ЧОУ ВО ЮУ
«Южный университет (Институт
управления бизнеса и права)»,
г. Ростов-на-Дону, Российская
Федерация
ORCID: 0000-0002-5220-860Х
e-mail: d.giliova@yandex.ru © Гилева Д.В., 2022.
Статья доступна по лицензии Creative Commons «Attribution» («Атрибуция») 4.0. всемирная (http://creativecommons.org/licenses/by/4.0/).
© Gileva D.V., 2022.
This is an open access article under the CC BY 4 0 license (http://creativecommons org/licenses/by/4 0/) Введение Преобразования, осуществляемые в нашей стране, направлены на формирование эффективной экономи-
ческой системы. В этом плане актуальным остается вопрос создания рынка на микроуровне, то есть на уров-
не деятельности предприятий. В данном отношении рынок следует рассматривать как экономическую сре-
ду, в которой функционирует предприятие. 108 Экономика: проблемы, решения и перспективы Новые условия организации реализации бизнеса потребовали, как внедрения принципиально новых спо-
собов хозяйствования, так и изменения взглядов на сущность и содержание основных функций, для обеспе-
чения ритмичной и доходной деятельности предприятия. Новые условия организации реализации бизнеса потребовали, как внедрения принципиально новых спо-
собов хозяйствования, так и изменения взглядов на сущность и содержание основных функций, для обеспе-
чения ритмичной и доходной деятельности предприятия. Экономика любой страны представляет собой сложный экономический механизм, различные элементы
которого взаимодействуют между собой и обусловливают взаимное развитие друг друга. Она представляет
собой огромное число хозяйствующих субъектов, создающих разнообразные товары и услуги. В современном информационном зависимом обществе, любые вопросы решаются только через инфор-
мационные системы, а прогресс требует перехода на новейшие технологии. О необходимости осуществлять
экономическую деятельность с помощью цифровых технологий уже никто не спорит, но вопрос о том, как
это реализовать и какой должна быть цифровая экономика остается открытым. Цифровизацию рассматривают как тренд мирового развития, в том случае, если данная трансформация
охватывает все сферы деятельности (производство, бизнес, наука, обычная жизнь граждан) [2]. Основным фактором, влияющим на деятельность бизнеса в условиях цифровой экономики, становится об-
работка значительных массивов данных, представленных в цифровом формате. Применение результатов ана-
лиза этих данных, может повысить эффективность бизнеса и вывести его на качественно новый уровень [7]. б
ф
б
б Теория и методы Стратегия цифровизации успешно реализуется по всему миру руководителями ИТ-организаций и пред-
приятий различного уровня. Она порождает технологический сдвиг, в короткие сроки преображая бизнес-
среду, выявляя новых лидеров, и заставляя компании расти и бороться на конкурирующем рынке [6]. Первым этапом цифровизации должно стать изменение бизнес процессов в компании. Следует изучить
слабые места в текущих решениях и составить список недостатков, для дальнейшей их ликвидации, от ко-
торых впоследствии отталкиваться при принятии решений о трансформации бизнеса [6]. Необходимо отметить, что в первую очередь цифровизация бизнеса необходима для компаний, работа-
ющих в жесткой конкурентной среде. Для таких компаний задержка в этапе цифровизации или отказ от нее,
может привести к поражению в конкурентной среде. Во-вторых, это компании, которые в своей отрасли при-
бегают к большим массивам данных [6]. На сегодняшний день стоимость цифровой трансформации бизнеса велика, однако она оправдывает за-
траты уже в первые месяцы после инноваций. Это происходит за счет уменьшения издержек на традицион-
ное обслуживание и ресурсозатраты со стороны персонала и материалов [6]. Значение контроля в условиях цифровой экономики усиливается, в связи с использованием в процессе
управления больших баз данных интернет носителей. Контроль является важным функциональным элемен-
том любой системы управления. В корпоративной системе управления внутренний контроль позволяет вы-
явить и оптимизировать мало результативные бизнес процессы [7]. Бухгалтерский учет, является значимым показателем экономической политики компании, и отражает
полную картину ее имущественного и финансового состояния. К ведению бухгалтерского учета на всех предприятиях, независимо от форм собственности предъявля-
ются одинаковые требования. Они вытекают из нормативно-правовых документов, так как время и научно-
технический прогресс не ведут себя размеренно, что обусловлено следующими признаками: автоматизация
и внедрение цифровизации в сферу бизнеса. Квалифицированный бухгалтерский учет способен предоставить информацию не только о структуре
имущества и финансовом положении фирмы, но и отразить эффективность финансово-хозяйственной дея-
тельности, и дать объективную оценку стоимости бизнеса. Исходя из вышеизложенного, в современных и быстроизменяющихся условиях развития экономики,
предприятия должны за короткое время уметь адаптироваться к новым способам ведения бизнеса. зложенного, в современных и быстроизменяющихся условиях развития экономики
за короткое время уметь адаптироваться к новым способам ведения бизнеса. Цифровизация бухгалтерского учета – это создание новых, и модернизация устаревших программ для
систематизации, анализа и расчета показателей [3]. Цифровизация сектора бухгалтерского учета способствует внедрению и раскрытию комплекса цифро-
вых технологий (рис. 1). Теория и методы 109 Вестник университета № 2, 2022 Цифровые технологии в бухгалтерском учете
Большие данные и
предиктивная аналитика
Блокчейн
Оптическое
распознавание
Смарт-пространство
Цифровые двойники
Электронный
документооборот
(ЭДО)
Квалифицированная
электронная
подпись (КЭП)
Скорость и качество обработки больших
данных влияет на эффективность и произво-
дительность компаний; предиктивная анали-
тика применяется для анализа больших
объемов данных и формирования прогнозов
Информация системы блокчейн не хранится
в одном месте, что в свою очередь говорит
о достоверности и безопасности данных
Технология предназначена для оптического
распознавания и цифровизации документо-
оборота компании
Физическая или цифровая среда, в которой
люди и технологические системы, взаимо-
действуют между собой
Синхронизированная виртуальная модель
предприятия, отражающее состояние
на текущую дату, а также предсказывает
будущее, основываясь на текущих данных
Документооборот между
предприятиями в электронной форме
Электронная подпись, с использованием
криптографических преобразований
Рис. 1. Цифровые технологии в бухгалтерском учете
Источник: [8] Технология предназначена для оптического
распознавания и цифровизации документо-
оборота компании Оптическое
распознавание Физическая или цифровая среда, в которой
люди и технологические системы, взаимо-
действуют между собой Синхронизированная виртуальная модель
предприятия, отражающее состояние
на текущую дату, а также предсказывает
будущее, основываясь на текущих данных Электронный
документооборот
(ЭДО) Рис. 1. Цифровые технологии в бухгалтерском учете В современном мире цифровая трансформация является одним из основных факторов мирового эко-
номического роста. Использование информационно-коммуникационных технологий, как ведущего инстру-
мента цифровизации во многих отраслях не только изменяет жизнь людей, но и трансформирует эконо-
мические структуры [4]. Одним из показателей успешности бизнеса является рост его стоимости, который не только обусловли-
вает долгосрочное и устойчивое функционирование компании, но и трансформируется в показатели соци-
ально-экономического благополучия всего общества. Основные результаты На основе приведенных сведений, основные препятствия цифровизации – это недостаточная зрелость
текущих процессов, низкий уровень автоматизации, отсутствие компетенций и низкий уровень ИТ-грамот-
ности сотрудников. Для решения выявленных проблем предприятиям необходимо разработать и внедрить «дорожную кар-
ту», повысить квалификацию сотрудников и ввести новую позицию руководителя программ цифровизации. Д
б Для решения выявленных проблем предприятиям необходимо разработать и внедрить «дорожную кар-
ту», повысить квалификацию сотрудников и ввести новую позицию руководителя программ цифровизации. «Дорожная карта» – это документ, в котором отображены основные этапы реализации стратегии, указа-
ны исполнители и сроки завершения каждого этапа у»,
о
с
ал ф
ац ю со руд
о
ес
о ую
оз ц ю ру о од
ел
ро ра
ц фро
зац
. «Дорожная карта» – это документ, в котором отображены основные этапы реализации стратегии, указа-
ны исполнители и сроки завершения каждого этапа. «Дорожная карта» – это документ, в котором отображены основные этапы реализации стратегии, указа-
ны исполнители и сроки завершения каждого этапа. А для дальнейшего урегулирования вопроса низкого уровня автоматизации и отсутствия бюджета су-
ществует программа поддержки цифровизации малого и среднего бизнеса, которая предоставляет возмож-
ность работы с более доступными программами-аналогами для цифрового учета. А для дальнейшего урегулирования вопроса низкого уровня автоматизации и отсутствия бюджета су-
ществует программа поддержки цифровизации малого и среднего бизнеса, которая предоставляет возмож-
ность работы с более доступными программами-аналогами для цифрового учета. Согласно исследованию Организации экономического развития и сотрудничества (OECD), выделяются
два ключевых фактора, которые оказывают влияние на скорость внедрения цифровых технологий [8]. Согласно исследованию Организации экономического развития и сотрудничества (OECD), выделяются
два ключевых фактора, которые оказывают влияние на скорость внедрения цифровых технологий [8]. Первый фактор – это внутренние способности организации, которые подразумевают процессы управ-
ления кадрами, наличие и правильное распределение ресурсов. Второй фактор, стимулирующий цифровую
трансформацию включает уровень конкуренции в индустрии, доступность технологий и капитала, а также
развитие законодательства [8]. Первый фактор – это внутренние способности организации, которые подразумевают процессы управ-
ления кадрами, наличие и правильное распределение ресурсов. Второй фактор, стимулирующий цифровую
трансформацию включает уровень конкуренции в индустрии, доступность технологий и капитала, а также
развитие законодательства [8]. Для успешной реализации цифровых инициатив необходимы сильные лидерские качества и современ-
ные практики управления. Эффективное слияние технологий и бизнес-процессов внутри компании требу-
ет базовых ИТ-навыков среди сотрудников, программ для поддержания и развития данных навыков, а так-
же соответствия характера работы их компетенциям [8]. Постановка проблемы На пути цифровой трансформации данной сферы, возникают следующие проблемы: отсутствие ком-
плексной программы цифровизации, недостаточность компетентных специалистов, низкий уровень автома-
тизации, отсутствие бюджета, низкий уровень ИТ-грамотности. Наряду с вышеперечисленными причинами для беспокойства в условиях модификации, выявляют ри-
ски информационной безопасности и снижение количества рабочих мест. 110 Экономика: проблемы, решения и перспективы Вестник университета № 2, 2022 Вестник университета № 2, 2022 Наиболее эффективный подход к реализации цифровой трансформации – это использование методи-
ки «от бизнес-задач», которая предполагает, что сначала определяется результат, который нужно увидеть,
и источники создания ценности, а лишь затем предприятия занимаются выбором конкретной технологии
для внедрения. Это в корне отличается от традиционной технологии большинства компаний, когда сначала
реализуют пилотный проект по той или иной технологии и только потом оценивают эффект от нее и ее не-
обходимость внедрения. Основные результаты Для большинства российских компаний, понятие цифровой трансформации связано с внедрением но-
вых технологий, ставших возможными в последние годы: аналитика больших данных, оптическое распозна-
вание документов, искусственный интеллект, интернет платформы, роботизация и другие. Предпосылками
для внедрения и эволюции цифровизации в России стала необходимость в хранении и аналитике больших
данных, скорости обработки процессов и повышения эффективности. Цифровая трансформация становит-
ся более доступной, за счет снижения стоимости зарубежных технологий, в следствии развития отечествен-
ных программ поддержки цифровизации малого и среднего бизнеса. Российские компании готовы проводить цифровую трансформацию бизнеса, однако следует учитывать
мнение руководителей о том, какой экономический эффект может быть получен по итогам внедрения [8]. Цифровизация – это новше-
ство, которое внесет изменения
в бухгалтерию, с помощью новых
технологий и сервисов [5]. Пути
усовершенствования цифровиза-
ции повышают эффективность
и качество работы бухгалтер-
ского учета компаний. Измене-
ния в данной области отражают-
ся в повышении эффективности
работы аппарата бухгалтерии. Рис. 2. Факторы цифровой трансформации
Источник: [8]
Цифровая трансформация
Внутренние способности организации
Стимулирующие факторы
– процессы управления;
– наличие ресурсов;
– грамотность в распределении
ресурсов
– уровень конкуренции;
– доступность технологий;
– развитие законодательства фф
дрения цифровых технологий
компании является повыше-
ние производительности и сокращение издержек. Наибольший экономический эффект, принесет решение
по созданию и развитию анализа больших данных и предиктивной аналитике, так как данная технология
позволяет повысить производительности и вывести на новый уровень качество принимаемых человеком
решений. Это находит свое отражение в большей эффективности взаимодействия с клиентами и лучше
качестве планирования. Факторы, влияющие на скорость цифровой трансформации, отразим на рисунке 2. Рис. 2. Факторы цифровой трансформации
Источник: [8] Рис. 2. Факторы цифровой трансформации 111 Заключение В современном мире цифровизация определяется, как преобразование информации в цифровой фор-
мат, что в свою очередь способствует повышению эффективности, снижению издержек предприятия и раз-
витию новых направлений. В цифровизации бухгалтерского учета выявлены положительные стороны. 1. Снижение затрат на содержание бухгалтерского аппарата. Стоит отметить, что данный показатель
не говорит о сокращении численности организации, наоборот, свидетельствует о повышении квалификации
или переквалификации сотрудника в профессию будущего, например из профессии бухгалтер переобучиться
на финансового инженера. Новые экономические и технологические условия требуют создания и реализа-
ции подходов по содействию гражданам в освоении ключевых компетенций цифровой экономики, обеспече-
нии массовой цифровой грамотности и персонализации образования. В этих целях реализуется федеральный
проект «Кадры для цифровой экономики» национальной программы «Цифровая экономика Российской Фе-
дерации». Основная цель федерального проекта – обеспечение подготовки высококвалифицированных ка-
дров для цифровой экономики [1]. 2. Эффективность финансово-хозяйственной деятельности. 3. Скорость обработки данных и информации. 4. Налаживание удаленной работы. 5. Информационная безопасность. С ростом цифровизации, возрастает необходимость в безопасности
и надежности хранения данных. В рамках национального проекта, разработано направление по кибербез-
опасности. В результате реализации данного направления будут обеспечены устойчивость и безопасность
информационной инфраструктуры, конкурентоспособность отечественных разработок и технологий ин-
формационной безопасности, и выстроена эффективная система защиты прав и законных интересов лич-
ности, бизнеса и государства от угроз информационной безопасности [1]. 5. Информационная безопасность. С ростом цифровизации, возрастает необходимость в безопасности
и надежности хранения данных. В рамках национального проекта, разработано направление по кибербез-
опасности. В результате реализации данного направления будут обеспечены устойчивость и безопасность
информационной инфраструктуры, конкурентоспособность отечественных разработок и технологий ин-
формационной безопасности, и выстроена эффективная система защиты прав и законных интересов лич-
ности, бизнеса и государства от угроз информационной безопасности [1]. Исходя из вышеизложенного видим необходимость цифровой трансформации бизнеса, и в особенно-
сти такого важного отдела, как бухгалтерия, ведь на основе своевременности учета финансовых данных
формируется достоверная информация как для инвесторов, так и собственников бизнеса о финансово-хо-
зяйственной деятельности компаний. Библиографический список 1. Национальная программа «Кадры в цифровой экономике» // Министерство цифрового развития, связи и массовых ком-
муникаций Российской Федерации [Электронный ресурс]. – Режим доступа: https://digital.gov.ru/ru/activity/directions/866/
(дата обращения: 09.01.2022). 2. Опубликован паспорт Национальной программы «Цифровая экономика Российской Федерации» // Правительство России
[Электронный ресурс]. – Режим доступа: http://government.ru/info/35568/ (дата обращения: 02.01.2022). ронный ресурс]. – Режим доступа: http://government.ru/info/35568/ (дата обращения: 02.01.2022). 3. Будович, Ю. И. Цифровизация корпоративного учета // Человеческий капитал в формате цифровой экономики. Между-
народная научная конференция, посвященная 90-летию С. П. Капицы, Москва, 16 февраля, 2018 г. – М.: Российский
новый университет, 2018. – С. 117–125. https://doi.org/10.25586/RNU.CONF.18.02.P.117 4. Евсеева, С. А., Евсеева, О. А., Кальченко О. А. Возможности цифровизации российского малого и среднего бизнеса
на региональном уровне // European Social Science Journal. – T. 2, № 12. – С. 136–142. 5. Ермилова, Ю. А. Аспекты учетно-аналитического обеспечения инновационной деятельности на предприятии // Регио-
нальная инновационная экономика: сущность, элементы, проблемы формирования, новые вызовы: сборник трудов Ше-
стой Всероссийской Научной Конференции с Международным Участием, Ульяновск, 26–28 октября, 2016 г. / ред. кол.:
Белый Е. М., Иванова Т. Ю., Рожкова Е. В. – Ульяновск: Ульяновский государственный университет, 2016. – С. 18–21. 112 Экономика: проблемы, решения и перспективы 6. Позмогов, А. И. Цифровая трансформация российского бизнеса: монография. – М.: Кнорус, 2019. – 456 с. 6. Позмогов, А. И. Цифровая трансформация российского бизнеса: монография. – М.: Кнорус, 2019. – 456 с. 7. Рогуленко, Т. М., Агеева, О. А., Горлов, В. В. и др. Цифровая экономика – драйвер развития бухгалтерского учета, аудита,
контроля: монография. – М.: ООО «Кнорус», 2020. – 230 с. 7. Рогуленко, Т. М., Агеева, О. А., Горлов, В. В. и др. Цифровая экономика – драйвер развития бухгалтерского учета, аудита,
контроля: монография. – М.: ООО «Кнорус», 2020. – 230 с. 8. Цифровые технологии в российских компаниях. Результаты исследования // КПМГ [Электронный ресурс]. – Режим
доступа: https://assets.kpmg/content/dam/kpmg/ru/pdf/2019/01/ru-ru-digital-technologies-in-russian-companies.pdf (дата об-
ращения: 02.01.2022). 8. Цифровые технологии в российских компаниях. Результаты исследования // КПМГ [Электронный ресурс]. – Режим
доступа: https://assets.kpmg/content/dam/kpmg/ru/pdf/2019/01/ru-ru-digital-technologies-in-russian-companies.pdf (дата об-
ращения: 02.01.2022). References 1. National Programme “Personnel in the Digital Economy”, Ministry of Digital Development, Communications and Mass Media
of the Russian Federation. Available at: https://digital.gov.ru/ru/activity/directions/866 / (accessed 09.01.2022). 1. National Programme “Personnel in the Digital Economy”, Ministry of Digital Development, Communications and Mass Media
of the Russian Federation. Available at: https://digital.gov.ru/ru/activity/directions/866 / (accessed 09.01.2022). 2. Passport of the National Programme “Digital Economy of the Russian Federation” has been published, The Russian Government. Available at: http://government.ru/info/35568/ (accessed 02.01.2022). 2. Passport of the National Programme “Digital Economy of the Russian Federation” has been published, The Russian Government. Available at: http://government.ru/info/35568/ (accessed 02.01.2022).i 3. Budovich Yu. I. The digitalization of corporate accounting, Human capital in the digital economy format. International Scientific
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2018, pp. 117–125. (In Russian). https://doi.org/10.25586/RNU.CONF.18.02.P.117 4. Evseeva S. A., Evseeva O. A., Kalchenko O. A. Digitalization possibilities of Russian small and medium business at the regional
level, European Social Science Journal, vol. 2, no. 12, pp. 136–142. (In Russian). 5. Ermilova Yu. A. Aspects of accounting and analytical support of innovation activity at the enterprise, Regional innovation econ-
omy: essence, elements, problems of formation, new challenges: Proceedings of the Sixth All-Russian Scientific Conference with
International Participation, Ulyanovsk, October 26–28, 2016, Ed. Board Belyi E. M., Ivanova T. Yu., Rozhkova E. V., Ulyanovsk,
Ulyanovsk State University, 2016, pp. 18–21. (In Russian). Pozmogov A. I. Digital transformation of Russian business: monograph, Moscow Knorus, 2019, 456 7. Rogulenko T. M., Ageeva O. A., Gorlov V. V. et al. Digital economy – the driver of accounting, audit, control development:
monograph, Moscow, OOO “Knorus”, 2020, 230 p. (In Russian). 8. Digital Technologies in Russian Companies. Results of the Study, KPMG. Available at: https://assets.kpmg/content/dam/kpmg/
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Ticagrelor versus clopidogrel in real-world patients with ST elevation myocardial infarction: 1-year results by propensity score analysis
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© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Vercellino et al. BMC Cardiovascular Disorders (2017) 17:97
DOI 10.1186/s12872-017-0524-3 Vercellino et al. BMC Cardiovascular Disorders (2017) 17:97
DOI 10.1186/s12872-017-0524-3 Ticagrelor versus clopidogrel in real-world
patients with ST elevation myocardial
infarction: 1-year results by propensity
score analysis Matteo Vercellino1*
, Federico Ariel Sànchez2, Valentina Boasi3, Dino Perri4, Chiara Tacchi5, Gioel Gabrio Secco1,
Stefano Cattunar4, Gianfranco Pistis6 and Giovanni Mascelli7 * Correspondence: matteovrc@gmail.com; matteo.vercellino@ospedale.al.it
1Interventional Cardiology, Santi Antonio, Biagio e Cesare Arrigo Hospital,
Alessandria, AL, Italy
Full list of author information is available at the end of the article * Correspondence: matteovrc@gmail.com; matteo.vercellino@ospedale.al.it
1Interventional Cardiology, Santi Antonio, Biagio e Cesare Arrigo Hospital,
Alessandria, AL, Italy
Full list of author information is available at the end of the article Methods A pre-post case-control study was performed using data
from the Cardio-STEMI Sanremo registry, a single-centre,
ongoing, observational cohort study conducted in Sanremo
Hospital, the hub for a population of 210,000 inhabitants,
with 2 spokes. Every adult patient (>18 years old) admitted
to the hub with a diagnosis of STEMI [18] was enrolled in
the registry. Exclusion criteria were: type 4a or 5 AMI according to
the universal definition of myocardial infarction [19]; high
probability of
being unavailable for follow-up visits
because of limited ability to cooperate or severe comor-
bidity with very short life expectancy. Ticagrelor is a novel oral, reversible P2Y12 inhibitor
belonging to the cyclopentyltriazolopyrimidine class. It has
a plasma half-life of 12 h. It is an active drug with more
rapid onset and offset of action than clopidogrel, so that
inhibition and recovery of platelet function is faster [4, 9]. For this study, all patients enrolled between February
2011 and June 2013 were examined. Ticagrelor became
available in addition to clopidogrel in May 2012, in both in-
hospital and pre-hospital settings. Since then, the choice
between the two drugs has been left to the cardiologist who
first makes the diagnosis of STEMI. Ticagrelor was intro-
duced into clinical practice independently from this obser-
vational study and in accordance with European guidelines
for the treatment of STEMI patients published in 2012
[20]. Patients were divided into the ticagrelor group and the
clopidogrel
group
according
to
the
P2Y12
inhibitor
received. Local guidelines recommend the early administra-
tion of 2PY12 inhibitors, acetylsalicylic acid and unfractio-
nated heparin immediately after a STEMI diagnosis. Therefore, in most patients presenting through the ambu-
lance service, 2PY12 inhibitors were administered during
the ambulance journey, usually after transmission of the
electrocardiogram report to the hub. Patients were treated
according to usual clinical practice at each institution, and
PCI was performed using standard techniques. All patients
received indication to continue DAPT for at least 1 year
after discharge, according to the current European and
American guidelines [20, 21]. Study endpoints were: (1)
TIMI flow grade before and after PCI [22] and ST
resolution 90 min after PCI; (2) the rate of hospital major
adverse
cardiovascular
events
(MACE;
cardiovascular A large randomized controlled trial (RCT) showed the
superiority of ticagrelor compared with clopidogrel in
ACS in patients with STEMI and non-ST-segment eleva-
tion myocardial infarction (NSTEMI) [6]. Background In
patients with STEMI, drug metabolism is hampered by
specific circulatory conditions, resulting in a delayed effect
of the drug compared with the time frame needed for
percutaneous coronary intervention (PCI) [7, 8]. Moreover,
this pathway is susceptible to genetic polymorphism, which
may lead to unexpected variations in drug activity. Abstract BMC Cardiovascular Disorders (2017) 17:97 Background DAPT have recently been examined. A recent meta-
analysis showed that patients treated with short DAPT
(<12 months) have similar survival (all-cause mortality
and cardiovascular mortality) than patients treated with
long DAPT (≥12 months). However, the RCTs collected in
the meta-analysis examined DAPT duration, not the type
of DAPT (ticagrelor vs clopidogrel), and the prevalence of
ACS was widely heterogeneous (32–77%). To date, we
have few data about the differences related to the type of
DAPT in a population of STEMI patients treated with
second-generation DESs. Patients with ST elevation myocardial infarction (STEMI)
represent 32% of patients with acute coronary syndrome
(ACS), with in-hospital mortality ranging from 5% to 15%
according to geographic and baseline differences [1]. Each
year in the United States alone, hospital costs related to
acute myocardial infarction (AMI) are estimated to be as
high as US$11.5 billion [2]. g
Dual antiplatelet therapy (DAPT) is a cornerstone of
therapy for patients with STEMI. Clopidogrel has been
used extensively worldwide for more than a decade; more
recently, new antiplatelet agents, prasugrel and ticagrelor,
have been developed and tested clinically, resulting in
faster, more potent and consistent antiplatelet action [3–6]. Clopidogrel, a second-generation thienopyridine, is now
available as a low-cost generic drug, with a favourable cost-
effectiveness ratio. A drawback of clopidogrel is that as a
pro-drug, it needs liver metabolism to be activated. In
patients with STEMI, drug metabolism is hampered by
specific circulatory conditions, resulting in a delayed effect
of the drug compared with the time frame needed for
percutaneous coronary intervention (PCI) [7, 8]. Moreover,
this pathway is susceptible to genetic polymorphism, which
may lead to unexpected variations in drug activity. Dual antiplatelet therapy (DAPT) is a cornerstone of
therapy for patients with STEMI. Clopidogrel has been
used extensively worldwide for more than a decade; more
recently, new antiplatelet agents, prasugrel and ticagrelor,
have been developed and tested clinically, resulting in
faster, more potent and consistent antiplatelet action [3–6]. We thus performed the present study to compare the
effect of ticagrelor and clopidogrel in a real-world popula-
tion of patients with STEMI. Clopidogrel, a second-generation thienopyridine, is now
available as a low-cost generic drug, with a favourable cost-
effectiveness ratio. A drawback of clopidogrel is that as a
pro-drug, it needs liver metabolism to be activated. Abstract Background: European guidelines recommend the use of ticagrelor versus clopidogrel in patients with ST elevation
myocardial infarction (STEMI). This recommendation is based on inconclusive results and subanalyses from clinical trials. Few data are available on the effects of ticagrelor in a real-world population. Methods: To compare the effects of ticagrelor and clopidogrel in a real-world STEMI population, we conducted a
pre-post case-control study examining all patients with STEMI included in the Cardio-STEMI Sanremo registry between
February 2011 and June 2013. Cases and controls were defined according to P2Y12 inhibitors, correcting the bias due
to lack of randomization by propensity score analysis. Ticagrelor was introduced in 2012 in both in-hospital and
pre-hospital settings independently of this study. Results: Of the 416 patients enrolled in the Cardio-STEMI registry, 401 with a definite diagnosis of STEMI were included
in this study. One hundred forty-two patients received ticagrelor and 259 received clopidogrel. Regarding clinical
presentation and procedural data, those in the ticagrelor group had lower CRUSADE scores (23 [14–36] vs 27 [18–38];
p = 0.015] but a higher proportion of radial access (33% vs 14%; p < 0.001), percutaneous coronary intervention
(PCI; 92% vs 81 %; p = 0.002) and primary PCI ≤12 h (82% vs 66%; p = 0.001). The patients in the ticagrelor group had
a higher procedural success rate (100% vs. 96%; p = 0.044). There was no difference in Bleeding Academic Research
Consortium bleeding and in unadjusted incidence of hospital major adverse cardiovascular events (MACE; cardiac
death, myocardial infarction, or stroke) but there was a significant reduction in unadjusted cardiac hospital
death in the ticagrelor group (0.7% vs 5.4%; p = 0.024). After correcting for propensity score, hospital death
(p = 0.22) and hospital MACE (p = 0.96) did not differ in both groups. The unadjusted survival at 1 year after
STEMI was higher in the ticagrelor group (97.8% vs 87.8%; p = 0.024), and this result was confirmed by
propensity score analysis (hazard ratio = 0.29 [0.08–0.99]; p = 0.048). Conclusions: In this real-word propensity score analysis, ticagrelor did not affect the risk of MACE during the
hospital phase, or the incidence of hospital bleeding in patients with STEMI. However, in this mono-centric
experience, ticagrelor resulted in improved 1-year survival, even after correction by propensity score. Keywords: Acute coronary syndrome, ST elevation myocardial infarction, Clopidogrel, Ticagrelor, Registry Page 2 of 11 Page 2 of 11 Vercellino et al. Methods This large RCT
led to a change in guideline recommendations in favour of
ticagrelor in patients with STEMI undergoing primary
PCI, largely reducing clopidogrel use in this setting. The
beneficial effects of ticagrelor were demonstrated to be
independent from the clinical presentation of STEMI,
because no interaction has been demonstrated in the
PLATO (Platelet Inhibition and Patient Outcomes) [8]. However, no specific randomized trial was designed to
detect the effect of ticagrelor in patients with STEMI,
leading to inconclusive and underpowered data [5, 10]. Although results from large real-world registries are
important to better understand the effectiveness, use and
outcomes of novel therapies [11–16], data on the benefits
of ticagrelor in a real-world population of patients with
STEMI are lacking. The sole large observational registry
about ticagrelor in STEMI population yielded results that
were in contrast with the PLATO trial, with no improve-
ment in ischaemic events and a higher rate of bleeding for
ticagrelor versus clopidogrel [17]. The safety and efficacy of drug-eluting stents (DESs)
based on the generation of the device and the duration of Vercellino et al. BMC Cardiovascular Disorders (2017) 17:97 Page 3 of 11 Vercellino et al. BMC Cardiovascular Disorders (2017) 17:97 Model discrimination was measured by the C statistic
and calibration by the Hosmer-Lemeshow goodness-of-fit
test [27]. The propensity score was used as a correction
factor in a binary logistic regression to calculate the
adjusted hospital outcomes and in a Cox regression
analysis to examine the adjusted 1-year survival. The Cox
regression results were expressed by hazard ratios (HR)
with 95% CIs. All tests were two-sided. A p value ≤0.05 was
considered statistically significant. Statistics were calculated
using SPSS version 22.0 (SPSS Inc., Chicago, IL, USA). death, AMI and stroke) and bleeding; (3) the 12-month
survival. MACE were defined according to the PLATO trial
criteria, with stroke defined as focal loss of neurologic
function caused by ischaemic or haemorrhagic events, with
symptoms lasting at least 24 h or leading to death [6]. Myocardial infarction (MI) was defined according to the
universal definition reported in the literature [19]. Bleeding
Academic Research Consortium (BARC) criteria were used
for bleeding assessment [23]. A paper-based case report form was prospectively
collected for each patient by the referring physician. All
the data were entered into an electronic database by 4
trained researchers, and the data entries were checked
periodically by a data manager. Methods The validation of in-
hospital study outcomes was performed through the
periodic revision of medical documents. Follow-up was
performed with the support of a local health registry and a
telephone interview with the patient, family physician or
relatives [24]. Only mortality was inquired about during
post-discharge follow-up, because this endpoint can be
reliably and easily collected, and it is a surrogate for the
most severe sequelae such as re-infarction, stroke or
bleeding occurring during follow-up. Results A total of 416 patients were enrolled in the Cardio-
STEMI Sanremo registry during the study period. Fifteen
patients (3.6%) were subsequently ruled out with condi-
tions mimicking MI, and were therefore excluded from
this study. The study flowchart is shown in Fig. 1. The
study population included 401 patients, 259 patients in
the clopidogrel group and 142 patients in the ticagrelor
group. From its introduction into clinical practice in May
2012, its use had peaked at 83% by the end of 2012. Data
on the adoption of ticagrelor in clinical practice are
reported in Fig. 2. The Cardiology Department of Sanremo Hospital served
as the data analysis centre. The study was conducted in
accordance with the principles of the Helsinki Declaration
and received local ethical committee approval before
recruitment. Demographic and baseline data are reported in Table 1. Comparing the baseline data of the 2 groups, there were no
significant differences between ticagrelor and clopidogrel,
except for a lower proportion of patients aged over 75 years
(21% vs 32%, respectively; p = 0.037), with no difference in
median age. In the ticagrelor group, all patients received the loading
dose, administered in 98% of cases before the cardiac
catheterization laboratory; in the clopidogrel group, 236
patients (91%) received the loading dose (42% >300 mg as
reported in Fig. 3), administered in 94% before the cardiac
catheterization laboratory. Statistical analysis Baseline
patient
and
procedural
characteristics
are
presented as means ± standard deviation, median (inter-
quartile range), or frequency (percentage) as appropriate. The normal distribution of continuous variables in the
study was determined by Shapiro-Wilks and Kolmogorov-
Smirnov tests. Normally distributed continuous variables
were compared using the Student t test and those with
skewed distributions using the Mann-Whitney U test. Categorical variables were compared using the χ2 test or
Fisher exact test. In-hospital nominal outcomes are
presented as proportions and odds ratios (OR) with
relative 95% confidence intervals (CI). We used Kaplan-
Meier curves for survival analysis and the log-rank test to
compare the treatment groups. With the aim of reducing
bias related to the lack of randomization, a propensity
score was calculated [25, 26] using a non-parsimonious,
multivariate, binary logistic regression model. With regard to clinical presentation and reperfusion
strategy, a lower CRUSADE score (23 [14–36] vs 27
[18–38]; p = 0.015], a higher rate of PCI (92% vs 80%;
p = 0.002) and primary PCI within 12 h (82% vs 66%;
p = 0.001) were found in the ticagrelor group compared
with the clopidogrel group. (Table 2). The laboratory tests
were similar between the 2 groups (Additional file 1). Procedural data and times are reported in Table 3. The
only significant difference was a higher frequency of the
radial access approach for PCI in the ticagrelor group
(33% vs 14%; p < 0.001). For patients undergoing
primary PCI, the frequency of thrombus aspiration (45%
vs 37%; p = 0.22) and the use of glycoprotein (GP) IIb/
IIIa receptor antagonists (13% vs 17%; p = 0.41) were
comparable in the ticagrelor and clopidogrel groups. With regard to the type of stent used, there were no
significant differences between the clopidogrel and tica-
grelor groups, although the use of bare metal stents was
nominally higher in the clopidogrel group (27% vs 15%). Variables presenting a p value ≤0.2 for the univariate
analysis and those judged to be of clinical importance,
biologically plausible or supported by previously published
data in the literature, were tested for inclusion in the
multivariable model building process. Variables with a
missing rate ≥5% were excluded. Vercellino et al. BMC Cardiovascular Disorders (2017) 17:97 Page 4 of 11 Fig. 1 Study flowchart. CABG coronary artery bypass grafting, DAPT dual antiplatelet treatment, ECG electrocardiogram, CI confidence interval,
NA not available Fig. 1 Study flowchart. Statistical analysis Data are
expressed as percentage (frequency), median (IQR), or mean (SD)
Ticagrelor
Clopidogrel
p
Call to emergency service
43.0 (61/142)
41.3 (107/259)
0.75
Secondary transfer by
emergency service
40.8 (58/142)
40.3 (104/258)
0.92
Hub vs spoke
67.1 (94/140)
64.5 (167/259)
0.65
Anterior AMI
46.5 (66/142)
39.8 (103/259)
0.21
New LBBB at first EKG
2.3 (5/142)
3.5 (6/256)
0.53
Cardiac frequency (bpm)
75 (65–88)
77 (63–94)
0.28
Systolic blood pressure (mmHg)
140 (120–160)
140 (120–160)
0.76
Killip class ≥3
5.6 (8/142)
8.1 (21/259)
0.42
Cardiac arrest
2.1 (3/142)
3.1 (8/259)
0.75
LV ejection fraction (%)
46 (40–55)
45 (40–55)
0.83
GFR Cockroft Gault (mL/min/m2)
72 (55–90)
67 (52–86)
0.08
GFR MDRD (mL/min/m2)
76 ± 25
72 ± 23
0.09
GRACE-in-hospital mortality
139 (122–157)
141 (121–167)
0.32
GRACE 6-month mortality
106 (86–128)
106 (87–125)
0.96
TIMI risk score
3 (2–5)
4 (2–5)
0.18
CRUSADE
23 (14–36)
27 (18–38)
0.015
Thrombolysis
1.8 (4/142)
4.6 (12/259)
0.44
Coronary angiography
100.0 (142/142)
93.1 (241/259)
0.001
PCI
92.3 (131/142)
80.7 (209/259)
0.002
Primary PCI (≤12 h after
first medical contact)
82.4 (117/142)
66.4 (172/259)
0.001
IABP
0.7 (1/142)
3.1 (8/259)
0.17
Italics: p value ≤0.05
AMI acute myocardial infarction, LBBB left bundle branch block, EKG,
electrocardiogram, LV left ventricle, GFR glomerular filtration rate, MDRD
modification of diet in renal disease, GRACE global registry of acute coronary
events, TIMI thrombolysis in myocardial infarction, CRUSADE can rapid risk
stratification of unstable angina patients suppress adverse outcome with early
implementation of ACC/AHA Guidelines, PCI percutaneous coronary
intervention, IABP intra-aortic balloon pump In each group, most of the DESs were second-generation
devices. The system-related delay, patient-related delay and the
time intervals were not different between the 2 groups. Discharge therapy was not significantly different between
the ticagrelor and clopidogrel groups (Additional file 2). 2,2%
55,8%
2,6%
39,4%
150 mg
300 mg
450 mg
600 mg
Fig. 3 Clopidogrel loading dose Procedural success, defined either as TIMI 3 or TIMI
2–3 with stenosis <50% after primary PCI, was higher in
the ticagrelor group than in the clopidogrel group (99% vs
90%, p = 0.001; 100.0% vs. 96%, p = 0.044), whereas the
proportion of patients with ≥50% ST resolution (78% vs
78%; p = 0.879) or ≥70% ST resolution (60% vs 67%;
p = 0.294) was similar between the groups. Data on
procedural outcomes are reported in Table 4. Statistical analysis Data are
expressed as percentage (frequency), median (IQR), or mean (SD)
Ticagrelor
Clopidogrel
p
Call to emergency service
43.0 (61/142)
41.3 (107/259)
0.75
Secondary transfer by
emergency service
40.8 (58/142)
40.3 (104/258)
0.92
Hub vs spoke
67.1 (94/140)
64.5 (167/259)
0.65
Anterior AMI
46.5 (66/142)
39.8 (103/259)
0.21
New LBBB at first EKG
2.3 (5/142)
3.5 (6/256)
0.53
Cardiac frequency (bpm)
75 (65–88)
77 (63–94)
0.28
Systolic blood pressure (mmHg)
140 (120–160)
140 (120–160)
0.76
Killip class ≥3
5.6 (8/142)
8.1 (21/259)
0.42
Cardiac arrest
2.1 (3/142)
3.1 (8/259)
0.75
LV ejection fraction (%)
46 (40–55)
45 (40–55)
0.83
GFR Cockroft Gault (mL/min/m2)
72 (55–90)
67 (52–86)
0.08
GFR MDRD (mL/min/m2)
76 ± 25
72 ± 23
0.09
GRACE-in-hospital mortality
139 (122–157)
141 (121–167)
0.32
GRACE 6-month mortality
106 (86–128)
106 (87–125)
0.96
TIMI risk score
3 (2–5)
4 (2–5)
0.18
CRUSADE
23 (14–36)
27 (18–38)
0.015
Thrombolysis
1.8 (4/142)
4.6 (12/259)
0.44
Coronary angiography
100.0 (142/142)
93.1 (241/259)
0.001
PCI
92.3 (131/142)
80.7 (209/259)
0.002
Primary PCI (≤12 h after
first medical contact)
82.4 (117/142)
66.4 (172/259)
0.001
IABP
0.7 (1/142)
3.1 (8/259)
0.17
Italics: p value ≤0.05
AMI acute myocardial infarction, LBBB left bundle branch block, EKG,
electrocardiogram, LV left ventricle, GFR glomerular filtration rate, MDRD
modification of diet in renal disease, GRACE global registry of acute coronary
events, TIMI thrombolysis in myocardial infarction, CRUSADE can rapid risk
stratification of unstable angina patients suppress adverse outcome with early
implementation of ACC/AHA Guidelines, PCI percutaneous coronary
intervention, IABP intra-aortic balloon pump Table 1 Baseline and demographic data. Data are expressed as
percentage (frequency) or median (IQR) Table 2 Clinical presentation and reperfusion strategy. Statistical analysis CABG coronary artery bypass grafting, DAPT dual antiplatelet treatment, ECG electrocardiogram, CI confidence interval,
NA not available Fig. 2 Introduction of ticagrelor in clinical practice Fig. 2 Introduction of ticagrelor in clinical practice Fig. 2 Introduction of ticagrelor in clinical practice Vercellino et al. BMC Cardiovascular Disorders (2017) 17:97 Page 5 of 11 Table 1 Baseline and demographic data. Data are expressed as
percentage (frequency) or median (IQR)
Ticagrelor
(n = 142)
Clopidogrel
(n = 259)
p
Age (years)
66 (56–73)
67 (56–67)
0.206
Age ≥75 years
21.8 (31)
32.0 (83)
0.037
Sex male
73.9 (105)
69.9 (81)
0.420
BMI (kg/m2)a
26 (24–30)
26 (24–29)
0.772
Hypertension
52.8 (75)
56.0 (145)
0.600
Dyslipidemia
36.6 (52)
39.0 (101)
0.668
Active smoke
45.8 (65)
37.8 (98)
0.137
Diabetes mellitus
22.5 (32)
18.5 (48)
0.362
Familiar of CAD
21.1 (30)
24.7 (64)
0.461
Previous stable angina
11.3 (16)
6.6 (17)
0.128
Previous unstable angina
3.5 (5)
4.6 (12)
0.797
Previous AMI
11.3 (16)
11.2 (29)
1.000
Previous PCI
7.0 (10)
9.3 (24)
0.574
Previous CABG
1.4 (2)
0.0 (0)
0.125
Previous CVA
4.9 (7)
7.3 (19)
0.403
PVD
16.2 (23)
11.2 (29)
0.164
Chronic kidney disease
7.0 (10)
4.6 (12)
0.361
COPD
8.5 (12)
8.9 (23)
1.000
Previous bleeding
0.7 (1)
3.9 (10)
0.106
Previous neoplasia
10.6 (15)
15.8 (41)
0.175
Italics: p value ≤0.05
BMI body mass index, CAD coronary artery disease, AMI acute myocardial
infarction, PCI percutaneous coronary intervention, CABG coronary artery
bypass grafting, CVA cerebrovascular accident, PVD peripheral vascular disease,
COPD chronic obstructive pulmonary disease
an = 139 for ticagrelor, 258 for clopidogrel In each group, most of the DESs were second-generation
devices. The system-related delay, patient-related delay and the
time intervals were not different between the 2 groups. Discharge therapy was not significantly different between
the ticagrelor and clopidogrel groups (Additional file 2). Procedural success, defined either as TIMI 3 or TIMI
2–3 with stenosis <50% after primary PCI, was higher in
the ticagrelor group than in the clopidogrel group (99% vs
90% p
0 001; 100 0% vs 96% p
0 044) whereas the
Table 1 Baseline and demographic data. Statistical analysis Data are expressed as
percentage (frequency) or median (IQR)
Ticagrelor
(n = 142)
Clopidogrel
(n = 259)
p
Age (years)
66 (56–73)
67 (56–67)
0.206
Age ≥75 years
21.8 (31)
32.0 (83)
0.037
Sex male
73.9 (105)
69.9 (81)
0.420
BMI (kg/m2)a
26 (24–30)
26 (24–29)
0.772
Hypertension
52.8 (75)
56.0 (145)
0.600
Dyslipidemia
36.6 (52)
39.0 (101)
0.668
Active smoke
45.8 (65)
37.8 (98)
0.137
Diabetes mellitus
22.5 (32)
18.5 (48)
0.362
Familiar of CAD
21.1 (30)
24.7 (64)
0.461
Previous stable angina
11.3 (16)
6.6 (17)
0.128
Previous unstable angina
3.5 (5)
4.6 (12)
0.797
Previous AMI
11.3 (16)
11.2 (29)
1.000
Previous PCI
7.0 (10)
9.3 (24)
0.574
Previous CABG
1.4 (2)
0.0 (0)
0.125
Previous CVA
4.9 (7)
7.3 (19)
0.403
PVD
16.2 (23)
11.2 (29)
0.164
Chronic kidney disease
7.0 (10)
4.6 (12)
0.361
COPD
8.5 (12)
8.9 (23)
1.000
Previous bleeding
0.7 (1)
3.9 (10)
0.106
Previous neoplasia
10.6 (15)
15.8 (41)
0.175
Italics: p value ≤0.05
BMI body mass index, CAD coronary artery disease, AMI acute myocardial
infarction, PCI percutaneous coronary intervention, CABG coronary artery
bypass grafting, CVA cerebrovascular accident, PVD peripheral vascular disease,
COPD chronic obstructive pulmonary disease
an = 139 for ticagrelor, 258 for clopidogrel
2,2%
Table 2 Clinical presentation and reperfusion strategy. Statistical analysis Data are expressed as percentage (frequency) or median (IQR)
Ticagrelor
Clopidogrel
p
Radial vs femoral
33.1 (47/142)
14.5 (35/241)
<0.001
Left main
1.4 (2/142)
2.1 (5/241)
1.00
Multi-vessel CAD
36.6 (52/142)
36.5 (88/241)
1.00
Culprit vessel
0.19
RCA
38.7 (53/137)
38.9 (91/234)
CX
10.9 (15/137)
17.9 (42/234)
LAD
48.9 (67/137)
41.5 (97/234)
Other
1.5 (2/137)
1.7 (4/234)
Complete revascularization
58.0 (76/131)
56.9 (119/234)
0.91
Total contrast amount (mL)
200 (147–268)
203 (148–284)
0.89
Primary PCI
Multi-vessel CAD
35.9 (42/117)
34.9 (88/241)
0.90
Culprit vessel
0.44
RCA
38.8 (45/116)
39.0 (67/172)
CX
11.2 (13/116)
15.7 (27/172)
LAD
48.2 (56/116)
43.0 (74/172)
Others
1.8 (2/116)
2.3 (4/172)
TIMI flow pre-PCI
1 (0–3)
1 (0–3)
0.09
Reperfusion before PCI
(TIMI 3)
26.5 (31/117)
16.9 (29/172)
0.047
Reperfusion before PCI
(TIMI 2 o 3)
45.3 (53/117)
37.8 (65/172)
0.22
Type B2-C ACC/AHA lesion
classification
73.6 (81/110)
74.5 (117/155)
0.78
Thrombus aspiration
44.8 (52/116)
37.2 (64/172)
0.22
GPIIb/IIIa receptor antagonist
12.8 (15/117)
16.9 (29/172)
0.41
Stent type
0.15
BMS
14.7 (17/116)
27.1 (46/170)
First-generation DES
0.9 (1/116)
0.6 (1/170)
Second-generation DES
75.0 (86/116)
61.8 (104/170)
BMS and DES
2.6 (3/116)
2.4 (4/170)
No stent
7.8 (9/116)
8.8 (15/170)
Symptom onset to EKG
(hh:min)
1:25 (0:40–2:26) 1:19 (0:50–2:41) 0.84
EKG to door hub (hh:min)
1:26 (1:14–2:10) 1:21 (1:06–1:51) 0.25
Transfer time (hh:min)
0:42 (0:36–0:52) 0:38 (0:34–0:48) 0.11
DTB min (hh:min)
1:46 (1:35–2:37) 1:49 (1:34–2:30) 0.75
DTB ≤60 min
3.5 (4/114)
3.0 (5/166)
1.00
DTB ≤90 min
20.2 (23/114)
21.7 (36/166)
0.88
DTB ≤120 min
52.6 (60/114)
52.4 (87/166)
1.00
Total ischaemic time(hh:min)
3:50 (2:45–5:34) 3:30 (2:42–5:39) 0.37
Italics: p value ≤0.05
CAD coronary artery disease, RCA right coronary artery, CX circumflex coronary
artery, LAD left anterior descending, ACC/AHA American College of Cardiology/
American Heart Association, GPIIb/IIIa glycoprotein IIb/IIIa, PCI percutaneous
coronary intervention, TIMI thrombolysis in myocardial infarction, BMS bare metal
stent DES drug eluting stent EKG electrocardiogram DTB door to balloon Table 4 Procedural and hospital outcomes. Statistical analysis In the unadjusted analysis, there was no difference in
hospital MACE
(cardiovascular death, non-fatal
MI,
stroke) between the ticagrelor and clopidogrel groups
(4.9% vs 6.9%; p = 0.520; OR, 0.69 [95% CI, 0.28–1.70]). However, the use of ticagrelor resulted in a significant
reduction of cardiovascular mortality (0.7% vs 5.4%;
p = 0.024; OR, 0.12 [95% CI, 0.02–0.95]). No difference
was found in new hospital non-fatal AMI (3.5% vs 1.2%, Vercellino et al. BMC Cardiovascular Disorders (2017) 17:97 Page 6 of 11 Table 3 Procedural data in patients with STEMI and in primary
PCI. Statistical analysis Data are expressed
as percentage (frequency) or median (IQR)
Ticagrelor
(n = 142)
Clopidogrel
(n = 259)
P
Procedural outcomes (unadjusted analysis)
TIMI flow post-PCI
3 (3–3)
3 (3–3)
0.002
Procedural success (TIMI 3
and stenosis <50%)
99.0 (115/116)
89.9 (151/168)
0.001
Procedural success (TIMI
2–3 and stenosis <50%)
100.0 (116/116)
95.8 (161/168)
0.044
STR%
89 (50–100)
100 (50–100)
0.482
STR ≥50%
78.1 (82/105)
78.2 (122/156)
0.879
STR% ≥70%
60.0 (63/105)
66.7 (104/156)
0.294
Hospital outcomes (unadjusted analysis)
In-hospital all-cause death
0.7 (1)
5.4 (14)
0.024
In-hospital CV death
0.7 (1)
5.4 (14)
0.024
In-hospital non-fatal AMI
3.5 (5)
1.2 (3)
0.14
Ischaemic CVA
0.4 (1)
0.7 (1)
1.00
Total CVA (ischaemic or
haemorrhagic)
0.4 (1)
0.7 (1)
1.00
Hospital MACE
4.9 (7)
6.9 (18)
0.52
Definite stent thrombosis
1.4 (2)
0.8 (2)
0.62
Final LVEF [%]
50 (40–55)
48 (40–55)
0.55
CPK peak [UI/L]
1347 (535–2590) 1376 (677–2491) 0.77
Hospital stay [days]
4 (4–6)
5 (4–6)
0.84
Italics: p value ≤0.05
TIMI thrombolysis in myocardial infarction, STR ST resolution, CV
cardiovascular, AMI acute myocardial infarction, CVA cerebrovascular accidents,
LVEF left ventricular ejection fraction, CPK creatine phosphokinase Table 3 Procedural data in patients with STEMI and in primary
PCI. Data are expressed as percentage (frequency) or median (IQR) Table 4 Procedural and hospital outcomes. Data are expressed
as percentage (frequency) or median (IQR) p = 0.14) or in cerebrovascular accidents (0.4% vs 0.7%
vs 0.8%; p = 1.000) (Table 4). No significant difference
between the ticagrelor and clopidogrel groups was found
in stent thrombosis (1.4% vs 0.8%, p = 0.62) and infarct
size, estimated by the peak creatine kinase value (1347 vs
1372 UI/L, p = 0.77), or for left ventricular ejection
fraction (50% vs 48%, p = 0.55) before hospital discharge. The causes of cardiovascular death are reported in
Additional file 3. BARC bleeding was similar between the 2 groups; there
was no difference in BARC categories for BARC ≥2 and
BARC ≥3. The lowest median haemoglobin value (12.2 vs
12.2 g/dL; p = 0.8) and the difference between the highest
and lowest haemoglobin value during hospital stay (2.2 vs
2.1 g/dL; p = 0.9) were similar between the ticagrelor and
clopidogrel groups. The risk of transfusion and the
amount of packed red blood cell units did not show any
significant difference (Additional file 4). Statistical analysis In the Kaplan-Meier analysis, unadjusted 1-year survival
probability was higher in the ticagrelor group than in the
clopidogrel group (97.8% vs 87.8%; log-rank p = 0.024)
(Fig. 4), as was the probability of 1-year survival free from Vercellino et al. BMC Cardiovascular Disorders (2017) 17:97 Page 7 of 11 cardiovascular death (97 8% vs 90 2%; log-rank p = 0 005)
Fig. 4 Kaplan-Meier survival analysis for ticagrelor (red) vs clopidogrel (black) for 1 year of follow- up cardiovascular death (97.8% vs 90.2%; log-rank p = 0.005)
(Additional file 5). Fig. 5 Forest plot for in-hospital major adverse cardiovascular events
(MACE), cardiovascular death and Bleeding Academic Research Consortium
(BARC) bleeding. Unadjusted (at the top) and propensity-adjusted
(at the bottom) odds ratio (OR) for in-hospital MACE, cardiovascular death
and BARC bleedings ≥2. CI confidence interval Propensity score analysis A total of 21 interfering variables were included in the
propensity score and are reported in Additional file 6. Candidate variables included sex, age, BMI, smoking,
diabetes, dyslipidemia, familiar history of coronary artery
disease, cerebrovascular accident (CVA), bleeding, previ-
ous AMI or PCI, glomerular filtration rate, TIMI risk
score, CRUSADE score, anterior AMI, first medical
contact through the ambulance service, admission to hub
vs spoke, left ventricular ejection fraction, Killip class III-
IV, radial access, primary PCI ≤12 h. The propensity score was calculated for 368 patients
(missing for 33, 8.2%) and showed good discrimination
(area under the curve =0.695 [0.640–0.751]; p < 0.01) and
good calibration (p = 0.355). Using propensity score regres-
sion, there were no differences in mortality, ischaemic or
haemorrhagic events during the hospital stay. As showed in
Fig. 5, after adjusting for the propensity score, hospital
cardiovascular mortality (OR, 0.27 [95% CI, 0.03–2.19];
p = 0.218) and hospital MACEs (OR, 1.02 [95% CI,
0.38–2.79];], p = 0.963) were similar in the ticagrelor and
clopidogrel groups. Fig. 5 Forest plot for in-hospital major adverse cardiovascular events
(MACE), cardiovascular death and Bleeding Academic Research Consortium
(BARC) bleeding. Unadjusted (at the top) and propensity-adjusted
(at the bottom) odds ratio (OR) for in-hospital MACE, cardiovascular death
and BARC bleedings ≥2. CI confidence interval However, the survival analysis adjusted by propensity
score demonstrated that all-cause mortality at 1 year after
STEMI remained significantly lower for the ticagrelor
group (HR, 0.29 [0.08–0.99]; p = 0.048). Vercellino et al. BMC Cardiovascular Disorders (2017) 17:97 Vercellino et al. BMC Cardiovascular Disorders (2017) 17:97 Vercellino et al. BMC Cardiovascular Disorders (2017) 17:97 Page 8 of 11 Page 8 of 11 Page 8 of 11 STEMI population is still the subgroup analysis in the
PLATO and TRITON-TIMI 38 trials [10]. There were 6 crossovers from clopidogrel to ticagrelor
(3 for STEMI occurring on clopidogrel therapy, 3 for
unknown cause), and 17 crossovers from ticagrelor to
clopidogrel (4 for high bleeding risk or vitamin K antagon-
ist [VKA], 2 for dyspnoea, 2 for low patient compliance, 4
for low ischaemic risk after coronary angiography, 4 for
unknown cause). Seven of 401 patients were proposed for
elective
coronary
artery
bypass
graft
(1.7%)
during
hospitalization, 5 in the clopidogrel group and 2 in the
ticagrelor group. Ten patients were discharged without
DAPT for high bleeding risk or contemporary VKA therapy
(Fig. 1). Discussion In real-world populations of patients with STEMI admit-
ted to an Italian hospital, treatment with ticagrelor
reduced the mortality rate at 1 year compared with
clopidogrel, with a risk of mortality 3.4 times higher for
clopidogrel than for ticagrelor. This was confirmed when
propensity analysis was used to reduce the risk of bias
because of lack of randomization. Ticagrelor did not
reduce hospital MACE after propensity score correction,
and it did not affect hospital bleeding according to the
BARC classification. (
p
)
Because the PLATO and TRITON-TIMI 38 trials are
heterogeneous with regard to patient characteristics and
treatments, their results could not be compared. The
TRITON-TIMI 38 trial enrolled clopidogrel-naive patients
undergoing
primary
and
secondary
PCI,
whereas in
PLATO almost 50% of patients were preloaded with open-
label clopidogrel (300 or 600 mg), reducing the benefit of
ticagrelor in the early phase of follow-up. Considering the
PLATO subgroup analysis, ticagrelor did not reduce the
primary endpoint at 12 months but conferred a survival
advantage after 30 days, and this may be the result of a
mechanism that differs from antiplatelet activity. Type 1
equilibrative
nucleoside
transporter
(ENT1)
protects
adenosine from intercellular metabolism. Inhibiting ENT1
increases the concentration and biological activity of adeno-
sine, particularly at sites of ischemia and tissue injury where
it is formed [31]. Furthermore, in patients with STEMI, the
beneficial effect of ticagrelor may be more pronounced in a
subpopulation of high-risk patients characterized by high
on-treatment platelet reactivity even after large loading
doses of clopidogrel [32]. In these patients, even a double
dose of clopidogrel did not reduce cardiovascular events
compared with the standard dose, as demonstrated by the
CURRENT-OASIS trial [33]. The new 2PY12 inhibitors, ticagrelor and prasugrel, are
known to have more favourable pharmacologic proprieties
for ACS than clopidogrel. Because of more predictable
pharmacokinetics and a more potent and constant effect
with faster onset, there is strong rationale for their use in
patients with STEMI [4, 9]. However, despite promising
pharmacological data and European guidelines recommen-
dations to administer the new 2PY12 inhibitors in patients
with STEMI [28], there is still uncertainty about their real
clinical efficacy compared with the older and more widely
adopted clopidogrel. g
The STEMI population differs from the larger ACS
population in several respects, because it is composed
of younger patients with less comorbidities and lower
procedural haemorrhage risk, but higher incidence of
hemodynamic instability at presentation. Propensity score analysis The TRITON-TIMI trial subgroup analysis showed that
prasugrel was superior to clopidogrel (300 mg loading
dose/75 mg maintenance dose) in 3534 patients with
STEMI undergoing primary or secondary PCI when
considering cardiovascular death, non-fatal MI, or non-
fatal stroke at 30 days (115 [6.5%] vs 166 [9.5%]; 0.68
[0.54–0.87]; p = 0.0017] and at 15 months (174 [10.0%] vs
216 [12.4%]; 0.79 [0.65–0.97]; p = 0.0221). A subanalysis
in the PLATO trial on 7544 patients with STEMI has
shown no superiority in MACE (cardiovascular death,
non-fatal MI and stroke) for ticagrelor versus clopidogrel
at 12 months follow-up, although there was a nominal
trend in favour of ticagrelor (HR, 0.87 [0.75–1.01];
p = 0.07). However, in this analysis, ticagrelor significantly
reduced secondary endpoints such as total mortality (HR
0.82; p = 0.05), MI (HR, 0.8; p = 0.03), and definite stent
thrombosis (HR 0.66; p = 0.03). Discussion Another Swiss observational study, the Swiss ACS
bleeding score, demonstrated that clopidogrel and prasu-
grel had similar safety profiles at 30 days (BARC 3, 4, 5 ad-
justed HR, 0.75 [0.42–136]) and at 1 year (BARC 3, 4, 5
adjusted HR, 0.67 [0.38–1.20]), without considering effi-
cacy [14]. The SCAAR registry from Sweden demon-
strated that ticagrelor increased survival and reduced
bleeding risk in selected patients with ACS at low risk of
bleeding [11]. Conversely, there is no European registry
on the effect of ticagrelor in the STEMI population. The
Greek GRAPE registry considered the clinical effect of
clopidogrel, prasugrel, and ticagrelor in the general ACS
population,
hindering
conclusions
on
the
STEMI
population. The
GRAPE
study
demonstrated
that
ticagrelor does not reduce the rate of MACE at 1 year
(HR,
0.78
[0.54–1.12]),
whereas
the
bleeding
rate
increased (HR, 1.81 [1.55–2.10]) [16]. g
p
g
[
]
In the present study, there was a 10% improvement in
the success rate of primary PCI in patients on ticagrelor,
with improvement in post-PCI TIMI 3 score (p = 0.001). This result came from crude data, without randomization
or propensity score correction. Therefore, the better angio-
graphic results in patients treated with ticagrelor could be
secondary to chance. The PLATO angiographic substudy
[36] showed that neither coronary flow nor myocardial
perfusion demonstrated a difference with ticagrelor versus
clopidogrel. However, in this substudy, the time interval
between randomization and angiography or PCI was really
short, particularly in the STEMI patients. In our institution,
many patients were pretreated before arrival at the
catheterization laboratory and the time for the pharmaco-
logical effect was longer. This fact, associated with greater
platelet inhibition with ticagrelor, could have improved
angiographic outcomes. Ticagrelor is also known to inhibit
cell uptake of adenosine [31], and it could also be hypothe-
sized that ticagrelor might increase the concentration of ad-
enosine
in
the
myocardium
more
than
clopidogrel,
inducing hyperemia and vasodilation, which is inconsistent
with the results of the PLATO angiographic subgroup,
where there was no improvement in coronary flow after
percutaneous revascularization [36]. We should underline
that this finding came from crude analysis and it could be
biased by the lack of randomization or propensity score
correction. However, in our sample, as in the PLATO
angiographic subgroup, ticagrelor and clopidogrel achieved
a similar proportion of ST resolution after PCI, with no
effects on cardiac reperfusion. Discussion Moreover,
conditions affecting drug bioavailability, such as reduced
gastrointestinal absorption, vomiting and morphine ad-
ministration, can reduce the effect of P2Y12 more in
patients with STEMI than other ACS [8, 29]. In an RCT
directly comparing ticagrelor and clopidogrel, patients
with STEMI had a delayed onset of both these P2Y12
inhibitors [30]. A delayed onset of action for ticagrelor
was also confirmed in a small randomized trial measuring
platelet reactivity after a loading dose of ticagrelor and
prasugrel in patients with STEMI [9]. Despite the large number of patients included, PLATO
and TRITON-TIMI 38 were not designed specifically to
assess the effect of P2Y12 in patients with STEMI, and both
trials are underpowered to reach a definitive conclusion;
the low quality of this conclusion is summarized in the B
level of evidence in the European guidelines [28]. In the absence of high-quality RCTs, however, there is
increasing interest in prospective observational registries. The Swiss ATACS registry analysed the effect of prasugrel
on a STEMI population, demonstrating that it is advanta-
geous over clopidogrel (in-hospital mortality 1.7% vs 4.4%) With no randomized trial focusing on the effect of
ticagrelor or prasugrel versus clopidogrel in a STEMI
population and the uncertainties about pharmacokinetics,
the source of evidence on the new 2PY12 inhibitors in the Vercellino et al. BMC Cardiovascular Disorders (2017) 17:97 Page 9 of 11 function, such as adenosine mimetic action [31]. More-
over, it may result from a favourable ratio between the
unavoidable
haemorrhagic
risk
and
protection
from
ischaemic events. The observational registry by Park et al. [17] on ticagrelor in a STEMI population yielded contrast-
ing results, showing that it did not reduce the risk of
ischaemic events but was associated with an increased risk
of bleeding compared with clopidogrel after controlling
for propensity score. That study was conducted in Korea,
thus it may lack external validity in Europe due to genetic
variations and difference in patient characteristics, such as
BMI or age. Furthermore, Park et al. considered only pa-
tients with STEMI undergoing PCI, excluding patients
with STEMI undergoing medical therapy, where the bene-
ficial effect of ticagrelor vs clopidogrel is increased [35]. at the expense of an increased bleeding risk (significantly
superior for prasugrel on adjusted but not crude analysis)
[15]. Ethics approval and consent to participate pp
p
p
This study was carried out according to ethical standards, national and
international guidelines. It was approved by the local ethical committee,
Comitato Etico Regione Liguria. Written informed consent was obtained
from each patient to participate in the study. Limitations (DOCX 51 kb) Additional file 6: Interfering variables retained in the propensity score
model. (DOCX 11 kb) Discussion Our study is one of the few available registries on the
effect of ticagrelor in a STEMI population and is the first
to focus on the European setting. It has confirmed the
findings from the PLATO trial. Our study demonstrated
no benefit for ticagrelor with regard to cardiovascular
death, AMI and stroke during the hospital phase. The
risk reduction observed for in-hospital unadjusted car-
diovascular mortality was not confirmed after propensity
score analysis. This may be because of the use of a high
clopidogrel loading dose (over 42% patients ≥300 mg) in
the pre-hospital phase through the ambulance service. These results are concordant with the PLATO trial,
where the beneficial effect of ticagrelor on MACE was
achieved only after 30 days. In our data, ticagrelor did not increase the risk of
bleeding according to the BARC classification. This may
depend on the lower haemorrhagic risk of the STEMI
population compared with the general ACS population. The low haemorrhagic risk could have reduced the rate of
clinically relevant bleeding even if the drug demonstrated
a larger antiplatelet effect. Both these findings are in
accordance with the PLATO subanalysis on STEMI. A recent meta-analysis found a 20% reduced rate of
MACE in patients on the new P2Y12 compared with
clopidogrel [34], at the expense of a 50% increase in the
risk of stroke. This was not confirmed in our study,
where the proportions of MACE and stroke were similar
in the 2 groups. Ticagrelor was discontinued in 2% of patients due to
dyspnoea, a previously reported complication [6]. Other pa-
tients were converted to clopidogrel because of compliance
problems, increased haemorrhagic risk or contemporary
VKA treatment. There was only one intra-hospital death in
the ticagrelor group (caused by cardiogenic shock), com-
pared with 14 intra-hospital deaths in the clopidogrel
group; this difference is relevant but it was not significant
after controlling for the propensity score. The main result of the present study, a reduction in 1-
year mortality for ticagrelor, is concordant with the results
of the PLATO study and the substudy in the STEMI
population. In our registry, limited by the small sample
size, this finding may be the result of statistical chance;
however, it may also result from pharmacodynamic pro-
prieties of ticagrelor that are distinct from its antiplatelet Page 10 of 11 Vercellino et al. BMC Cardiovascular Disorders (2017) 17:97 Funding g
Cardio-STEMI is an independent study. English language editing and styling assistance was provided by Edra spa,
and unconditionally funded by AstraZeneca. Authors’ contributions FAS and MV contributed to conception and study design. FAS designed the
software to support data collection and acted as data manager. FAS, MV, VB,
CT, DP, SC collected data. FAS, MV, VB and GGS helped in data analysis and
interpretation. MV performed statistical analysis and acted as corresponding
author. MV, FAS, VB were the major contributors in drafting the manuscript. GM and GP revised the manuscript critically for important intellectual
t
t
d
i
d th d
l
t
f th
k All
th
d
d content and supervised the development of the work. All authors read and
approved the final manuscript. Abbreviations ACS: Acute coronary syndrome; AMI: Acute myocardial infarction;
BARC: Bleeding Academic Research Consortium; BMI: Body mass index;
CI: Confidence interval; CRUSADE: Can rapid Risk stratification of Unstable
angina patients Suppress ADverse outcome with Early implementation of
ACC/AHA Guidelines; CVA: Cerebrovascular accident; DAPT: Dual antiplatelet
treatment; DES: Drug-eluting stent; HR: Hazard ratio; IQR: Interquartile range;
MACE: Major adverse cardiovascular event; MI: Myocardial infarction;
NSTEMI: Non-ST-segment elevation myocardial infarction; OR: Odds ratio;
PCI: Percutaneous coronary intervention; RCT: Randomized controlled trial;
STEMI: ST-segment elevation myocardial infarction; TIMI score: Thrombolysis
in myocardial infarction score; VKA: Vtamin K antagonist Consent for publication
Not applicable. Not applicable. Limitations Additional file 2: Discharge therapy. Data are expressed as percentage
(frequency). (DOCX 11 kb) This study has the limitations of a prospective case-control
study. There were some differences between baseline and
procedural data. Cases and controls were enrolled in differ-
ent time periods, and this may contribute to confounding
and to the different sizes of the study groups. However, we
used propensity scores to minimize the bias related to lack
of randomization, a strategy commonly used in other
studies based on similar prospective registries. Power may
be limited by sample size, however to our knowledge this
study is the largest real-world registry on ticagrelor in
Europe, even though the study was not powered for hard
endpoints. The use of radial access was lower than the
current standard and this issue could have had an unfavor-
able impact, increasing the bleeding risk in our sample. However, the type of access was included in the propensity
score model in order to reduce the interference related to
the differences between ticagrelor and clopidogrel groups. One further limitation is the lack of data collection on
MACE and bleeding after the hospital phase. This may
have influenced the perception of the favourable effects on
the rate of MACE, which may become apparent only after
30 days [6]. However, this choice is often used during
follow-up data collection within observational registries, as
mortality is more easily and reliably collected after dis-
charge, and it may be considered a surrogate outcome of
complications including MACE and bleeding. Although
the 2012 ESC guidelines recommended the use of ticagre-
lor or prasugrel in place of clopidogrel, in our institution
prasugrel was not introduced during the time window
examined in this study. This is mainly because prasugrel
should not be administered in patients older than 75 years,
with low body weight (≤60 kg) or with a history of previ-
ous CVA. So its introduction in our STEMI network,
particularly in the pre-hospital setting, was considered
difficult. We have no data on the duration of DAPT;
however, each patient received indication to continue
DAPT for at least 12 months after STEMI. Additional file 3: Intra-hospital causes of death. Data are expressed as
percentage (frequency). (DOCX 11 kb) Additional file 4: Data on BARC bleeding. Data are expressed as
percentage (frequency) or median (IQR). (DOCX 12 kb) Additional file 5: Unadjusted Kaplan-Maier analysis on cardiovascular
mortality at 1 year. Availability of data and materials
h d
l
d d
h The datasets analysed during the current study are available from the
corresponding author on reasonable request. Conclusions The authors declare that they have no competing interests. Editorial support, in the form of technical editing and language editing and
proofreading was provided by Edra spa, and unconditionally funded by
AstraZeneca, manufacturer of ticagrelor. In this real-world single-centre experience, ticagrelor
resulted in improved survival at 1 year versus clopidogrel
in patients with STEMI, even after propensity score
correction. Ticagrelor did not reduce the composite
outcome of in hospital MACE, and it did not increase the
risk of in-hospital bleeding. Although these results con-
firm data from previous subanalysis in STEMI patients,
large RCTs are warranted to confirm the positive effect of
ticagrelor shown in this population. Competing interests Cardio-STEMI is an independent study. Additional file 1: Baseline laboratory data. (DOCX 15 kb) References Ticagrelor versus clopidogrel in patients with ST-elevation acute coronary
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presenting with ST-segment elevation. Eur Heart J. 2012;33:2569–619. 3. Wallentin L, Varenhorst C, James S, Erlinge D, Braun OO, Jakubowski JA, et al. Prasugrel achieves greater and faster P2Y12 receptor-mediated platelet
inhibition than clopidogrel due to more efficient generation of its active
metabolite in aspirin-treated patients with coronary artery disease. Eur Heart J. 2008;29:21–30. 23. O'Gara PT, Kushner FG, Ascheim DD, Casey DE, Chung MK, de Lemos JA, et al. 2013 ACCF/AHA guideline for the management of ST-elevation myocardial
infarction: executive summary. Circulation. 2013;127:529–55. 24. Cutlip DE, Windecker S, Mehran R, Boam A, Cohen DJ, van Es G-A, et al. Clinical
end points in coronary stent trials: a case for standardized definitions. Circulation. 2007;115:2344–51. 4. Gurbel PA, Bliden KP, Butler K, Tantry US, Gesheff T, Wei C, et al. Randomized
double-blind assessment of the ONSET and OFFSET of the antiplatelet effects
of ticagrelor versus clopidogrel in patients with stable coronary artery disease:
the ONSET/OFFSET study. Circulation. 2009;120:2577–85. 4. References Ticagrelor
versus clopidogrel in patients with acute coronary syndromes intended for
non-invasive management: substudy from prospective randomised PLATelet
inhibition and patient Outcomes (PLATO) trial. BMJ. 2011;342:d3527. 36. Kunadian V, James SK, Wojdyla DM, Zorkun C, Wu J, Storey RF, et al. Angiographic outcomes in the PLATO trial (Platelet Inhibition and Patient
Outcomes). JACC Cardiovasc Interv. 2013;6:671–83. 16. Alexopoulos D, Xanthopoulou I, Deftereos S, Hamilos M, Sitafidis G,
Kanakakis I, et al. Contemporary antiplatelet treatment in acute coronary
syndrome patients undergoing percutaneous coronary intervention: 1-year
outcomes from the GReek AntiPlatElet (GRAPE) Registry. J Thromb Haemost
2016;14:1146–54. 16. Alexopoulos D, Xanthopoulou I, Deftereos S, Hamilos M, Sitafidis G,
Kanakakis I, et al. Contemporary antiplatelet treatment in acute coronary
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outcomes from the GReek AntiPlatElet (GRAPE) Registry. J Thromb Haemost. 2016;14:1146–54.
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https://openalex.org/W2411070939
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https://journal.ipb.ac.id/index.php/jupe/article/download/2178/1207
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Indonesian
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KEMANDIRIAN PEMBUDIDAYA IKAN PATIN DI LAHAN GAMBUT DI DESA TANGKIT BARU, KEC. KUMPE ULU, KABUPATEN MUARO JAMBI PROVINSI JAMBI
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Jurnal penyuluhan
| 2,008
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cc-by
| 4,060
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Abstract Self reliance of the patin fish farmers in Tangkit Baru Village is very important to
promote better quality of life. Self reliance of the fish farmers reflected ability of them in coping
the problems and finding out of alternatives solution. The objectives of the study were: (1) To
indentify the characteristics of patin fish farmers in peatlands, (2) To analyze self reliance of
the patin fish farmers in managing of aquaculture, (3) To analyze the relationship between the
self reliance of the patin fish farmers and their characteristics. Population of this study were the
patin fish farmers in Tangkit Baru. The number of the respondens were 69 patin fish farmers in
Tangkit Baru which were taken by simple random sample. The data were collected on May to
July 2007. The data were analyzed by using Correlation Spearman. The results of the study
were: (1) Cultivation of patin fish in Tangkit Baru village is very successful. The fish farmers
could modify their business well, as they conducted their budiness independently, (2) Patin fish
farmers have been done a good action in capital management, production process and
marketing, (3) There were significant relation between self reliance of the patin fish farmers and
their characteristics, namely in formal education, motivation, family dependent members,
cosmopolite, man power, and experience in business. Key words: self reliance, patin fish cultivator, peatlands, patin fish memperbaiki kondisi rumah tangga, usaha,
dan aspek sosial kemasyarakatan. 58-2664 September 2008, Vol. 4 No. 2
KEMANDIRIAN PEMBUDIDAYA IKAN PATIN DI LAHAN GAMBUT
DI DESA TANGKIT BARU, KEC. KUMPE ULU,
KABUPATEN MUARO JAMBI PROVINSI JAMBI
(SELF RELIANCE OF THE PATIN FISH FARMER IN PEATLANDS
IN TANGKIT BARU VILLAGE, SUBDISTRICT OF KUMPE ULU,
DISTRICT OF MUARO JAMBI, PROVINCE OF JAMBI) ISSN: 1858-2664 ISSN: 1858-2664 ISSN: 1858-2664 Aprolita, Siti Amanah, dan Djoko Susanto Metode Penelitian gambut dimanfaatkan sebagai media tanaman
dan
ternyata
pembudidaya
mampu
menjalankan usaha budidaya ikan patin di
lahan usaha yang kritis itu. Populasi
penelitian
adalah
pembudidaya ikan patin di Kecamatan Kumpe
Ulu Kabupaten Muaro Jambi Provinsi Jambi. Pemilihan lokasi tersebut didasarkan pada
potensi pengembangan usaha budidaya ikan
patin di kolam dan karakteristik lahan yakni
berupa lahan gambut dengan jenis ikan patin
yang dibudidayakan
adalah jenis
siam. Pengumpulan data dilakukan pada bulan Mei
sampai Juli 2007. Di Desa Tangkit Baru saat ini telah
berkembang kolam ikan sebanyak 559 unit ( ±
18 ha atau 10% dari potensi yang ada) yang
dikelola oleh 230 kepala keluarga. Tingkat
produksi yang dicapai saat ini adalah 1 – 2
ton/unit/musim tanam untuk luas kolam antara
200 – 400 m²/ unit, sehingga dari kawasan ini
setiap hari rata-rata dapat dihasilkan ikan
patin sebanyak 2-3 ton. Saat ini kebutuhan
ikan patin di pasaran Provinsi Jambi setiap
hari sebesar 5 ton. Penentuan sampel dilakukan dengan
acak sederhana yakni cara pengambilan
sampel
dari
anggota
populasi
dengan
menggunakan acak tanpa memperhatikan
strata (tingkatan) dalam anggota populasi
tersebut. Pada lokasi penelitian jumlah
pembudidaya ikan patin sebanyak 230 orang
dengan jumlah sampel yang diambil sebanyak
69 orang pembudidaya di lokasi penelitian. Untuk itulah penelitian ini menjadi
penting dilakukan, mengingat ikan patin
merupakan komoditas unggulan. Selain itu
upaya pembudidaya untuk mengelola lahan
gambut sebagai lokasi pembudidayaan ikan
patin
dapat
dikembangkan
di
kondisi
lingkungan yang serupa. Berdasarkan hal di
atas, maka permasalahan yang diajukan dalam
penelitian ini adalah sebagai berikut: Apa
yang dilakukan pembudidaya ikan patin di
lahan gambut? Faktor-faktor apa saja yang
berhubungan
dengan
kemandirian
pembudidaya ikan patin dalam mengelola
usaha budidayanya? Bagaimana langkah-
langkah
atau
upaya
untuk
menjamin
keberlanjutan atau kemandirian pembudidaya
dalam mengelola usaha budidaya ikan patin? Penelitian
ini
dirancang
sebagai
sebuah
penelitian
survei,
di
mana
pengambilan data primer dilakukan melalui
wawancara
dan
pengisian
kuesioner. Penelitian ini merupakan penelitian deskriptif
korelational yang mempelajari huubungan
antara peubah bebas dengan terikat. Analisis
hubungan
antara
peubah
penelitian
menggunakan korelasi Spearman (Nazir. 1998), dan untuk memudahkan pengolahan
data digunakan program SPSS. Kemandirian pembudidaya ikan patin
dapat dilihat dari pencapaian keberhasilan
dalam usaha budidaya yang berdampak
langsung pada hasil budidaya yang diperoleh
per masa tanam, maka tujuan dari penelitian
ini
adalah
untuk:
(1)
Mengidentifikasi
keragaan pengelolaan budidaya ikan patin
yang dilakukan oleh pembudidaya ikan patin
di
lahan
gambut. (2)
Menganalisis
kemandirian pembudidaya ikan patin dalam
mengelola usaha budidayanya, yang meliputi
aspek
modal/keungan,
produksi,
dan
pemasaran. Metode Penelitian (3) Mengkaji hubungan antara
kemandirian
pembudidaya
dengan
karakteristik yang dimiliki pembudidaya ikan
patin. Aprolita, Siti Amanah, dan Djoko Susanto/
Jurnal Penyuluhan September 2008, Vol. 4 No. 2 Aprolita, Siti Amanah, dan Djoko Susanto/
Jurnal Penyuluhan September 2008, Vol. 4 No. 2 Metode Penelitian Pendahuluan Kemandirian pembudidaya ikan patin
di desa Tangkit Baru tidak terlepas dari
keinginan untuk hidup lebih baik dengan
pendapatan
yang
semakin
meningkat. Masyarakat dikatakan mandiri apabila telah
dapat
menolong
dirinya
sendiri
dalam
mengidentifikasi masalah dan mencari jalan
keluar penyelesaiannya. Pembudidaya Ikan
patin dapat dikatakan berhasil apabila mereka
dapat menjaga kelangsungan usaha budidaya
dengan baik, sehingga dapat membantu Dalam konteks pembangunan sikap
mandiri
harus
dijadikan
tolok
ukur
keberhasilan,
yakni apakah rakyat atau
masyarakat menjadi lebih mandiri (bebas)
atau malah semakin bergantung. Misalnya
apakah pembudidaya ikan patin lebih bebas
dalam melakukan usaha budidayanya. Pembudidaya ikan patin di Desa
Tangkit Baru memiliki keunikan dari sisi
lahan usaha bila dibandingkan dengan yang
ada di Jawa. Di Desa Tangkit Baru lahan 127 Kemandirian Pembudidaya dalam Usaha
Budidaya Ikan Patin di Lahan Gambut Kemandirian
pembudidaya
dalam
usaha budidaya ikan patin terdiri atas: Tingkat
kemandirian dalam proses budidaya ikan patin
yang baik dan benar, tingkat kemandirian
dalam pengelolaan modal dan keuangan,
tingkat kemandirian dalam pemasaran hasil
budidaya ikan patin (Tabel 2). Untuk melihat tingkat kemandirian
pembudidaya ikan patin maka digunakan
kategori berdasarkan nilai tengah simpangan
baku dari sebaran data yang diperoleh. Tabel 2. Distribusi Responden Berdasarkan Tingkat Kemandirian
dalam Budidaya Ikan Patin
No
Peubah
Kategori
Jumlah
Persentase
1. 2. 3. Tingkat kemandirian
dalam pengelolaan
modal
Tingkat kemandirian
dalam proses budidaya
Tingkat kemandirian
dalam pemasaran hasil
Rendah (jumlah skor ≤ 25)
Sedang (jumlah skor 25 – 30)
Tinggi (jumlah skor ≥ 30)
Rendah (jumlah skor ≤ 18)
Sedang (jumlah skor 18 – 23)
Tinggi (jumlah skor ≥ 23)
Rendah (jumlah skor ≤ 33)
Sedang (jumlah skor 33 – 38)
Tinggi (jumlah skor ≥ 38)
52
11
6.0
1.0
15
53
0.5
16.5
52
75.4
15.9
8.70
1.50
21.7
76.8
0.75
23.85
75.4 Tabel 2. Distribusi Responden Berdasarkan Tingkat Kemandirian
dalam Budidaya Ikan Patin Permodalan sesungguhnya merupakan
salah satu persoalan utama yang dikeluhkan
oleh responden. Selama ini pembudidaya ikan
patin mengandalkan modal sendiri yang
jumlahnya terbatas. Modal tersebut cukup
untuk melakukan usaha budidaya ikan patin
(untuk beberapa kolam) dan sisanya untuk
memenuhi kebutuhan hidup, namun tidak
cukup sebagai modal untuk mengembangkan
usaha budidaya ikan patin yang lebih besar. Keragaan Budidaya Ikan Patin
di Lahan Gambut Kemandirian pembudidaya ikan patin
yang diamati dalam penelitian ini adalah:
terdiri dari faktor internal yaitu: 1. umur, 2. motivasi, 3. pengalaman usaha, 4. tingkat
pendidikan formal, 5. tanggungan keluarga, 6. tingkat kekosmopolitan, 7. kontak dengan
penyuluh, 8. tenaga kerja, 9. akses kredit, dan
10. kontak dengan penyuluh (Tabel 1). 128 Aprolita, Siti Amanah, dan Djoko Susanto/
Jurnal Penyuluhan September 2008, Vol. 4 No. 2 Tabel 1. Distribusi Pembudidaya Ikan Patin
pada Sejumlah Karakteristik yang Diamati Tabel 1. Distribusi Pembudidaya Ikan Patin
pada Sejumlah Karakteristik yang Diamati
No
Karakteristik Pembudidaya
Kategori
Jumlah
n
%
1. Umur
Muda
Sedang
Tua
17
21
31
24.6
30.4
44.9
2. Motivasi
Rendah
Sedang
Tinggi
17
24
28
24.6
34.8
40.6
3. Pengalaman Usaha
Kurang
Sedang
Banyak
21
22
26
30.4
31.9
37.7
4. Tingkat Pendidikan
Rendah
Sedang
Tinggi
20
27
22
29.0
39.1
31.9
5. Tanggungan Keluarga
Sedikit
Sedang
Banyak
20
22
27
29.0
31.9
39.1
6. Kekosmopolitan
Rendah
Sedang
Tinggi
24
25
20
34.8
36.2
29.0
7. Kontak dengan Penyuluh
Kurang
Sedang
Sering
30
20
19
43.5
29.0
27.5
8. Tenaga Kerja
Sedikit
Sedang
Banyak
28
20
21
40.6
29.0
30.4
9. Akses Kredit
Sulit
Sedang
Mudah
26
21
22
37.7
30.4
31.9 Berdasarkan keragaan budidaya ikan
patin di Desa Tangkit Baru ada beberapa hal
yang menarik untuk dibahas, ini dapat dilihat
pada tabel di bawah, bahwa kemandirian
pembudidaya ikan patin di lokasi penelitian
tidaklah
tergantung
pada
umur,
dan
pendidikan, yang sangat mendukung dalam
kemandirian pembudidaya ikan patin di Desa
Tangkit Baru adalah motivasi untuk hidup
lebih
baik
lagi
sehingga
terpenuhinya
kebutuhan hidup sehari-hari. Hal ini senada
dengan
apa
yang
diungkapkan
Padmowihardjo
(1994)
bahwa
motivasi
merupakan
usaha
yang
dilakukan
oleh
manusia
untuk
menimbulkan
dorongan
berbuat
atau
melakukan
tindakan
yang
menyebabkan seseorang berbuat sesuatu guna
mencapai tujuan yang diinginkan. menjalankan usaha budidaya ikan patin ini. Walaupun tingkat kekosmopolitan dan kontak
dengan penyuluh rendah, tetapi pembudidaya
sangat aktif dalam kelompok yang mereka
dirikan. Mereka selalu mengadakan diskusi
dan saling membantu apabila diperlukan atau
bila menemui kesulitan dalam melakukan
budidaya. Hal
ini
menunjukan
bahwa
kemandirian bukanlah tergantung kepada
orang lain tetapi kemandirian akan timbul
apabila terjadinya saling tolong menolong
antar
sesama
pembudidaya
ikan
patin. Menurut
Slamet
(1995)
kemandirian
diarahkan
agar
dengan
kekuatan
dan
kemampuannya,
mereka
berupaya
untuk
bekerjasama dalam mencapai segala yang
dibutuhkan dan diinginkan. Pembudidaya ikan patin memiliki
banyak pengalaman sehingga mereka dapat 129 Aprolita, Siti Amanah, dan Djoko Susanto/
Jurnal Penyuluhan September 2008, Vol. 4 No. 2 Aprolita, Siti Amanah, dan Djoko Susanto/
Jurnal Penyuluhan September 2008, Vol. 4 No. 2 Kemandirian Pembudidaya dalam Usaha
Budidaya Ikan Patin di Lahan Gambut Kemandirian Pembudidaya dalam Usaha
Budidaya Ikan Patin di Lahan Gambut Tingkat Kemandirian Pembudidaya Ikan Patin
dalam Pengelolaan Modal Jumlah
skor
tingkat
kemandirian
responden dalam permodalan dapat dilihat
pada Tabel 2. yang memberi gambaran bahwa
pembudidaya memiliki tingkat kemandirian
dalam permodalan rendah yakni sebanyak 52
pembudidaya memiliki skor rendah yakni
kurang dari skor 25. Para pembudidaya berkeinginan untuk
meningkatkan hasil, selain dengan cara
memperbanyak kolam, juga dapat melakukan
pengolahan
hasil
budidaya
untuk
meningkatkan nilai jual. Untuk itu diperlukan
modal yang tidak sedikit. Menurut Suratiyah
(2006) modal adalah barang ekonomi yang
dapat digunakan untuk memproduksi kembali,
mempertahankan
atau
meningkatkan
pendapatan. Sehingga
modal
merupakan
faktor penunjang utama dalam kegiatan
budidaya. Tanpa adanya modal, pembudidaya Dalam melakukan usaha budidaya
ikan patin, responden memanfaatkan modal
yang dimiliki sendiri. Modal yang didapat dari
luar berupa fasilitas pinjaman dari Bank
diberikan pada beberapa orang saja. Hal ini
disebabkan
agunan
yang
diberikan
pembudidaya kepada Bank dianggap tidak
layak atau tidak mencukupi oleh pihak Bank. Biasanya pembudidaya mengagunkan lahan
usahanya. 130 Aprolita, Siti Amanah, dan Djoko Susanto/
Jurnal Penyuluhan September 2008, Vol. 4 No. 2 Aprolita, Siti Amanah, dan Djoko Susanto/
Jurnal Penyuluhan September 2008, Vol. 4 No. 2 kehitaman, hal ini karena air tersebut berasal
dari lahan gambut. akan
sulit
budidayanya. akan
sulit
mengembangkan
usaha
budidayanya. Pada seleksi dan penebaran benih,
pembudidaya tidak melakukan penyeleksian
benih karena mereka membeli benih yang
telah siap tebar lewat Balai Benih Air Tawar
(BBAT) Sei. Gelam dan penyedia bibit ikan
patin siam perorangan yang terdapat di
Provinsi Jambi. Penebaran benih dilakukan
pada suhu air rendah yakni pada pagi hari atau
sore
hari. Biasanya
benih
dimasukan
perlahan-lahan pada kotak-kotak kecil yang
dibuat dalam kolam. Hal ini dilakukan agar
benih dapat beradaptasi dengan kondisi air
kolam. Dalam melakukan usaha budidaya
ikan patin, responden tidak pernah mencatat
keluar masuknya uang atau pendapatan rumah
tangga. Mereka tidak melakukan perencanaan
usaha, apalagi melakukan analisis untung rugi
usaha. Sebagian kecil mereka yang melakukan
pencatatan uang masuk dan keluar dan
menganalisis untung dan rugi, hal ini
dilakukan oleh pembudidaya yang memiliki
tingkat pendidikan yang tergolong tinggi. Keputusan-keputusan yang berkaitan dengan
keuangan dan modal usaha budidaya ikan
patin
umumnya
diambil
berdasarkan
kebiasaan
atau
informasi
dari
sesama
pembudidaya ikan patin. Kemandirian pembudidaya ikan patin
pada tahap pengelolaan pakan, biasanya
pembudidaya patin mendapatkan pakan dari
kelompok budidaya yang membuat pakan
sendiri. Pembuatan pakan sendiri di lokasi
penelitian bertujuan untuk menekan harga
pakan, sehingga pembudidaya tidak begitu
terbebani
dengan
harga
pakan
dalam
pembudidayaan ikan patin, karena ikan patin
ini terkenal sangat rakus makanan untuk
membesarkan tubuhnya. Tingkat Kemandirian Pembudidaya Ikan Patin
dalam Pengelolaan Modal Pemberian pakan
dilakukan pembudidaya pada waktu pagi hari
dan pada waktu sore hari. Tingkat Kemandirian dalam
Proses Produksi Jumlah
skor
tingkat
kemandirian
responden dalam proses produksi dapat dilihat
pada Tabel. 2 yang memberi gambaran bahwa
pembudidaya memiliki tingkat kemandirian
dalam proses produksi tinggi yakni sebanyak
53 pembudidaya memiliki skor tinggi diatas
23. Pada tahap pengamatan kesehatan dan
penyakit pembudidaya melakukan sendiri
melalui
informasi
yang
didapat
dari
pembudidaya lain, buku, media massa,
maupun dari pengalaman yang sudah mereka
dapati selama melakukan usaha budidaya ikan
patin. Pengamatan kesehatan dan penyakit
berguna sekali, hal ini menurut responden
agar mereka cepat tanggap dalam mengambil
tindakan yang tepat pada saat ikan patin yang
ada pada kolam pembesaran memperlihatkan
gejala-gejala
yang
tidak
biasanya,
dan
pembudidaya
juga
bisa
mencari
solusi
terhadap musuh alami dalam budidaya ikan
patin yakni: berang-berang, biawak, ataupun
ikan gabus yang suka memakan benih patin
yanng berukuran kecil. Hampir
seluruh
responden
telah
mengetahui dengan baik teknik budidaya ikan
patin. Pengetahuan tersebut mereka dapatkan
baik melalui kelompok, media massa, dari
mulut ke mulut di antara sesama pembudidaya
ikan patin, atau dari pembudidaya yang
menjadi panutan pembudidaya lain di lokasi
penelitian. Setiap proses produksi umumnya
dilakukan sendiri oleh pembudidaya ikan
patin, kecuali pada saat persiapan lahan yakni
penggalian tanah yang dilakukan dengan
menggunakan
exavator
dan
pada
pembudidaya yang banyak memiliki kolam
mereka biasanya mengupah tenaga kerja
dengan sistem bagi hasil. Sedangkan pengairan, pembudidaya
mendapatkan air dari mata air yang berasal
dari kolam itu sendiri. Mata air akan didapat
bila penggalian kolam kurang lebih 1 meter. Air
untuk
kolam
ini
berwarna
coklat Kemandirian
pembudidaya
dalam
pengendalian hama dan penyakit sangat
membantu dalam proses budidaya ini. Hal ini
disebabkan mereka sangat paham bila terdapat 131 Aprolita, Siti Amanah, dan Djoko Susanto/
Jurnal Penyuluhan September 2008, Vol. 4 No. 2 Aprolita, Siti Amanah, dan Djoko Susanto/
Jurnal Penyuluhan September 2008, Vol. 4 No. 2 Aprolita, Siti Amanah, dan Djoko Susanto/
Jurnal Penyuluhan September 2008, Vol. 4 No. 2 gejala-gejala ikan patin yang terserang hama
dan
penyakit
dan
bagaimana
cara
pengobatannya. Disisi lain, pembudidaya ikan
patin dalam pengendalian hama dan penyakit
juga mendapat bantuan dari pembudidaya lain
yanng lebih tahu dan paham bagaimana cara
penanggulangannya. Terlihat di sini bahwa
pembudidaya yang mandiri bukanlah mereka
yang melakukan semua hal dengan sendiri
tanpa bantuan orang lain. dalam menjaring pembeli yang datang ke
lokasi agar mereka mau menjadi langganan
tetap. Kelemahan dari pemasaran ini menurut
responden adalah bila mereka tidak pintar
memperlakukan pembeli bisa saja pembeli
akan mencari ikan patin ke pembudidaya lain
yang dianggap dapat bekerjasama dengan
baik. Tingkat Kemandirian dalam
Proses Produksi Di lokasi penelitian pembudidaya
sangatlah
kompak
dalam
melakukan
usahanya,
karena
selain
mereka
membudidayakan
ikan
patin
secara
perorangan mereka juga tergabung dalam
kelompok
usaha
budidaya
ikan
patin. Kelompok
budidaya
ini
mengadakan
pertemuan setiap tanggal 10, untuk membahas
masalah harga, pemasaran, dan hal-hal yang
menyangkut usaha budidaya ikan patin. Proses pengolahan ikan patin menjadi
abon di lokasi penelitian masih dilakukan
secara berkelompok oleh para wanita desa. Pengolahan ikan patin menjadi abon adalah
salah satu usaha agar produksi yang melimpah
saat ini tidak sia-sia atau terbuang pada saat
panen. Pengolahan
hasil
ini
juga
meningkatkan pendapatan keluarga. Tingkat Kemandirian
dalam Pemasaran Hasil Hubungan
antara
karakteristik
responden dengan kemandiriannya dalam
usaha budidaya ikan patin dapat dilihat pada
Tabel. 3 Jumlah
skor
tingkat
kemandirian
responden dalam pemasaran hasil dapat
dilihat pada Tabel 2. yang memberi gambaran
bahwa
pembudidaya
memiliki
tingkat
kemandirian dalam pemasaran hasil tinggi
yakni sebanyak 52 pembudidaya memiliki
skor tinggi di atas skor 38. Umur dengan kemandirian responden
dalam usaha budidaya ikan patin di lahan
gambut tidak memiliki hubungan yang nyata. Dengan
kata
lain,
bahwa
umur
tidak
mempengaruhi kemandirian responden dalam
kegiatan usaha budidaya ikan patin, baik pada
aspek pengelolaan modal, proses produksi,
maupun pemasaran. Pada lokasi penelitian
umur pembudidaya tidaklah menjadi aspek
penghalang pembudidaya dalam kemandirian
usaha budidaya ikan patin. Dimana usia muda,
sedang, dan tua masing-masing memiliki
kemandirian yang sama dalam melakukan
usaha budidaya ikan patin ini. Begitupula
dengan pengelolaan modal dan pemasaran. Kemandirian pembudidaya ikan patin
dalam aspek pemasaran tergolong tinggi. Pemasaran yang dilakukan oleh pembudidaya
ikan
patin
yakni
pedagang
pengumpul
langsung membeli ikan patin ke pembudidaya,
bila harga telah disepakati maka ikan patin
pun akan dipanen dan langsung diambil
pedagang
pengumpul. Dari
pedagang
pengumpul ini akan dijual ke pasar-pasar
tradisional yang ada di Provinsi Jambi. Menurut Oedjoe (2001) banyaknya saluran
pemasaran akan menentukan besarnya harga
komoditi yang bersangkutan. Tingkat pendidikan formal memiliki
koefisien korelasi yang tinggi dengan tingkat
kemandirian dalam modal. Hal ini berarti ada
kecenderungan
semakin
tinggi
tingkat
pendidikan formal responden, maka tingkat
kemandirian dalam modal semakin tinggi. Hal
ini senada dengan hasil penelitian (Suyono, Pemasaran
ikan
patin
di
lokasi
penelitian memiliki rantai yang pendek. Pembudidaya tak perlu melakukan penjualan
ke pasar induk yang ada di Jambi, tetapi
pedaganglah yang menjemput hasilnya. Disini
pembudidaya ikan patin dituntut kejeliannya Aprolita, Siti Amanah, dan Djoko Susanto/
Jurnal Penyuluhan September 2008, Vol. 4 No. 2
1
Tabel. 3 Hubungan Karakteristik Pembudidaya
Ikan Patin dengan Kemandirian
No
Peubah
Tingkat Kemandirian Responden dalam:
Pengelolaan
Modal
Proses Produksi
Pemasaran
1. Umur
- 0.05
- 0.08
0.080
2. Pendidikan Formal
0.352**
0.199
0.233
3. Motivasi
- 0.169
- 0.25
-0.230
4. Jumlah Tanggungan
Keluarga
0.18*
- 0.037
0.155
5. Kosmopolitan
0.363**
- 0.05
0.127
6. Pengalaman Usaha
0.418**
0.013
0.099
7. Tenaga Kerja
0.146
0.24*
0.235
8. Kontak dengan Penyuluh
0.051
- 0.066
-0.230
9. Akses Kredit
0.425**
0.178
0.112
Keterangan:
* = terdapat hubungan yang nyata pada α = 0.05
**= terdapat hubungan yang nyata pada α = 0.01 132 Aprolita, Siti Amanah, dan Djoko Susanto/
Jurnal Penyuluhan September 2008, Vol. 4 No. Tingkat Kemandirian
dalam Pemasaran Hasil 2
133 133 Aprolita, Siti Amanah, dan Djoko Susanto/
Jurnal Penyuluhan September 2008, Vol. 4 No. 2 Aprolita, Siti Amanah, dan Djoko Susanto/
Jurnal Penyuluhan September 2008, Vol. 4 No. 2 Aprolita, Siti Amanah, dan Djoko Susanto/
Jurnal Penyuluhan September 2008, Vol. 4 No. 2 modal. Mereka
yang
memiliki
banyak
pengalaman cenderung berhati-hati dalam
mengeluarkan uang untuk usaha budidayanya,
mereka takut membelanjakan uangnya untuk
modal dalam usaha budidaya ikan patin ini
karena resiko kegagalan dapat saja terjadi bila
tidak
hati-hati
dalam
melakukan
usaha
budidaya
ikan
patin
ini. Sedangkan
pengalaman tidak mempengaruhi tingkat
kemandirian
pembudidaya
dalam
proses
produksi dan pemasaran hasil. proses produksi sehingga tugas pembudidaya
dengan
banyaknya
tenaga
kerja
dapat
mengurangi tugas pembudidaya sehingga
pembudidaya dapat melakukan aktifitas lain
yang menunjang keberhasilan usaha ini
seperti: mencari alternatif-alternatif lain dalam
memecahkan masalah yang sedang dihadapi
dalam budidaya atau mengadakan kontak
dengan pihak lain. Akses kredit pembudidaya berkorelasi
positif nyata dengan tingkat kemandirian
pembudidaya pada aspek pengelolaan modal
dan
keuangan. Di
lokasi
penelitian
pembudidaya yang mendapatkan akses kredit
lebih baik dalam pengelolaan modal dan
keuangan,
hal
ini
disebabkan
mereka
menyadari bahwa modal yang digunakan
dalam melakukan usaha budidaya ikan patin
ini bukan milik pribadi sehingga perlu
pengelolaan keuangan yang benar agar uang
pinjaman tersebut dapat dikembalikan setiap
bulannya. Tingkat kekosmopolitan pembudidaya
ikan patin sangat berkorelasi postif nyata
dalam kemandirian pembudidaya ikan patin
dalam hal permodalan. Hal ini terlihat dari
modal yang diperoleh pembudidaya yang
memiliki sifat kosmopolit tinggi, mereka
dapat
meminjam
ke
Bank
untuk
pengembangan
usahanya. Sedangkan
pembudidaya yang tidak memiliki sifat
kosmopolit mereka menggunakan modal
sendiri sehingga sulit dalam mengembangkan
usaha budidaya ikan patin ini. Di lokasi
penelitian
mereka
yang
memiliki
sifat
kosmopolit ini hanya beberapa orang saja,
mereka adalah pembudidaya-pembudidaya
yang tangguh dalam melakukan usahanya. Kekosmopolitan
pembudidaya
tidaklah
memiliki pengaruh terhadap kemandirian
pembudidaya pada aspek produksi dan
pemasaran. Tingkat Kemandirian
dalam Pemasaran Hasil 2 Tabel. 3 Hubungan Karakteristik Pembudidaya
Ikan Patin dengan Kemandirian 2006) bagi mereka yang memiliki tingkat
pendidikan yang memadai, jalan menuju
keberdayaan lebih terbuka lebar ketimbang
mereka
yang
tidak
berpendidikan. Pengalaman
di
negara-negara
maju
membuktikan,
bahwa
manusia-manusia
mandiri sebagian besar datang dari mereka
yang berpendidikan cukup tinggi. Sedangkan
hubungan pendidikan formal dengan proses
produksi dan pemasaran tidak berhubungan
dengan kemandirian pembudidaya. Motivasi pembudidaya ikan patin tidak
memiliki hubungan nyata dengan tingkat
kemandirian dalam proses produksi dengan
koefiesien korelasi. Ini berbeda dengan hasil
penelitian Sumardjo (1999) dan Agussabti
(2002) yang menyatakan bahwa besarnya
motivasi berpengaruh positif terhadap tingkat
kemandirian petani. proses produksi, hal ini disebabkan usaha
budidaya ikan patin ini merupakan usaha yang
masih tergolong baru bagi mereka, sehingga
pembudidaya masih banyak tergantung pada
pembudidaya lain yang dianggap berhasil
dalam melakukan usaha budidaya ikan patin. Hal ini menyebabkan tingkat kemandiriannya
terukur rendah. 2006) bagi mereka yang memiliki tingkat
pendidikan yang memadai, jalan menuju
keberdayaan lebih terbuka lebar ketimbang
mereka
yang
tidak
berpendidikan. Pengalaman
di
negara-negara
maju
membuktikan,
bahwa
manusia-manusia
mandiri sebagian besar datang dari mereka
yang berpendidikan cukup tinggi. Sedangkan
hubungan pendidikan formal dengan proses
produksi dan pemasaran tidak berhubungan
dengan kemandirian pembudidaya. Jumlah
tanggungan
keluarga
berkorelasi positif nyata terhadap kemandirian
pembudidaya
dalam
permodalan
untuk
melakukan usaha budidaya ikan patin ini. Hal
ini sangat jelas kaitannya karena pembudidaya
tidak hanya mengeluarkan biaya untuk usaha
ini
tetapi
pembudidaya
juga
harus
mengeluarkan
uang
untuk
mencukupi
kebutuhan keluarganya baik pangan, sandang,
pendidikan dan lain-lain, sehingga uang yang
digunakan
terbagi-bagi
dalam
penggunaannya. Sedangkan dalam proses
produksi dan pemasaran jumlah tanggungan
keluarga
tidak
mempengaruhi
tingkat
kemandirian pembudidaya. Motivasi pembudidaya ikan patin tidak
memiliki hubungan nyata dengan tingkat
kemandirian dalam proses produksi dengan
koefiesien korelasi. Ini berbeda dengan hasil
penelitian Sumardjo (1999) dan Agussabti
(2002) yang menyatakan bahwa besarnya
motivasi berpengaruh positif terhadap tingkat
kemandirian petani. Dalam penelitian ini ditemukan bahwa
pembudidaya yang memiliki motivasi yang
tinggi dalam melakukan usaha budidaya ikan
patin tetapi kemandiriannya dalam proses
budidaya rendah. Pembudidaya memiliki
keinginan yang kuat dalam melakukan usaha
budidaya
ikan
patin
sehingga
mereka
memiliki
motivasi
yang
tinggi
untuk
meningkatkan penghasilannya dalam usaha
budidaya ikan patin ini, tetapi pada dasarnya
mereka belumlah mandiri dalam melakukan Pengalaman
usaha
budidaya
responden berkorelasi positif nyata dengan
tingkat kemandirian pembudidaya pada aspek
pengelolaan modal. Dengan kata lain, bahwa
pengalaman sangat mempengaruhi mereka
dalam mencari modal dan menggunakan Aprolita, Siti Amanah, dan Djoko Susanto/
Jurnal Penyuluhan September 2008, Vol. 4 No. Kesimpulan Berdasarkan
hasil
penelitian
dan
pembahasan dapat dikemukakan kesimpulan
sebagai berikut: 1. Keragaan pengelolaan budidaya ikan patin
adalah sebagai berikut: Kontak dengan penyuluh tidaklah
memiliki
korelasi
positif
nyata
pada
kemandirian pembudidaya ikan patin baik
kemandirian dalam modal, proses produksi,
maupun pemasaran. Keadaan yang terjadi di
lokasi penelitian adalah penyuluh belum
memegang
peranan
penting
dalam
mendukung usaha budidaya ikan patin ini,
tetapi pemudidaya sendirilah yang sangat kuat
keinginannya
untuk
meningkatkan
penghasilannya dengan melakukan usaha
budidaya ikan patin. -
Tidak tergantung pada lahan usaha -
Dapat
mengelola
lahan
gambut
menjadi produktif secara mandiri -
Pengembangan usaha secara lebih
berkelanjutan -
Saling bekerjasama dalam melakukan
usaha budidaya ikan patin 2. Kemandirian Pembudidaya ikan patin
dalam melakukan usaha budidayanya
meliputi aspek : -
Modal: memiliki tingkat kemandirian
yang rendah, hal ini disebabkan modal
yang digunakan adalah modal sendiri,
sebagian kecil mereka yang mendapat
pinjaman dari Bank. Penggunaan tenaga kerja dalam usaha
budidaya ikan patin ini berkorelasi positif
nyata pada kemandirian pembudidaya pada
aspek
produksi. Pembudidaya
yang
menggunakan banyak tenaga kerja dalam
usaha budidayanya semakin terbantu pada -
Proses produksi: tingkat kemandirian
pembudidaya dalam proses produksi
masuk dalam kategori tinggi. 134 Aprolita, Siti Amanah, dan Djoko Susanto/
Jurnal Penyuluhan September 2008, Vol. 4 No. 2 Aprolita, Siti Amanah, dan Djoko Susanto/
Jurnal Penyuluhan September 2008, Vol. 4 No. 2 Aprolita, Siti Amanah, dan Djoko Susanto/
Jurnal Penyuluhan September 2008, Vol. 4 No. 2 Aprolita, Siti Amanah, dan Djoko Susanto/
Jurnal Penyuluhan September 2008, Vol. 4 No. 2 -
Pemasaran:
Tingkat
kemandirian
pembudidaya
ikan
patin
dalam
pemasaran hasil dikategorikan tinggi,
pemasaran
ikan
patin
di
lokasi
penelitian
memiliki
rantai
yang
pendek, pembeli langsung datang ke
lokasi. Nazir M. 1998. Metode Penelitian. Bandung:
Ghalia Indonesia. Padmowihardjo, S. 1994. Psikologi Belajar
Mengajar. Jakarta: Universitas Terbuka. Slamet. M. 1995. Memantapkan Posisi dan
Meningkatkan
Peran
Penyuluhan
Pembangunan
dalam
Membangun. Disampaikan dalam Seminar Nasional
Pemberdayaan Sumber Daya Manusia
Menuju Terwujudnya Masyarakat Madani. Bogor. Sumardjo. 1999. Transformasi
Model
Penyuluhan
Pertanian
Menuju
Pengembangan
Kemandirian
Petani
(disertasi). Bogor: Program Pascasarjana,
Institut Pertanian Bogor. Slamet. M. 1995. Memantapkan Posisi dan
Meningkatkan
Peran
Penyuluhan
Pembangunan
dalam
Membangun. Disampaikan dalam Seminar Nasional
Pemberdayaan Sumber Daya Manusia
Menuju Terwujudnya Masyarakat Madani. Bogor. Sumardjo. 1999. Transformasi
Model
Penyuluhan
Pertanian
Menuju
Pengembangan
Kemandirian
Petani
(disertasi). Bogor: Program Pascasarjana,
Institut Pertanian Bogor. Slamet. M. 1995. Memantapkan Posisi dan
Meningkatkan
Peran
Penyuluhan
Pembangunan
dalam
Membangun. Disampaikan dalam Seminar Nasional
Pemberdayaan Sumber Daya Manusia
Menuju Terwujudnya Masyarakat Madani. Bogor. 3. Kesimpulan Dari seluruh faktor yang diteliti tampak
bahwa
pendidikan
formal,
jumlah
tanggungan
keluarga,
kekosmopolitan,
pengalaman usaha, akses kredit, tenaga
kerja memiliki hubungan positif yang
nyata
dengan
kemandirian
dalam
mengelola usaha. Sumardjo. 1999. Transformasi
Model
Penyuluhan
Pertanian
Menuju
Pengembangan
Kemandirian
Petani
(disertasi). Bogor: Program Pascasarjana,
Institut Pertanian Bogor. Daftar Pustaka Agussabti. 2002. Kemandirian Petani dalam
Pengambilan Keputusan Adopsi Inovasi
(Kasus Petani Sayuran di Propinsi Jawa
Barat)
(disertasi). Bogor:
Program
Pascasarjana: Institut Pertanian Bogor. Haryono
Suyono. 2006. Pemberdayaan
Masyarakat
(Mengantar
Manusia
Mandiri, Demokratis dan Berbudaya). Jakarta: Khanata. Ken Suratiyah. 2006. Ilmu Usaha Tani. Jakarta: Penebar Swadaya.
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https://www.qeios.com/read/KLSYJV/pdf
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English
| null |
Capecitabine/Oxaliplatin/Trastuzumab Regimen
|
Definitions
| 2,020
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cc-by
| 74
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Open Peer Review on Qeios Open Peer Review on Qeios Capecitabine/Oxaliplatin/Trastuzumab
Regimen National Cancer Institute National Cancer Institute Qeios ID: KLSYJV · https://doi.org/10.32388/KLSYJV Qeios · Definition, February 2, 2020 Source National Cancer Institute. Capecitabine/Oxaliplatin/Trastuzumab Regimen. NCI
Thesaurus. Code C160542. Thesaurus. Code C160542. A regimen consisting of capecitabine, oxaliplatin, and trastuzumab that may be used in
the treatment of human epidermal growth factor receptor 2 (HER2)-positive advanced
gastric cancers. Qeios ID: KLSYJV · https://doi.org/10.32388/KLSYJV 1/1
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https://openalex.org/W2028600070
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https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0104771&type=printable
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English
| null |
NLRP3 Gene Silencing Ameliorates Diabetic Cardiomyopathy in a Type 2 Diabetes Rat Model
|
PloS one
| 2,014
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cc-by
| 10,436
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Abstract This is an open-access article distributed under the terms of the Creative Commons Attribution License, whic
e, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by the National Natural Science Foundation of China (81370325). The funders had no role in study design, data collection and
analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * Email: anfengshuang@aliyun.com Competing Interests: The authors have declared that no competing interests exist. * Email: anfengshuang@aliyun.com * Email: anfengshuang@aliyun.com Inflammasomes are multi-protein platforms that interact with
various immune and cell death pathways [10,11]. Different
inflammasomes have been identified, including nucleotide-binding
oligomerization domain-like receptors (NLRs) and absent in
melanoma 2 (AIM2) [12]. NLRP3, the most extensively studied
NLRs, forms a complexes comprised of the apoptosis associated
speck like protein (ASC), and the serine protease caspase-1 [13]. On activation, NLRP3 forms a complex with its adaptor ASC,
which facilitates the autocatalytic activation of pro-caspase-1 and
the formation of an active caspase-1 p10/20 tetramer [11]. The
activated caspase-1 can process pro-IL-1b into its mature form,
which is important in cardiomyocyte apoptosis [11,14]. Beibei Luo, Bo Li, Wenke Wang, Xiangjuan Liu, Yanfei Xia, Cheng Zhang, Mingxiang Zhang, Yun Zhang,
Fengshuang An* The Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education and Chinese Ministry of Health, Shandong University Qilu
Hospital, Jinan, Shandong, China The Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education and Chinese Ministry of Health, Shandong University Qilu
Hospital Jinan Shandong China Abstract Background: Nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome is associated with
metabolic disorder and cell death, which are important triggers in diabetic cardiomyopathy (DCM). We aimed to explore
whether NLRP3 inflammasome activation contributes to DCM and the mechanism involved. Methods: Type 2 diabetic rat model was induced by high fat diet and low dose streptozotocin. The characteristics of type 2
DCM were evaluated by metabolic tests, echocardiography and histopathology. Gene silencing therapy was used to
investigate the role of NLRP3 in the pathogenesis of DCM. High glucose treated H9c2 cardiomyocytes were used to
determine the mechanism by which NLRP3 modulated the DCM. The cell death in vitro was detected by TUNEL and EthD-III
staining. TXNIP-siRNA and pharmacological inhibitors of ROS and NF-kB were used to explore the mechanism of NLRP3
inflammasome activation. Results: Diabetic rats showed severe metabolic disorder, cardiac inflammation, cell death, disorganized ultrastructure,
fibrosis and excessive activation of NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain
(ASC), pro-caspase-1, activated caspase-1 and mature interleukin-1b (IL-1b). Evidence for pyroptosis was found in vivo, and
the caspase-1 dependent pyroptosis was found in vitro. Silencing of NLRP3 in vivo did not attenuate systemic metabolic
disturbances. However, NLRP3 gene silencing therapy ameliorated cardiac inflammation, pyroptosis, fibrosis and cardiac
function. Silencing of NLRP3 in H9c2 cardiomyocytes suppressed pyroptosis under high glucose. ROS inhibition markedly
decreased nuclear factor-kB (NF-kB) phosphorylation, thioredoxin interacting/inhibiting protein (TXNIP), NLRP3 inflamma-
some, and mature IL-1b in high glucose treated H9c2 cells. Inhibition of NF-kB reduced the activation of NLRP3
inflammasome. TXNIP-siRNA decreased the activation of caspase-1 and IL-1b. Conclusion: NLRP3 inflammasome contributed to the development of DCM. NF-kB and TXNIP mediated the ROS-induced
caspase-1 and IL-1b activation, which are the effectors of NLRP3 inflammasome. NLRP3 gene silencing may exert a
protective effect on DCM. Editor: Philip Michael Bauer, University of Pittsburgh School of Medicine, United States of America Editor: Philip Michael Bauer, University of Pittsburgh School of Medicine, United States of America Received February 16, 2014; Accepted July 15, 2014; Published August 19, 2014 Copyright: 2014 Luo et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
use, distribution, and reproduction in any medium, provided the original author and source are credited. pyright: 2014 Luo et al. August 2014 | Volume 9 | Issue 8 | e104771 NLRP3 Gene Silencing Ameliorates Diabetic Cardiomyopathy of cell death, is dependent on caspase-1 activity [16]. The
morphology of pyroptosis shares the unique characteristics with
both apoptosis and necrosis [15]. As in apoptotic cell, pyroptotic
cells incur DNA damage and become positive in the terminal
deoxynucleotidyl transferase-mediated dUTP nick end-labeling
(TUNEL) staining. Similar to necrosis, pyroptosis results in pore
formation in the cell membrane, release of pro-inflammatory
cytosolic content, and cell lysis. Therefore, membrane impermeant
dyes such as EthD-III stain pyroptotic cells by entering through
the pores, but do not stain apoptotic cells [17,18]. Pyroptosis is
initially described in macrophages and dendritic cells infected with
different pathogens [19,20]. Recent studies showed that pyroptosis
could also occur in non-myeloid cells induced by non-infectious
stimuli [21,22,23]. Electron microscopy studies of myocardium in
diabetic mice and rats showed that the majority of dying cells had
swollen fibril and mitochondria, which are the characteristics of
cell swelling and lysis in pyroptosis [24,25,26]. Activated caspase-
1, the executor caspase of pyroptosis, is found to be elevated in
DCM in a rat model. However, it is not clear whether pyroptosis
participates in hyperglycemia-induced cardiomyocyte death. of cell death, is dependent on caspase-1 activity [16]. The
morphology of pyroptosis shares the unique characteristics with
both apoptosis and necrosis [15]. As in apoptotic cell, pyroptotic
cells incur DNA damage and become positive in the terminal
deoxynucleotidyl transferase-mediated dUTP nick end-labeling
(TUNEL) staining. Similar to necrosis, pyroptosis results in pore
formation in the cell membrane, release of pro-inflammatory
cytosolic content, and cell lysis. Therefore, membrane impermeant
dyes such as EthD-III stain pyroptotic cells by entering through
the pores, but do not stain apoptotic cells [17,18]. Pyroptosis is
initially described in macrophages and dendritic cells infected with
different pathogens [19,20]. Recent studies showed that pyroptosis
could also occur in non-myeloid cells induced by non-infectious
stimuli [21,22,23]. Electron microscopy studies of myocardium in
diabetic mice and rats showed that the majority of dying cells had
swollen fibril and mitochondria, which are the characteristics of
cell swelling and lysis in pyroptosis [24,25,26]. Activated caspase-
1, the executor caspase of pyroptosis, is found to be elevated in
DCM in a rat model. However, it is not clear whether pyroptosis
participates in hyperglycemia-induced cardiomyocyte death. Serum measurements After rats fasted overnight, serum triglycerides (TG) and total
cholesterol (TC) were determined by use of an automatic analyzer
(Roche, Basel, Switzerland). Serum level of insulin was measured
by enzyme-linked immunosorbent assay (Jiancheng, Nanjing,
China). Measurement of myocardial fibrosis Masson’s trichrome and Sirius red staining were used for
detecting collagen. The fibrotic area fraction was analyzed by
automated image analysis (Image-Pro Plus, Media Cybemetics,
US). Echocardiography Transthoracic echocardiography involved the Vevo 770 imag-
ing system with RMB710 transducer (VisualSonics, Toronto,
Canada). Images were obtained from rats anesthetized with 10%
chloral
hydrate. The
derived
echocardiography
parameters
included left ventricular end-diastolic dimension (LVEDd), left
ventricular ejection fraction (LVEF), fractional shortening (FS),
peak E to peak A ratio (E/A), and early (E9) to late (A9) diastolic
velocity ratio (E9/A9). p
p
yp
g y
y
y
Recent studies indicate that NF-kB mediated the ROS-induced
NLRP3 inflammasome by promoting the transcription of NLRP3
inflammasome [27,28]. Thioredoxin interacting/inhibiting pro-
tein (TXNIP) can bind NLRP3 directly and lead to NLRP3
inflammasome assembly under oxidative stress [6,29]. However,
little is known about whether NF-kB and TXNIP participate in
the regulation of NLRP3 in hyperglycemia-treated cardiomyocyte. In this study, we hypothesized that pyroptosis, regulated by
NLRP3 inflammsome, might participate in the pathogenesis of
DCM. We also hypothesized that NF-kB and TXNIP might be
links between ROS and NLRP3 inflammasome activation. Recent studies indicate that NF-kB mediated the ROS-induced
NLRP3 inflammasome by promoting the transcription of NLRP3
inflammasome [27,28]. Thioredoxin interacting/inhibiting pro-
tein (TXNIP) can bind NLRP3 directly and lead to NLRP3
inflammasome assembly under oxidative stress [6,29]. However,
little is known about whether NF-kB and TXNIP participate in
the regulation of NLRP3 in hyperglycemia-treated cardiomyocyte. Histological examination In this study, we hypothesized that pyroptosis, regulated by
NLRP3 inflammsome, might participate in the pathogenesis of
DCM. We also hypothesized that NF-kB and TXNIP might be
links between ROS and NLRP3 inflammasome activation. The hearts were arrested with 10% KCl, and heart weight was
measured immediately. The images of whole heart and cross
sections from the midpoint of heart were obtained by camera
(Canon, Tokyo, Japan). NLRP3 Gene Silencing Ameliorates Diabetic Cardiomyopathy For the analysis of NLRP3 inflammasome and IL-1b expres-
sion, cardiac samples of DM rats were collected at 0, 4, 8, 12, 16
and 20 weeks (n = 3) after STZ injection, and control rats (n = 3)
were collected at 20 weeks after diabetic rats received STZ (Fig. 1). The experiments complied with the Animal Management Rule of
Chinese Ministry of Health (documentation no. 55, 2001), and
experimental protocols were approved by the Shandong Univer-
sity Animal Care Committee. Real-time RT-PCR Real-time RT-PCR was as described previously [34]. The
primer sequences are shown in Table 1. The relative expression of
genes was calculated by the 22DDCT method. Transmission electron microscopy (TEM)
3 About 1 mm3 tissue was obtained from the left ventricle of rats
and fixed in 2.5% glutaraldehyde in 0.1 mol/L sodium cacodylate
buffer (pH 7.4) for 2 h. The following steps were as described
previously [32]. Finally, the specimens were studied by transmis-
sion electron microscopy (TEM, JEM-1200EX, Japan). Animal study y
We randomized 120 Sprague-Dawley rats (100–120 g) into 4
groups (n = 30 per group): control, diabetes mellitus (DM), DM +
vehicle, DM + NLRP3-miRNA. All rats were housed at 22uC with
12 h light-dark cycles. The control group was fed the basal diet,
and the other groups a high fat diet (HF diet, 16% fat and 0.25%
cholesterol). 4 weeks later, the intraperitoneal glucose tolerance
test (IPGTT) and intraperitoneal insulin tolerance test (IPITT)
were performed to determine insulin resistance. Diabetes was
induced by a single intraperitoneal injection of streptozotocin
(STZ; 35 mg/kg, Solarbio, China) to rats in the groups of DM,
DM + vehicle and DM + NLRP3-miRNA. After one week of STZ
injection, blood glucose levels were measured (Roche, Germany)
after rats fasted overnight. Only rats with blood glucose level $
11.1 mmol/L were used in the study. According to previous
studies, diabetic rats showed onset of cardiac dysfunction after 8
weeks of STZ injection [30,31]. Our gene silencing treatment
occurred after 8 weeks of STZ injection (Fig. 1). The groups of
DM + vehicle and DM + NLRP3-miRNA rats in our study
received a jugular-vein injection of a total lentivector dose of
16108 TU/rat vehicle (empty virus) or NLRP3-miRNA (Invitro-
gen, Shanghai, China). Sequences for rat NLRP3-miRNA oligos
were: forward, 59- TGCTGATAAGAAGTTCTCTCCTGGT-
TGTTTTGGCCACTGACTGACAACCAGGAGAACTTCTT-
AT-39; and reverse, 59- CCTGATAAGAAGTTCTCCTGGT-
TGTCAGTCAGTGGCCAAAACAACCAGGAGAGAACTTC-
TTATC -39. After 8 weeks of lentivector injection, treated rats and
age-matched control and DM rats were killed. The heart was
excised and immediately frozen to determine the transfection
efficiency under fluorescence microscope. Immunohistochemistry Immunohistochemistry was as described previously [33]. We
used antibodies against caspase-1 (1:50, Abcam, UK) and IL-1b
(1:200). Data were analyzed by Image-Pro Plus 6.0 (Media
Cybemetics, US). Immunohistochemistry was as described previously [33]. We
used antibodies against caspase-1 (1:50, Abcam, UK) and IL-1b
(1:200). Data were analyzed by Image-Pro Plus 6.0 (Media
Cybemetics, US). Introduction Diabetic cardiomyopathy (DCM), characterized by consistent
diastolic dysfunction and increased ventricular mass, is the leading
cause of mortality among patients with diabetes [1,2]. Hypergly-
cemia-induced reactive oxygen species (ROS) generation is
considered to be responsible for progression and development of
DCM [3,4]. The increased ROS could induce a number of
cytokine and inflammatory factors, such as nuclear factor-kB (NF-
kB), thioredoxin interacting/inhibiting protein (TXNIP), and
inflammasome [5,6,7]. Although inflammasome was shown to be
involved in the pathogenic mechanisms of type 2 diabetes and its
complications [8,9], the potential role and regulatory mechanism
of inflammasome in DCM has remained largely unexplored. In addition to resulting in the maturation of IL-1b, activated
caspase-1 can induce a distinct form of programmed cell death
called ‘‘pyroptosis’’ [15]. Pyroptosis, a highly inflammatory form PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 1 August 2014 | Volume 9 | Issue 8 | e104771 August 2014 | Volume 9 | Issue 8 | e104771 Caspase-1 activity assay Caspase-1 activity was measured by using colorimetric assay
(Beyotime, China). This assay was based on the ability of caspase-1
to change acetyl-Tyr-Val-Ala-Asp p-nitroaniline (Ac-YVAD-pNA)
into the yellow formazan product p-nitroaniline (pNA). 50 ug of
total cytosolic protein was incubated in a 96-well microtiter plate
with 20 nmol Ac-YVAD-pNA overnight at 37uC. The absorbance
values of pNA at 405 nm, OD405, were tested using spectropho-
tometer Varioscan Flash and SkanIt Software Version 2.4.3. RE
(Thermo Fisher Scientific Inc, USA). A standard curve of pNA Immunofluorescence Immunofluorescence was as described previously [36]. We used
antibodies against caspase-1 (1:50). The fluorescence was visual-
ized by confocal microscopy (LSM710, Carl Zeiss, Germany) and
analyzed by Image-Pro Plus 6.0 (Media Cybemetics, US). Statistical analysis Data
are
expressed
as
mean6SEM. Differences
among
experimental groups were analyzed by ANOVA, followed by the
Tukey-Kramer post hoc test and independent samples t test. Analysis involved use of SPSS v18.0 (SPSS, Chicago, IL, USA). Significance was defined as p,0.05. TUNEL assay IL-1b, IL-18, TNF-a and IL-6 in cultured cardiomyocyte
supernatants
were
measured
using
commercial
ELISA
kits
(Jiancheng, Nanjing, China). Detection of DNA cleavage was performed using an ApopTag
in situ apoptosis detection kit (Millipore, USA). Deparaffinized
sections were treated with 3% hydrogen peroxide in methanol for
10 min. After adding the equilibration buffer, sections were
treated with TdT-enzyme at 37uC for 1 h. The sections were then
incubated with digoxigenin-conjugated antibodies at 37uC for
30 min. Then sections were colorized with DAB (ZSGB-BIO,
China). Finally, the stained sections were examined under
microscope (Olympus, Japan). Results NLRP3 inflammasome and IL-1b were activated in DCM
When compared with the control rats, diabetic rats showed
elevated mRNA and protein levels of NLRP3, ASC, caspase-1 and
IL-1b beginning at 4 or 8 weeks of diabetes (Fig. 2A–C, all p,
0.01). Moreover, the mRNA and protein levels of NLRP3
inflamamsome components and IL-1b achieved the highest levels
at 8 or 12 weeks of diabetes except pro-IL-1b (Fig. 2A–C, all p,
0.01). Lentivirus transfection H9c2 cardiomyocytes plated at a density of 16105/cm2 were
infected with vehicle or lentivrius-NLRP3-miRNA at 10 multi-
plicity of infection (MOI) in serum-free media for 2 h, followed by
incubation with DMEM containing 10% FBS for an additional
48 h before processing. The transfection efficiency was observed
by fluorescence microscope. For each experiment, NLRP3
knockdown was assessed in transfected cells, and cells were used
only if NLRP3 mRNA was decreased by 70% compared with
vehicle. siRNA transfection H9c2 cardiomyocytes were transfected with vehicle (empty
plasmid) or TXNIP-siRNA plasmid (JiMa, Shanghai, China) by
using Lipofectamin 2000 (Invitrogen, Shanghai, China). Sequenc-
es for rat TXNIP-siRNA were: sense, 59- UGGUCACGUC-
GAAAUGAAUTT-39;
antisense,
59-TTACCAGUGCAG-
CUUUACUUA-39. ROS levels Intracellular generation of ROS was tested by peroxide-sensitive
fluorescent probe 29,79-dichlorofluorescein diacetate (DCFH-DA,
Sigma, Shanghai, China). The stimulated cells were washed twice
with cold PBS and incubated with DCFH-DA at 37uC for 30 min
in dark. Fluorescent signal of 6 fields per groups was recorded by
using a fluorescence microscopy (488 nm filter, Olympus, Japan). Fluorescence intensity was analyzed with HMIAS-2000 software. Cell death assay Cell death was assessed by TUNEL assay and EthD-III/calcein
AM staining. For TUNEL assay, cells cultured in chamber slide
were fixed with 4% paraformaldehyde for 30 min. Cells were
permeabilized with immunol staining wash buffer (Beyotime,
China) for 2 min on ice. The following steps were as introduced
above. EthD-III/calcein
AM
staining
(Viability/Cytotoxicity
Assay Kit, Biotium, USA) was also used for cell death assay. The AM produces a bright green fluorescence in live cells. EthD-
III enters dead cells, thereby producing red fluorescence in dead
cells. Cells were simultaneously stained with 2 mM calcein AM and
4 mM EthD-III at 37uC for 45 min. Then cells were incubated
with Hoechst 33342 (Beyotime, China) at 37uC for 30 min. Coverslips were finally sealed by a drop of antifade mounting
medium (Beyotime, China). The fluorescence was visualized by
confocal microscopy (LSM710, Carl Zeiss, Germany). The
percentage of dead cell was achieved by counting the ratio of
red positive cells to of blue positive cells. Cell culture and treatment H9c2 cardiomyocytes culture was as described previously [35]. During the treatment period, H9c2 cells were cultured in normal
glucose medium with minimal essential medium for 12 h, followed
by the exposure of control glucose (Ctrl, 5.5 mM), medium glucose
(MG, 25 mM), high glucose (HG, 33.3 mM), and osomotic control
(OC, 27.5 mM mannose) for 24, 36, or 48 h. In ROS inhibition
experiment, cells were treated with HG for 36 h in the presence of
10 mmol/L N-acetylcysteine (NAC, Beyotime, China) or PBS. For
NF-kB inhibition, cells were pretreated with 10 mmmol/L BAY
11-7082 or DMSO before HG stimulation. Western blot The protein bands were developed by the use of
chemiluminescence (Millipore, USA), and quantified by densito-
metric analysis (Quantity One, Bio-Rad, USA). was performed. The production of pNA in tested samples
indicated the level of caspase-1 activation. Western blot Western blot was as described previously [34]. We used
antibodies against NF-kB (1:700, t-NF-KB p65 ab131485, p-NF-
kB p65, ab28856, Abcam, UK), TXNIP (1:700, ab86983), NLRP3
(1:500, ab109314), ASC (1:700, ab64808), caspase-1 (1:500,
ab1872), IL-1b (1:1000, ab9722), collagen I (1:1000, ab34710),
collagen III (1:500, ab7778), and b-actin (1:1000, sc8432, Santa Western blot was as described previously [34]. We used
antibodies against NF-kB (1:700, t-NF-KB p65 ab131485, p-NF-
kB p65, ab28856, Abcam, UK), TXNIP (1:700, ab86983), NLRP3
(1:500, ab109314), ASC (1:700, ab64808), caspase-1 (1:500,
ab1872), IL-1b (1:1000, ab9722), collagen I (1:1000, ab34710),
collagen III (1:500, ab7778), and b-actin (1:1000, sc8432, Santa PLOS ONE | www.plosone.org August 2014 | Volume 9 | Issue 8 | e104771 2 NLRP3 Gene Silencing Ameliorates Diabetic Cardiomyopathy Figure 1. The information of experimental design. The euthanized rats in the yellow box were used to explore the expression levels of NLRP3
inflammasome in different stages of DCM. The sacrificed rats in the green box were used in the rest of experiments in vivo. IPGTT: intraperitoneal
glucose tolerance test; IPITT: intraperitoneal insulin tolerance test; HF: high fat diet; Ctrl: control rats; DM: diabetes rats; DM+NLRP3-miRNA: diabetes
rats received lentiviral NLRP3-miRNA; DM+vehicle: diabetes rats received empty lentiviral vector. doi:10.1371/journal.pone.0104771.g001
NLRP3 Gene Silencing Ameliorates Diabetic Cardiomyopathy Figure 1. The information of experimental design. The euthanized rats in the yellow box were used to explore the expression levels of NLRP3
inflammasome in different stages of DCM. The sacrificed rats in the green box were used in the rest of experiments in vivo. IPGTT: intraperitoneal
glucose tolerance test; IPITT: intraperitoneal insulin tolerance test; HF: high fat diet; Ctrl: control rats; DM: diabetes rats; DM+NLRP3-miRNA: diabetes
rats received lentiviral NLRP3-miRNA; DM+vehicle: diabetes rats received empty lentiviral vector. doi:10.1371/journal.pone.0104771.g001 Table 1. Primer sequences for real-time RT-PCR. Symbol
Forward primer
Reverse primer
NF-kB
GAGATTGTGCCAAGAGTGAC
CTTGTCTTCCATGGTGGATG
TXNIP
GCTCAATCATGGTGATGTTCAAG
CTTCACACACTTCCACTGTCAC
NLRP3
GTGGAGATCCTAGGTTTCTCTG
CAGGATCTCATTCTCTTGGATC
ASC
CTCTGTATGGCAATGTGCTGAC
GAACAAGTTCTTGCAGGTCAG
Caspase-1
GAGCTGATGTTGACCTCAGAG
CTGTCAGAAGTCTTGTGCTCTG
IL-1b
TGCTGTCTGACCCATGTGAG
GTCGTTGCTTGTCTCTCCTTG
b-actin
AGACCTTCAACACCCCAG
CACGATTTCCCTCTCAGC
TXNIP, thioredoxin interacting/inhibiting protein; NLRP3, Nucleotide-binding oligomerization domain-like receptor protein 3; ASC, apoptosis-associated speck-like
protein containing a caspase recruitment domain; IL-1b, interleukin-1b. doi:10.1371/journal.pone.0104771.t001 Table 1. Primer sequences for real-time RT-PCR. August 2014 | Volume 9 | Issue 8 | e104771 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 3 NLRP3 Gene Silencing Ameliorates Diabetic Cardiomyopathy Cruz, USA). The protein bands were developed by the use of
chemiluminescence (Millipore, USA), and quantified by densito-
metric analysis (Quantity One, Bio-Rad, USA). Cruz, USA). NLRP3 gene silencing improved metabolism
abnormalities In the DM group, blood glucose, TC, TG and INS were higher
than the control (Table 2, all p,0.01). ISI in DM group was lower
than control group (Table 2, p,0.01). NLRP3 gene silencing was
insufficient to improve the systemic metabolism disorder. August 2014 | Volume 9 | Issue 8 | e104771 PLOS ONE | www.plosone.org 4 NLRP3 Gene Silencing Ameliorates Diabetic Cardiomyopathy Figure 2. NLRP3 inflammasome was activated in myocardium after 4 weeks of diabetes. (A) The relative mRNA expression of NLRP3 (A1),
ASC (A2), caspase-1(A3) and IL-1b (A4). (B and C) The protein levels of NLRP3 (C1), ASC (C2), pro-caspase-1 (C3), activated caspase-1(C4), pro-IL-1b (C5)
and mature IL-1b (C6). Data were presented as means6SEM, n = 3. *p,0.05, **p,0.01 vs. Ctrl at 20 w; #p,0.05, ##p,0.01 vs. DM at 0 w. Ctrl:
control rats sacrificed at 20 weeks after diabetes rats received STZ injection; DM: diabetes rats; w: weeks; Pro-Casp-1: pro-caspase-1; Casp-1 p20:
activated caspase-1; IL-1b p17: mature IL-1b. doi:10.1371/journal.pone.0104771.g002 Figure 2. NLRP3 inflammasome was activated in myocardium after 4 weeks of diabetes. (A) The relative mRNA expression of NLRP3 (A1),
ASC (A2), caspase-1(A3) and IL-1b (A4). (B and C) The protein levels of NLRP3 (C1), ASC (C2), pro-caspase-1 (C3), activated caspase-1(C4), pro-IL-1b (C5)
and mature IL-1b (C6). Data were presented as means6SEM, n = 3. *p,0.05, **p,0.01 vs. Ctrl at 20 w; #p,0.05, ##p,0.01 vs. DM at 0 w. Ctrl:
control rats sacrificed at 20 weeks after diabetes rats received STZ injection; DM: diabetes rats; w: weeks; Pro-Casp-1: pro-caspase-1; Casp-1 p20:
activated caspase-1; IL-1b p17: mature IL-1b. doi:10.1371/journal.pone.0104771.g002 mRNA and protein levels of cardiac NLRP3 (Fig. 3A–C, both
p,0.01). In addition, the protein levels of activated caspaspe-1
and mature IL-1b were lower in NLRP3-miRNA treated diabetic
rats than the vehicle treated rats (Fig. 3B, E and G, both p,0.01). NLRP3-miRNA did not have effect on the protein levels of pro- Cardiac NLRP3 expression was suppressed by gene
silencing NLRP3 Gene Silencing Ameliorates Diabetic Cardiomyopathy Figure 3. The inhibition of NLRP3 was effective in vivo. Relative mRNA expression of NLRP3 (A) and protein levels of NLRP3, pro-caspase-1,
activated caspase-1, pro-IL-1b and mature IL-1b (B–G) in cardiac tissue with NLRP3-miRNA treatment. Immunohistochemical staining (H) and a semi-
quantitative analysis of positive staining (I–J) of caspase-1 and IL-1b; scale bar: 20 mm. Data were presented as means6SEM, n = 6. **p,0.01 vs. Ctrl;
1p,0.05, 11p,0.01 vs. DM+vehicle. Ctrl: control rats sacrificed at 16 weeks after diabetes rats received STZ injection; DM : diabetes rats sacrificed at 16
weeks after STZ injection; Casp-1 p20: activated caspase-1; IL-1b p17: mature IL-1b. doi:10.1371/journal.pone.0104771.g003 Figure 3. The inhibition of NLRP3 was effective in vivo. Relative mRNA expression of NLRP3 (A) and protein levels of NLRP3, pro-caspase-1,
activated caspase-1, pro-IL-1b and mature IL-1b (B–G) in cardiac tissue with NLRP3-miRNA treatment. Immunohistochemical staining (H) and a semi-
quantitative analysis of positive staining (I–J) of caspase-1 and IL-1b; scale bar: 20 mm. Data were presented as means6SEM, n = 6. **p,0.01 vs. Ctrl;
1p,0.05, 11p,0.01 vs. DM+vehicle. Ctrl: control rats sacrificed at 16 weeks after diabetes rats received STZ injection; DM : diabetes rats sacrificed at 16
weeks after STZ injection; Casp-1 p20: activated caspase-1; IL-1b p17: mature IL-1b. doi:10.1371/journal.pone.0104771.g003 caspase-1 and pro-IL-1b (Fig. 3B, D and F). Immunohistochem-
istry revealed that increased caspase-1 predominantly localized in
perinuclear area, while elevated IL-1b showed diffused distribu-
tion pattern in DCM (Fig. 3H–J, p,0.05,p,0.01). disorganized cristae, excess glycogen lysis, and accumulated lipids
(Fig. 5E). Moreover, the diabetic group showed increased inter-
stitial cardiac fibrosis as compared with the control (Fig. 5F and G,
p,0.01). Coincident with cardiac fibrosis, the protein levels of
collagen I and collagen III, and the collagen I-to-III ratio were
higher in DM group than the control (Fig. 5H–K, all p,0.01). gene silencing reversed myocardial remodeling g
g
y
g
The DM group showed the phenotype of eccentric ventricular
hypertrophy (Fig. 5A). The ratio of heart weight to body weight
was larger for DM than the control group (Fig. 5B, p,0.01). TUNEL result showed that the percentage of dead cell was higher
in DM group than control (Fig. 5C and D, p,0.01). The
ultrastructure of cardiomyocyte in control rats showed typical
symmetric myofibrils, well-organized Z lines with sarcomeres, and
packed mictochontria beside the fibers (Fig. 5E). In contrast, DM
rats showed severe damage of the left ventricular ultrastructure,
including destruction of myofibrils, swollen mitochondria with NLRP3 gene silencing alleviated left ventricular
dysfunction in DCM With NLRP3 gene silencing, heart weight to body weight ratio
and cell death were decreased in the NLRP3-miRNA group versus
the vehicle group (Fig. 5B, C and D, both p,0.01). NLRP3-
miRNA treatment normalized alterations in myofilaments and
mitochondria, along with reduced glycogen lysis and lipid
accumulation in diabetic rats (Fig. 5E). Moreover, cardiac fibrosis
area, collagen I, collagen III, and the collagen I to III ratio were
lower in the DM + NLRP3-miRNA group than vehicle group
(Fig. 5F–K, all p,0.01). The results of echocardiography showed that LVEDd of DM
rat was larger than control (Fig. 4A and B, p,0.01). LVEF, FS,
E/A and E9/A9 were lower in DM than control (Fig. 4A, C–F,
both p,0.01). When compared with vehicle, increased LVEF, FS,
E/A, E9/A9 and decreased LVEDd were observed in NLRP3-
miRNA group (Fig. 4A–F, p,0.05,p,0.01). Cardiac NLRP3 expression was suppressed by gene
silencing After 8 weeks of NLRP3 silencing treatment, transfection
efficiency was checked in all groups (Fig. S1A). As compared with
vehicle treatment, NLRP3-miRNA treatment decreased the Table 2. NLRP3 gene silencing did not attenuate metabolic abnormalities. Ctrl
DM
DM + Vehicle
DM + NLRP3-miRNA
Blood glucose (mmol/L)
5.0860.38
18.8060.39**
18.5560.65**
18.5160.82**
TC (mmol/L)
1.6360.05
2.7060. 08**
2.7360.09**
2.7160.04**
TG (mmol/L)
0.6560.06
2.7260.08**
2.7160.15**
2.6760.05**
INS (mmol/L)
14.1060.34
16.1960.12**
16.0960.12**
16.1360.34**
ISI
24.2660.09
25.7160.02**
25.6960. 04**
25.6860. 05**
The blood samples were collected at the end of the experiment. Ctrl, control; DM, diabetes mellitus; Blood glucose, blood glucose tested at the end of the experiment;
TG, triglyceride; TC, total cholesterol; INS, insulin tested at the end of the experiment; ISI, insulin sensitivity index. Data are mean6SEM, n = 6–8 per group. **p,0.01 vs. control. doi:10.1371/journal.pone.0104771.t002 Table 2. NLRP3 gene silencing did not attenuate metabolic abnormalities. The blood samples were collected at the end of the experiment. Ctrl, control; DM, diabetes mellitus; Blood glucose, blood glucose tested at the end of the experiment;
TG, triglyceride; TC, total cholesterol; INS, insulin tested at the end of the experiment; ISI, insulin sensitivity index. Data are mean6SEM, n = 6–8 per group. ** ,0 01
t
l The blood samples were collected at the end of the experiment. Ctrl, control; DM, diabetes mellitus; Blood glucose, blood glucose tested at the end of the experiment;
TG, triglyceride; TC, total cholesterol; INS, insulin tested at the end of the experiment; ISI, insulin sensitivity index. Data are mean6SEM, n = 6–8 per group. **p,0.01 vs. control. doi:10.1371/journal.pone.0104771.t002 August 2014 | Volume 9 | Issue 8 | e104771 PLOS ONE | www.plosone.org 5 NLRP3 Gene Silencing Ameliorates Diabetic Cardiomyopathy The increased expression of NLRP3 inflammasome and
IL-1b were induced by high glucose Different levels of glucose caused a concentration-dependent
increase of NLRP3, ASC, caspase-1 and IL-1b mRNA in H9c2
cells in 36 to 48 h (Fig. 6A, all p,0.01). Likewise, the protein
levels of all NLRP3 inflammasome components, pro- IL-1b and
mature IL-1b were increased at high glucose as compared with
control and medium glucose in 36 and 48 h (Fig. 6B and C, all p,
0.01). Moreover, the increase of NLRP3, ASC and mature IL-1b August 2014 | Volume 9 | Issue 8 | e104771 PLOS ONE | www.plosone.org 6 NLRP3 Gene Silencing Ameliorates Diabetic Cardiomyopathy Figure 4. NLRP3 gene silencing improved cardiac dysfunction in diabetes rats. (A) The cardiac function of rats in different groups shown by
echocardiography. Representative images of 2D echocardiograms (A1), M-mode echocardiograms (A2), pulse-wave Doppler echocardiograms of
mitral inflow (A3), tissue Doppler echocardiograms (A4). (B–F) Evaluation of LVEDd, LVEF, FS, E/A and E9/A9. Data were presented as means6SEM,
n = 6. **p,0.01 vs. Ctrl; 1p,0.05, 11p,0.01 vs. DM+vehicle. LV: left ventricle; IVS: interventricular septum; LVEDd: left ventricular end-diastolic
dimension; LVEF: left ventricular ejection fraction; FS: fractional shortening; E/A: peak E to peak A ratio; E9/A9: early (E9) to late (A9) diastolic velocity
ratio. doi:10 1371/journal pone 0104771 g004 Figure 4. NLRP3 gene silencing improved cardiac dysfunction in diabetes rats. (A) The cardiac function of rats in different groups shown by
echocardiography. Representative images of 2D echocardiograms (A1), M-mode echocardiograms (A2), pulse-wave Doppler echocardiograms of
mitral inflow (A3), tissue Doppler echocardiograms (A4). (B–F) Evaluation of LVEDd, LVEF, FS, E/A and E9/A9. Data were presented as means6SEM,
n = 6. **p,0.01 vs. Ctrl; 1p,0.05, 11p,0.01 vs. DM+vehicle. LV: left ventricle; IVS: interventricular septum; LVEDd: left ventricular end-diastolic
dimension; LVEF: left ventricular ejection fraction; FS: fractional shortening; E/A: peak E to peak A ratio; E9/A9: early (E9) to late (A9) diastolic velocity
ratio. doi:10.1371/journal.pone.0104771.g004 Figure 4. NLRP3 gene silencing improved cardiac dysfunction in diabetes rats. (A) The cardiac function of rats in different groups shown by
echocardiography. Representative images of 2D echocardiograms (A1), M-mode echocardiograms (A2), pulse-wave Doppler echocardiograms of
mitral inflow (A3), tissue Doppler echocardiograms (A4). (B–F) Evaluation of LVEDd, LVEF, FS, E/A and E9/A9. Data were presented as means6SEM,
n = 6. **p,0.01 vs. Ctrl; 1p,0.05, 11p,0.01 vs. DM+vehicle. NLRP3 was involved in cell death induced by high
glucose To explore the role of NLRP3 in high glucose-induced cell
death of H9c2, we inhibited the expression of NLRP3 by lentivirial
NLRP3-miRNA. The transfection efficacy of lentivirial NLRP3-
miRNA in H9c2 cardiomyocytes reached 80% (Fig. S1D). Both
the mRNA and protein levels of NLRP3 in cells transfected with
NLRP3-miRNA were lower than the vehicle (Fig. 8A and B, both
p,0.01). After inhibiting the expression of NLRP3, the protein
levels of activated caspase-1 and mature IL-1b induced by high
glucose decreased as compared with vehicle (Fig. 8E and G, p,
0.05,p,0.01). In addition, the secreted levels of IL-1b, IL-18,
TNF-a and IL-6 in cultured supernatant of H9c2 cells were lower
in HG+NLRP3-miRNA group than HG+vehicle (Fig. 8H–K, p, The increased expression of NLRP3 inflammasome and
IL-1b were induced by high glucose LV: left ventricle; IVS: interventricular septum; LVEDd: left ventricular end-diastolic
dimension; LVEF: left ventricular ejection fraction; FS: fractional shortening; E/A: peak E to peak A ratio; E9/A9: early (E9) to late (A9) diastolic velocity
ratio. doi:10 1371/journal pone 0104771 g004 doi:10.1371/journal.pone.0104771.g004 doi:10.1371/journal.pone.0104771.g004 damaged cell membrane as compared with control and medium
glucose (Fig. 7E and F, p,0.01). protein showed the highest level at 36 h (Fig. 6B, C1, C2 and C6,
all p,0.01). Thus, we chose high glucose as the stimulation, and
chose 36 h as the stimulated time in subsequent experiments. The
expression of NLRP3 inflammasome and mature IL-1b in H9c2
cells with control glucose or isotonic mannose had no significant
change at the same time point tested (Fig.S1 B and C). NLRP3 Gene Silencing Ameliorates Diabetic Cardiomyopathy NLRP3-miRNA did not have effect on the protein
levels of pro-caspase-1 and pro-IL-1b (Fig. 8B, D and F). In
keeping with these observations, the dead cell rate detected by
TUNEL was lower in HG + NLRP3-miRNA than HG + vehicle
(Fig. 8L and M, p,0.01). To avoid the interference of lentivector
fluorescence, we did not use calcein AM in the detection of live
cells. The high glucose induced-cell-death rate detected by EthD-
III were decreased in NLRP3-miRNA treated cells as compared
with vehicle (Fig. 8N and O, p,0.01). 0.05,p,0.01). NLRP3-miRNA did not have effect on the protein
levels of pro-caspase-1 and pro-IL-1b (Fig. 8B, D and F). In
keeping with these observations, the dead cell rate detected by
TUNEL was lower in HG + NLRP3-miRNA than HG + vehicle
(Fig. 8L and M, p,0.01). To avoid the interference of lentivector
fluorescence, we did not use calcein AM in the detection of live
cells. The high glucose induced-cell-death rate detected by EthD-
III were decreased in NLRP3-miRNA treated cells as compared
with vehicle (Fig. 8N and O, p,0.01). NAC inhibited high glucose-induced increase in intracellular ROS
activity (Fig. 9E, p,0.01). Intriguingly, the levels of NF-kB
phosphorylation and TXNIP were also lower in the NAC treated
group than the PBS treated group (Fig. 9F–H, all p,0.01). In
keeping with the decreased expression of NF-kB and TXNIP, the
expression of all component of NLRP3, ASC, pro-caspaspe-1,
activated caspase-1, pro- IL-1b and mature IL-1b were down-
regulated with pretreatment of NAC as compared with PBS
(Fig. 9I–O, all p,0.01). Pretreatment of cells with NF-kB inhibitor BAY 11-7082
attenuated the mRNA expression of NF- kB. The protein levels
of NLRP3 inflammasome, pro-IL-1b and mature IL-1b were
decreased in HG+BAY 11-7082 group than in HG+DMSO group
(Figure 10B–G, p,0.05,p,0.01). NLRP3 Gene Silencing Ameliorates Diabetic Cardiomyopathy NLRP3 Gene Silencing Ameliorates Diabetic Cardiomyopathy Figure 5. NLRP3 gene silencing alleviated myocardial cell death, ultrastructral disorder and fibrosis in diabetic group. Heart size (A1)
and cross sections of hearts at the papillary muscle level (A2); scale bar: 5 mm. (B) Quantitative data for heart weight to body weight ratio. TUNEL
staining of myocardium (D, scale bar: 20 mm) and quantitative analysis of TUNEL positive cells (C) in each group. (E) Transmission electron microscopy
of rat hearts; normal myofibrils (M); normal mitochondria (MT); normal Z-lines (Z); disorganized myofibrils (black asterisk); swollen mitochondria
(white asterisk); accumulated lipid (white arrow); excessive glycogen lysis (black arrow); scale bar: 1 mm. (F) Cardiac fibrosis in rats; (F1) Masson
staining; fibrotic areas stained green and normal cardiac myocytes stained red; bright field (F2) and dark field (F3) with Sirius red staining; collagen
was stained dark red in bright field; collagen I was stained red and collagen III green in dark field; scale bar: 50 mm. (G) Quantitative analysis of the
fibrotic area to total area ratio. (H–K) Western blot analysis of collagen I and III. Data were presented as means6SEM, n = 4–6. **p,0.01 vs. Ctrl; 11p,
0.01 vs. DM+vehicle. doi:10 1371/journal pone 0104771 g005 Figure 5. NLRP3 gene silencing alleviated myocardial cell death, ultrastructral disorder and fibrosis in diabetic group. Heart size (A1)
and cross sections of hearts at the papillary muscle level (A2); scale bar: 5 mm. (B) Quantitative data for heart weight to body weight ratio. TUNEL
staining of myocardium (D, scale bar: 20 mm) and quantitative analysis of TUNEL positive cells (C) in each group. (E) Transmission electron microscopy
of rat hearts; normal myofibrils (M); normal mitochondria (MT); normal Z-lines (Z); disorganized myofibrils (black asterisk); swollen mitochondria
(white asterisk); accumulated lipid (white arrow); excessive glycogen lysis (black arrow); scale bar: 1 mm. (F) Cardiac fibrosis in rats; (F1) Masson
staining; fibrotic areas stained green and normal cardiac myocytes stained red; bright field (F2) and dark field (F3) with Sirius red staining; collagen
was stained dark red in bright field; collagen I was stained red and collagen III green in dark field; scale bar: 50 mm. (G) Quantitative analysis of the
fibrotic area to total area ratio. (H–K) Western blot analysis of collagen I and III. Data were presented as means6SEM, n = 4–6. **p,0.01 vs. Ctrl; 11p,
0.01 vs. DM+vehicle. d
l doi:10.1371/journal.pone.0104771.g005 doi:10.1371/journal.pone.0104771.g005 0.05,p,0.01). High glucose induced caspase-1 activation and cell death
in H9c2 cells High glucose increased the level of activated caspase-1 as
compared with control and medium glucose (Fig. 7A, p,
0.05,p,0.01). Immunofluorescence result showed the accumu-
lation of caspase-1 in the cytoplasm of H9c2 cells incubated with
medium and high glucose (Fig. 7B). Coincident with caspase-1
activation in high glucose, TUNEL result revealed increased cell
death with nucleus DNA damage (Fig. 7C and D, p,0.01), and
EthD-III/calceim AM staining showed elevated cell death with PLOS ONE | www.plosone.org August 2014 | Volume 9 | Issue 8 | e104771 PLOS ONE | www.plosone.org 7 NF-kB and TXNIP mediated the ROS-induced NLRP3
inflammasome activation Glucose treatment promoted ROS generation in H9c2. The
increase of ROS was dose dependent (Fig. 9A, all p,0.01). Coincidence with ROS production, the phosphorylation of NF-kB
p65 was increased in medium and high glucose treated cells. And
the expression of TXNIP also exhibited a dose-dependent
response (Fig. 9B–D, all p,0.01). Pretreatment of cells with We inhibited the expression of TXNIP by TXNIP-siRNA
plasmid. The transfection efficacy of TXNIP-siRNA plasmid into
H9c2 cells reached 70% (Fig. S1E). Both the mRNA and protein
levels of TXNIP in cells treated with TXNIP-siRNA were lower August 2014 | Volume 9 | Issue 8 | e104771 PLOS ONE | www.plosone.org 8 NLRP3 Gene Silencing Ameliorates Diabetic Cardiomyopathy Figure 6. High glucose induced NLRP3 inflammasome and IL-1b expression in H9c2 cardiomyocytes. (A) The relative mRNA expression
of NLRP3 (A1), ASC (A2), caspase-1(A3) and IL-1b (A4). (B–C) Western blot analysis of NLRP3 (C1), ASC (C2), pro-caspase-1(C3), activated caspase-1(C4),
pro-IL-1b (C5) and mature IL-1b (C6). Data were presented as means6SEM, from 3 independent experiments. The significance among Ctrl, MG and
HG within the same stimulated time was analyzed by one way ANOVA. The significance among HG groups within different stimulated time was
analyzed by one way ANOVA. **p,0.01 vs. Ctrl in 24 h, 36 h or 48 h; ##p,0.01 vs. MG in 24 h, 36 h or 48 h; 1p,0.05, 11p,0.01 vs. HG for 36 h. Ctrl:
control glucose, 5.6 mM glucose; MG: medium glucose, 25 mM glucose; HG: high glucose, 33.3 mM glucose; Pro-Casp-1: pro-caspase-1; Casp-1 p20:
activated caspase-1; IL-1b p17: mature IL-1b. doi:10.1371/journal.pone.0104771.g006 Figure 6. High glucose induced NLRP3 inflammasome and IL-1b expression in H9c2 cardiomyocytes. (A) The relative mRNA expression
of NLRP3 (A1), ASC (A2), caspase-1(A3) and IL-1b (A4). (B–C) Western blot analysis of NLRP3 (C1), ASC (C2), pro-caspase-1(C3), activated caspase-1(C4),
pro-IL-1b (C5) and mature IL-1b (C6). Data were presented as means6SEM, from 3 independent experiments. The significance among Ctrl, MG and
HG within the same stimulated time was analyzed by one way ANOVA. The significance among HG groups within different stimulated time was
analyzed by one way ANOVA. **p,0.01 vs. Ctrl in 24 h, 36 h or 48 h; ##p,0.01 vs. MG in 24 h, 36 h or 48 h; 1p,0.05, 11p,0.01 vs. HG for 36 h. NF-kB and TXNIP mediated the ROS-induced NLRP3
inflammasome activation Ctrl:
control glucose, 5.6 mM glucose; MG: medium glucose, 25 mM glucose; HG: high glucose, 33.3 mM glucose; Pro-Casp-1: pro-caspase-1; Casp-1 p20:
activated caspase-1; IL-1b p17: mature IL-1b. doi:10.1371/journal.pone.0104771.g006 than the vehicle (Fig. 10H–J, p,0.05,p,0.01). After inhibiting
the expression of TXNIP, the protein levels of activated caspase-1
and mature IL-1b induced by high glucose decreased as compared
with vehicle (Fig. 10I, M and O, both p,0.01). TXNIP-siRNA
did not attenuate the protein levels of NLRP3, pro-caspase-1 and
pro-IL-1b (Fig. 10I, K, L and N). group were increased at 4 weeks, and reached to the highest levels
at 8 or 12 weeks, and maintained at moderate high levels until 20
weeks of diabetes. The increase of NLRP3 inflammasome at early
stage of diabetes was accompanied with the decrease in cardiac
function in diabetic rats, which indicated the importance of
NLRP3 inflammasome in the pathogenesis of DCM. The decrease
of cardiac NLRP3 inflammasome in late stage of diabetes might be
due to the adaptive protection of myocardium under the consistent
stimuli in diabetes. Therefore, activation of NLRP3 inflammasome
might contribute to the development and progression of DCM. Discussion The present study provided new evidence to show the direct
contribution of NLRP3 inflammasome in the pathogenesis of
DCM, which was associated with the IL-1b-related metabolic
disorder and caspase-1-mediated pyroptosis in myocardium. In
addition, we found that the NF-kB and TXNIP were involved in
the
ROS-induced
NLRP3
inflammasome
activation. Thus,
NLRP3 inflammasome has a pivotal role in DCM and could be
an attractive drug target for treating type 2 diabetes. In light of the pivotal role of NLRP3 inflammasome in DCM,
we used NLRP3-miRNA in vivo to explore whether downregu-
lation of NLRP3 could prevent the pathogenesis of DCM. According to our preliminary study, we used 1610‘8 TU/rat
lentivirial vehicle or NLRP3-miRNA in this study. In our study,
NLRP3-miRNA treatment produced no notable adverse effects
and no deaths in rats. NLRP3 mRNA and protein levels were
significantly decreased with NLRP3-miRNA treatment. More-
over, activated caspase-1 and mature IL-1b, the important
effectors
of
NLRP3
inflammasome,
were
also
significantly
decreased in NLRP3-miRNA treated diabetic rats. In present study, we have successfully established type 2 diabetic
rat models, which were characterized by severe metabolic
disturbance, cardiac cell death, progressive myocardial dysfunc-
tion and remodeling. In keeping with previous studies [30,31], we
found early onset of diastolic dysfunction in diabetic rats at 8 weeks
after diabetes in this study (data not shown). Intriguingly, the
cardiac NLRP3 inflammasome and IL-1b expression in DM In current study, our diabetic rats showed severe metabolic
disorders. However, the NLRP3 gene silencing therapy had little
effect on improving these systemic metabolic disorders. Therefore, PLOS ONE | www.plosone.org August 2014 | Volume 9 | Issue 8 | e104771 August 2014 | Volume 9 | Issue 8 | e104771 9 NLRP3 Gene Silencing Ameliorates Diabetic Cardiomyopathy ll death in H9c2 cells. (A) Analysis of caspase-1 activity using Ac-YVAD- Figure 7. Pyroptosis is implicated in the high glucose-induced cell death in H9c2 cells. (A) Analysis of caspase-1 activity using Ac-YVAD-
pNA, the activity of caspase-1 was reflected by the production of pNA. (B) Immunofluorescence analysis of caspase-1in H9c2 cardiomyocytes; scale
bar: 20 mm. (C) TUNEL staining of medium and high glucose-treated H9c2 cells, positive cell indicated by black arrow; scale bar: 20 mm. (D)
Quantitative analysis of TUNEL positive cell in each group. (E) EthD-III (red) and calcein AM (green) staining; scale bar: 50 mm. (F) Quantitative analysis
of EthD-III positive cell. Data were presented as means6SEM, from 3 independent experiments. NLRP3 Gene Silencing Ameliorates Diabetic Cardiomyopathy Figure 8. NLRP3 gene silencing suppressed activated caspase-1, inflammatory cytokines, and pyroptosis in high glucose-treated
H9c2 cells. Relative mRNA expression of NLRP3 (A) and protein levels of NLRP3, pro-caspase-1, activated caspase-1, pro-IL-1b and mature IL-1b (B–G)
in H9c2 cardiomyocytes with treatment. (H–K) The protein levels of IL-1b, IL-18, TNF-a and IL-6 in the cultured supernatants of H9c2 cardiomyocytes. (L) TUNEL staining of H9c2 cells, positive cell indicated by black arrow; scale bar: 20 mm. (M) Quantitative analysis of TUNEL positive cell in each group. (O) EthD-III (red) staining; scale bar: 50 mm. (N) Quantitative analysis of EthD-III positive cell. Data were presented as means6SEM, from 3 independent
experiments. *p,0.05, **p,0.01 vs. Ctrl; 1p,0.05, 11p,0.01 vs. HG+vehicle. doi:10.1371/journal.pone.0104771.g008 Figure 8. NLRP3 gene silencing suppressed activated caspase-1, inflammatory cytokines, and pyroptosis in high glucose-treated
H9c2 cells. Relative mRNA expression of NLRP3 (A) and protein levels of NLRP3, pro-caspase-1, activated caspase-1, pro-IL-1b and mature IL-1b (B–G)
in H9c2 cardiomyocytes with treatment. (H–K) The protein levels of IL-1b, IL-18, TNF-a and IL-6 in the cultured supernatants of H9c2 cardiomyocytes. (L) TUNEL staining of H9c2 cells, positive cell indicated by black arrow; scale bar: 20 mm. (M) Quantitative analysis of TUNEL positive cell in each group. (O) EthD-III (red) staining; scale bar: 50 mm. (N) Quantitative analysis of EthD-III positive cell. Data were presented as means6SEM, from 3 independent
experiments. *p,0.05, **p,0.01 vs. Ctrl; 1p,0.05, 11p,0.01 vs. HG+vehicle. doi:10.1371/journal.pone.0104771.g008 cemia-induced cell death in DCM. NLRP3 was an important
factor in the regulation of pyroptosis. plasma membrane, which presumably contributes to protein
secretion at early stages of pyrotosis, can be the initiator of later
cell death due to ruptures caused by cytoplasmic swelling [41]. Moreover, pyroptosis may lead to DNA damage in the nuleus
[17]. Intriguingly,
we
found
the
pivotal
characteristics
of
pyroptosis in myocardium of DCM in vivo, including activated
caspase-1, cytoplasmic swelling, and nucleus DNA damage. These
data suggested that pyroptosis participate in the pathogenesis of
DCM. In vitro, the activated NLRP3 inflammasome lead to
cytoplasmic caspase-1 activation, along with permeabilization of
plasma membrane and nucleus DNA damage in high glucose-
treated H9c2 cardiomyocytes. The result supported our conjecture
that pyroptosis was present in cardiomyocyte. NLRP3 gene
silencing in vivo and in vitro markedly suppressed caspase-1
activation, which was paralleled with decreased cell death
characterized by integrity of cell membrane, cytoplamic swelling
and DNA damage. Discussion **p,0.01 vs. Ctrl; #p,0.05 vs. MG. Ctrl: control
glucose, 5.6 mM glucose for 36 h; MG: medium glucose, 25 mM glucose for 36 h; HG: high glucose, 33.3 mM glucose for 36 h; Casp-1: activated
caspase-1. doi:10 1371/journal pone 0104771 g007 Figure 7. Pyroptosis is implicated in the high glucose-induced cell death in H9c2 cells. (A) Analysis of caspase-1 activity using Ac-YVAD-
pNA, the activity of caspase-1 was reflected by the production of pNA. (B) Immunofluorescence analysis of caspase-1in H9c2 cardiomyocytes; scale
bar: 20 mm. (C) TUNEL staining of medium and high glucose-treated H9c2 cells, positive cell indicated by black arrow; scale bar: 20 mm. (D)
Quantitative analysis of TUNEL positive cell in each group. (E) EthD-III (red) and calcein AM (green) staining; scale bar: 50 mm. (F) Quantitative analysis
of EthD-III positive cell. Data were presented as means6SEM, from 3 independent experiments. **p,0.01 vs. Ctrl; #p,0.05 vs. MG. Ctrl: control
glucose, 5.6 mM glucose for 36 h; MG: medium glucose, 25 mM glucose for 36 h; HG: high glucose, 33.3 mM glucose for 36 h; Casp-1: activated
caspase-1. d
l doi:10.1371/journal.pone.0104771.g007 In addition
to improving
cardiac
inflammation, NLRP3
silencing could reduce cardiac cell death in DCM. Cell death
usually occurs through apoptosis or necrosis. The apoptosis of
cardiomyocyte is an important pathological change, which causes
loss of myocardial contractile unit and finally results in cardiac
dysfunction in DCM [38]. Recent studies found that there are
many other forms of cell death, one of which is pyroptosis
[21,23,39,40]. Pyroptosis, a proinflamatory form of programmed
cell death, is caspase-1 dependent [17]. Pore formation of the we could evaluate the effects of NLRP3 inflammasome on DCM
without the influence of systemic changes. In the diabetic state, induction of proinflammatory cytokines by
hyperglycemia leads to the persistent inflammation in myocardi-
um, which contributes to diabetic cardiac dysfunction [37]. Consistent with previous studies, we found an increased expression
of IL-1b in the left ventricle of diabetes rats. The NLRP3-miRNA
treatment effectively alleviated the cardiomyocyte inflammation by
suppressing the release of IL-1b, IL-18, TNF-a and IL-6. August 2014 | Volume 9 | Issue 8 | e104771 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 10 NLRP3 Gene Silencing Ameliorates Diabetic Cardiomyopathy August 2014 | Volume 9 | Issue 8 | e104771 NLRP3 Gene Silencing Ameliorates Diabetic Cardiomyopathy The protein levels of pNF-kB (F
and G), TXNIP (G and H), NLRP3 (I and J), ASC (I and K), pro-caspase-1 (I and L), activated caspase-1(I and M), pro-IL-1b (I and N) and mature IL-1b (I and
O). Data were presented as means6SEM, from 3 independent experiments. **p,0.01 vs. Ctrl; ##p,0.01 vs. MG; 11p,0.01 vs. HG+vehicle. doi:10.1371/journal.pone.0104771.g009 Figure 9. ROS induced NLRP3 inflammsome activation. (A) Quantitative analysis of the ROS production in H9c2 cells treated with medium and
high glucose. (B–D) Western blot analysis of pNF-kB and TXNIP. (E) The production of cellular ROS after NAC treatment. The protein levels of pNF-kB (F
and G), TXNIP (G and H), NLRP3 (I and J), ASC (I and K), pro-caspase-1 (I and L), activated caspase-1(I and M), pro-IL-1b (I and N) and mature IL-1b (I and
O). Data were presented as means6SEM, from 3 independent experiments. **p,0.01 vs. Ctrl; ##p,0.01 vs. MG; 11p,0.01 vs. HG+vehicle. doi:10.1371/journal.pone.0104771.g009 lipid accumulation, and less cardiac fibrosis; thus, NLRP3
silencing attenuated DCM. glucose-induced NF-kB and TXNIP were markedly reduced. In
keeping with these observations, the expression of NLRP3
inflammasome and mature IL-1b were decreased within ROS
inhibition. Intriguingly, the ROS-induced NLRP3 inflammasome
up-regulation was suppressed by NF-kB inhibitor, which indicated
NF-kB
was
an
essential
link
between
ROS
and
NLRP3
inflammasome. One research of type 2 diabetes has shown that
the maturation of caspase-1 and IL-1b, the sign of NLRP3
inflammation activation, did not required TXNIP in bone
marrow-derived macrophages [48]. In contrast, some researches
have indicated that TXNIP silencing could reduce NLRP3
inflammasome-mediated
caspase-1
and
IL-1b
activation
by
affecting the TXNIP-NLRP3 interaction rather than decreasing
the expression levels of NLRP3, pro-caspase-1 and pro-IL-1b
[29,49]. In our study, TXNIP-siRNA abrogated the maturation of
caspase-1 and IL-1b without alteration of NLRP3 expression level
in high glucose-treated H9c2 cardiomyocytes. The result indicated Three distinct signal pathways have been proposed to be
involved in NLRP3 inflammasome activation, including puriner-
gic receptor P2X7, cathepsin B, and ROS related pathways [29]. NLRP3 activation can be blocked by ROS scavengers [44]. Additional levels of crosstalk between ROS and NLRP3 inflam-
masome may involve ROS-induced NF-kB activation of NLRP3
transcription, thereby facilitating the overexpression of NLRP3
inflammasome and maturation of caspase-1 and IL-1b [45,46]. The activation of NLRP3 under oxidative stress also involves
TXNIP, which can directly bind NLRP3 and induce NLRP3 to
interact with ASC and caspase-1 [29,47]. NLRP3 Gene Silencing Ameliorates Diabetic Cardiomyopathy Together, these data suggested that the
caspase-1-dependent pyroptosis is a contributor to the hypergly- plasma membrane, which presumably contributes to protein
secretion at early stages of pyrotosis, can be the initiator of later
cell death due to ruptures caused by cytoplasmic swelling [41]. Moreover, pyroptosis may lead to DNA damage in the nuleus
[17]. Intriguingly,
we
found
the
pivotal
characteristics
of
pyroptosis in myocardium of DCM in vivo, including activated
caspase-1, cytoplasmic swelling, and nucleus DNA damage. These
data suggested that pyroptosis participate in the pathogenesis of
DCM. In vitro, the activated NLRP3 inflammasome lead to
cytoplasmic caspase-1 activation, along with permeabilization of
plasma membrane and nucleus DNA damage in high glucose-
treated H9c2 cardiomyocytes. The result supported our conjecture
that pyroptosis was present in cardiomyocyte. NLRP3 gene
silencing in vivo and in vitro markedly suppressed caspase-1
activation, which was paralleled with decreased cell death
characterized by integrity of cell membrane, cytoplamic swelling
and DNA damage. Together, these data suggested that the
caspase-1-dependent pyroptosis is a contributor to the hypergly- Our diabetic rats showed obvious lipid accumulation in the
myocardium of DCM on TEM examination. The increased lipid
droplets, which can induce cardiac lipotoxicity, is a contributor to
the DCM [1,31,42]. After NLRP3 gene silencing, the cardiac lipid
accumulation was markedly reversed. Diabetic rats showed abnormal collagen deposition in interstitial
areas on Masson and Sirius red staining. Intensive cardiac fibrosis,
which can induce LV stiffness, is a common finding in progressed
DCM [43]. Consistent with this, echocardiography revealed
diastolic dysfunction. NLRP3 silencing reduced the aberrant
interstitial collagen accumulation, collagen I and III content, and
collagen I to III ratio in DCM. Furthermore, echocardiography
results of NLRP3-miRNA treated diabetes rats revealed that both
the LV systolic and diastolic dysfunction were improved, which
were attributed to decreased cardiac cell death, reduced cardiac August 2014 | Volume 9 | Issue 8 | e104771 11 PLOS ONE | www.plosone.org NLRP3 Gene Silencing Ameliorates Diabetic Cardiomyopathy li id
l ti
d l
di
fib
i
th
NLRP3
l
i d
d NF kB
d TXNIP
k dl
d
d I
Figure 9. ROS induced NLRP3 inflammsome activation. (A) Quantitative analysis of the ROS production in H9c2 cells treated with medium and
high glucose. (B–D) Western blot analysis of pNF-kB and TXNIP. (E) The production of cellular ROS after NAC treatment. Conclusion Our results suggest that the NLRP3 inflammasome is implicated
in DCM. The activation of NLRP3 inflammasome and its
effectors was regulated by NF-kB and TXNIP. Caspase-1
dependent pyroptosis had a distinct role in the process of DCM. NLRP3 gene silencing ameliorated the development of DCM in
type 2 diabetic rat, by reducing cardiac inflammation, cardiomy-
ocyte pyroptosis and left ventricular fibrosis. The cardioprotective
effects with NLRP3 silencing suggest a potential role for NLRP3
antagonists in treating DCM in type 2 diabetes. NLRP3 Gene Silencing Ameliorates Diabetic Cardiomyopathy TXNIP was an important regulator of the activation of caspase-1
and IL-1b, which are the pivotal effecter of NLRP3 inflamma-
some. Acknowledgments We thank the editor and anonymous reviewers, who helped to improve the
manuscript. NLRP3 Gene Silencing Ameliorates Diabetic Cardiomyopathy In line with these
findings, our study showed glucose-treated H9c2 cardiomyocyte
produced excessive ROS in a concentration-dependent manner. The levels of pNF-kB and TXNIP showed a similar expression
pattern in response to glucose. With the ROS inhibition, the high August 2014 | Volume 9 | Issue 8 | e104771 PLOS ONE | www.plosone.org 12 NLRP3 Gene Silencing Ameliorates Diabetic Cardiomyopathy Figure 10. NF-kB and TXNIP induced NLRP3 inflammasome activation. Relative mRNA expression of NF-kB (A) and protein levels of NLRP3,
pro-caspase-1, activated caspase-1, pro-IL-1b and mature IL-1b (B-G) in H9c2 cardiomyocytes with treatment. Relative mRNA expression of TXNIP (H)
and protein levels of TXNIP, NLRP3, pro-caspase-1, activated caspase-1, pro-IL-1b and mature IL-1b (I-O) in H9c2 cardiomyocytes. Data were presented
as means6SEM, from 3 independent experiments. *p,0.05, **p,0.01 vs. Ctrl; 11p,0.01 vs. HG+vehicle. doi:10.1371/journal.pone.0104771.g010
NLRP3 Gene Silencing Ameliorates Diabetic Cardiomyopathy Figure 10. NF-kB and TXNIP induced NLRP3 inflammasome activation. Relative mRNA expression of NF-kB (A) and protein levels of NLRP3,
pro-caspase-1, activated caspase-1, pro-IL-1b and mature IL-1b (B-G) in H9c2 cardiomyocytes with treatment. Relative mRNA expression of TXNIP (H)
and protein levels of TXNIP, NLRP3, pro-caspase-1, activated caspase-1, pro-IL-1b and mature IL-1b (I-O) in H9c2 cardiomyocytes. Data were presented
as means6SEM, from 3 independent experiments. *p,0.05, **p,0.01 vs. Ctrl; 11p,0.01 vs. HG+vehicle. doi:10 1371/journal pone 0104771 g010 Figure 10. NF-kB and TXNIP induced NLRP3 inflammasome activation. Relative mRNA expression of NF-kB (A) and protein levels of NLRP3,
pro-caspase-1, activated caspase-1, pro-IL-1b and mature IL-1b (B-G) in H9c2 cardiomyocytes with treatment. Relative mRNA expression of TXNIP (H)
and protein levels of TXNIP, NLRP3, pro-caspase-1, activated caspase-1, pro-IL-1b and mature IL-1b (I-O) in H9c2 cardiomyocytes. Data were presented
as means6SEM, from 3 independent experiments. *p,0.05, **p,0.01 vs. Ctrl; 11p,0.01 vs. HG+vehicle. doi:10.1371/journal.pone.0104771.g010 vehicle transfection (scale bar: 50 mm); n = 6. (B and C) Western
blot analysis of NLRP3 inflammsome and IL-1b with 5.6 mM
glucose or 27.5 mM mannose for 24 to 48 h; Data were presented
as means6SEM, from 3 independent experiments. (D) Transfec-
tive efficiency of vehicle or NLRP3-miRNA in H9c2 cells. Bright
green cells indicate GFP with lentivirus-NLRP3-miRNA or vehicle
transfection (scale bar: 50 mm). (E) Transfective efficiency of
vehicle or TXNIP-siRNA in H9c2 cells. Bright green cells indicate
GFP with TXNIP-siRNA plasmid or vehicle transfection (scale
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y
p
7. Author Contributions Figure S1
Lentiviral vehicle and NLRP3-miRNA were
effectively transfected in vivo and in vitro. Mannose could
not activate NLRP3 inflammsome. (A) Transfective efficiency of
vehicle or NLRP3-miRNA in myocardium. Bright green points
(white arrow) indicate GFP with lentivirus-NLRP3-miRNA or Conceived and designed the experiments: B. Luo. Performed the
experiments: B. Luo. Analyzed the data: B. Luo. Contributed reagents/
materials/analysis tools: B. Luo B. Li WW XL YX. Wrote the paper: B. Luo. Revised the paper: CZ MZ YZ FA. August 2014 | Volume 9 | Issue 8 | e104771 PLOS ONE | www.plosone.org 13 NLRP3 Gene Silencing Ameliorates Diabetic Cardiomyopathy References (2013)
NLRP3 inflammasome activation results in hepatocyte pyroptosis, liver
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mitochondria by overexpression of MnSOD reduces diabetic cardiomyopathy. Diabetes 55: 798–805. 47. Martinon F (2010) Signaling by ROS drives inflammasome activation. Eur J Immunol 40: 616–619. 48. Masters SL, Dunne A, Subramanian SL, Hull RL, Tannahill GM, et al. (2010)
Activation of the NLRP3 inflammasome by islet amyloid polypeptide provides a
mechanism for enhanced IL-1beta in type 2 diabetes. Nat Immunol 11: 897–
904. 25. Van Linthout S, Spillmann F, Riad A, Trimpert C, Lievens J, et al. (2008)
Human apolipoprotein A-I gene transfer reduces the development of
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26. Yu W, Wu J, Cai F, Xiang J, Zha W, et al. (2012) Curcumin alleviates diabetic
cardiomyopathy in experimental diabetic rats. PLoS One 7: e52013. 26. Yu W, Wu J, Cai F, Xiang J, Zha W, et al. (2012) Curcumin alle 49. Mohamed IN, Hafez SS, Fairaq A, Ergul A, Imig JD, et al. (2014) Thioredoxin-
interacting protein is required for endothelial NLRP3 inflammasome activation
and cell death in a rat model of high-fat diet. Diabetologia 57: 413–423. cardiomyopathy in experimental diabetic rats. PLoS One 7: e520 27. Bauernfeind FG, Horvath G, Stutz A, Alnemri ES, MacDonald K, et al. (2009)
Cutting edge: NF-kappaB activating pattern recognition and cytokine receptors August 2014 | Volume 9 | Issue 8 | e104771 PLOS ONE | www.plosone.org 14
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English
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Analysis of Agricultural Products Logistics in Hunan Province and Green Development Countermeasures
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Journal of management and sustainability
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cc-by
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Journal of Management and Sustainability; Vol. 5, No. 2; 2015
ISSN 1925-4725 E-ISSN 1925-4733
Published by Canadian Center of Science and Education Journal of Management and Sustainability; Vol. 5, No. 2; 2015
ISSN 1925-4725 E-ISSN 1925-4733
Published by Canadian Center of Science and Education Received: March 4, 2015 Accepted: March 29, 2015 Online Published: May 31, 2015
doi:10.5539/jms.v5n2p146 URL: http://dx.doi.org/10.5539/jms.v5n2p146 This work is supported by the funds project under the Education Department of Hunan Province for outstanding
young people under Grant No.13B152 and the general project under the Education Department of Hunan
Province under Grant No. 13C1165. Abstract Hunan is an agricultural province with abundant output of agricultural products and frequent agricultural
products logistics activities. However, the level of its agricultural products logistics is still low and the “negative
external effect” of high energy consumption, big loss, environment pollution and waste of resources still exists in
the logistics process. The agricultural products logistics of Hunan Province needs to take a green development
way with attributes of efficient energy-saving and low wastage. Based on factor analysis and cluster analysis,
this paper analyzed agricultural products logistics conditions of 14 cities of Hunan province, from the
perspective of supply, demand and energy consumption. Relevant development countermeasures were given
according to the results of empirical analysis. Keywords: agricultural products logistics, green development, factor analysis, cluster analysis, Hunan Province
1. Introduction Analysis of Agricultural Products Logistics in Hunan Province and
Green Development Countermeasures Dan Tan1 & Yajiao Tang1
1 College of Economics, Central South University of Forestry and Technology, Changsha, China
Correspondence: Dan Tan, College of Economics, Central South University of Forestry and Technology, No.498
South Shaoshan Road, Tianxin District, Changsha 410004, China. Tel: 86-137-8741-8695. E-mail:
tan_dan@126.com Dan Tan1 & Yajiao Tang1
1 College of Economics, Central South University of Forestry and Technology, Changsha, China
Correspondence: Dan Tan, College of Economics, Central South University of Forestry and Technology, No.498
South Shaoshan Road, Tianxin District, Changsha 410004, China. Tel: 86-137-8741-8695. E-mail:
tan_dan@126.com Received: March 4, 2015 Accepted: March 29, 2015 Online Published: May 31, 2015
doi:10.5539/jms.v5n2p146 URL: http://dx.doi.org/10.5539/jms.v5n2p146 2. Literature Review There are some scholars have studied the agricultural products logistics in Hunan Province. HUANG Fuhua and
ZHOU Min (2008) analyzed the situation and problem of agricultural products logistics in Hunan Province from
the perspective of joint logistics and proposed the countermeasures of developing joint logistics of agricultural
products. PANG Yan and WANG Zhongwei (2009) put forward the thoughts of constructing modern logistics
system of agricultural products of Hunan Province. ZHOU Xiaomei and LI Qianlan (2010) came up with the
recommendation of the industrialized operation of agricultural products logistics in Hunan Province, from the
perspective of logistics circle construction, information sharing, logistics channel establishment and
demonstrative agricultural products wholesale market construction. LI Ming (2011) conducted the data statistics
of the operation situation of agricultural products logistics of Hunan Province, and raised the development
suggestions from the aspects of awareness, legal system, standard, principal part, environment, human resource
and technology. LIU Changjun (2011) put forward the security mechanism of the development of agricultural
products logistics in Hunan Province. These researches have established the good foundation of the development
of agricultural products logistics of Hunan Province. However, there is a lack of research on evaluating the
agricultural products logistics in Hunan. 1. Introduction Hunan Province is located in the south of the Yangtze River, which belongs to the middle reaches of the Yangtze
River area, since the ancient times to enjoy the reputation of “a land flowing with milk and honey”. The region's
GDP reached 2,215.42 billion Yuan in 2012, with the growth of 11.3%, and ranked 10th in the whole country. At
the end of 2011, the provincial highway traffic total mileage reached 232,200 kilometers, and the road network
density is 106.36 km/hundred square kilometers. The road has reached 100% of the villages and towns in the
whole province. Its shipping conditions are very superior. The existing navigable rivers in this province are 373,
and the provincial traffic mileage reaches 11,900 kilometers, accounting for about 1/10 of the inland river
navigable mileage of the country, ranking the third in the nation. Hunan is a major agricultural province, with
abundant agricultural products. The total output value of agricultural products in 2012 is 490.41 billion Yuan,
with the increase of 3% over the previous year. With its special geographic position, Hunan Province has huge
inflow and outflow of agricultural products every year. Although the agricultural products logistics activities in
Hunan Province are frequent, yet the level of logistics activities is still relatively low. The circulation channel of
agricultural products is complex and unstable. The normative systematic management and control of the
procurement, transportation, processing, warehousing, packaging, and distribution of agricultural products does
not establish. The safety, health and environment protection have not been paid enough attention in the process
of agricultural products circulation. The “negative external effect” of high energy consumption, big loss, exhaust
emission, noise pollution and waste of resources does exist. The agricultural products logistics of Hunan
Province needs to take a green development way with attributes of efficient energy-saving and low wastage. Therefore, this paper makes the evaluation on the situation of the agricultural products logistics of 14 cities in
Hunan Province by using the factor analysis and cluster analysis method, and provides the development
measures according to the empirical results. 146 Vol. 5, No. 2; 2015 Journal of Management and Sustainability www.ccsenet.org/jms 3. Methodology The factor analysis method is developed by Karl Pearson and Charles Spearmen at the beginning of 20th century,
based on the statistics and analysis of the intelligence test. It is a mathematical procedure used to reduce a large
amount of data into a structure that can be more easily studied. It takes the minimal loss of information as the
premise, starting from the internal dependence of the correlation matrix, integrating many variables into a few
indicators, which called factor. In other words, it summarizes information contained in a large number of
variables and condenses it into a smaller number of factors containing variables that are interrelated. Cluster Analysis was originated in anthropology by Driver and Kroeber in 1932 and introduced to psychology by
Zubin in 1938 and Robert Tryon in 1939 and famously used by Cattell beginning in 1943 for trait theory
classification in personality psychology. It is a multivariate analysis technique that seeks to organize information
about variables so that relatively homogeneous groups, or “clusters”, can be formed. 4.1 The Selection of Sample Indicators and Data The factor analysis requires that the number of samples should be more than the number of indicators and the
KMO test should also be passed. In general, the value of KMO should be higher than 0.6, then the factor analysis
can be conducted. At present, the system of statistical methods for regional agricultural products logistics level
has not yet formed in China. Based on the principle of relevance, according to the influencing factors of regional
agricultural products logistics indicators, the evaluation indicators of factor analysis on agricultural products
logistics in Hunan province were preselected through reading books or statistical yearbook, consulting the
relevant departments, web search and other means. 36 indicators were initially identified to represent the level of
agricultural products logistics in Hunan province. The data statistics of some indicators were found incomplete
or could not be acquired during the process of investigation. Since the research object is the 14 cities in Hunan
Province, the number of sample is 14, which means the number of factors must be restricted within 13 indicators. Therefore, the 36 indicators have been screened and those indicators which are similar to each other or reflect the
same aspect of agricultural products logistics were deleted. 12 indicators which can better reflect the level of
regional agricultural products logistics and energy consumption have been chosen to construct the evaluation
system of agricultural products logistics in Hunan Province. These indicators are: X1(Regional GDP),
X2(Population), X3(Gross output value of farming, forestry, animal husbandry and fishery), X4(Total output of
agricultural products), X5(Employees in traffic, transport, storage and post), X6(Number of civil vehicles owned),
X7(Total length of highways), X8(Freight traffic), X9(Revenue from postal and telecommunication Service),
X10(Investment in transport, storage and post), X11(Number of internet users), X12(Opposite number of energy
consumption of unit GDP).Taking the opposite number of energy consumption of unit GDP as one of the
indicators is in order to reflect the energy consumption situation of agricultural products logistics activities. The
less energy the agricultural products logistics activities consume, the higher the opposite number of energy
consumption is, which shows that the green degree of agricultural products logistics activities is higher. 4. Study process and results 4.1 The Selection of Sample Indicators and Data 4.2 The Raw Data of Indicators In the evaluation indicators system, the raw data of indicators of the 14 cities is shown as table 1. The data are
obtained from the “Hunan statistical yearbook 2012” and the yearbook 2012 of each city and has been processed. 147 Journal of Management and Sustainability Vol. 5, No. 2; 2015 www.ccsenet.org/jms The agricultural products mentioned in X4(Total output of agricultural products) contain grain, oil, fruit, meat,
dairy, aquatic products, vegetables and cotton. Table 1. The raw data of factor analysis of agricultural products logistics in Hunan Province Table 1. The raw data of factor analysis of agricultural products logistics in Hunan Province
Name
of
the city
X1
(100,000,
000 yuan)
X2
(10,000
person)
X3
(10,000
yuan)
X4
(10,000
ton)
X5
(10,000
person)
X6
(unit)
X7
(km)
X8
(10,000
ton)
X9
(100,000,
000 yuan)
X10
(10,000
yuan)
X11
(10,000
subscribers)
X12
(ton SCE/
10,000 Yuan
Changsha
5619.33
709.07
3877162.00
836.19
1.66
1193649.00
15438.21
25651.00
106.31
2125515.00
111.54
-0.640
Zhuzhou
1564.27
388.08
1917342.00
462.02
0.32
526021.00
13531.15
14760.00
29.91
143557.00
41.97
-0.964
Xiangtan
1124.14
276.45
1718823.00
356.50
0.50
400108.00
7784.08
8220.69
22.45
206275.00
24.10
-1.301
Hengyang
1734.30
716.60
4746623.00
840.89
0.36
616240.00
20569.04
17680.00
34.55
271870.00
44.34
-0.887
Shaoyang
907.23
710.72
3185620.00
664.75
1.36
547924.00
21609.36
15610.80
29.73
506529.00
34.47
-0.935
Yueyang
1899.49
548.53
3805034.00
748.04
1.01
432391.00
20076.05
16542.49
32.36
120771.00
40.58
-1.006
Changde
1811.19
573.26
4692046.00
817.81
0.36
748829.00
22160.17
10401.00
34.14
337058.00
41.13
-0.727
Zhangjiajie
298.04
149.01
615189.20
148.55
0.34
171804.00
8739.67
1755.00
10.76
79814.00
13.42
-0.700
Yiyang
883.63
431.44
2902927.00
612.33
0.38
464119.00
15819.18
9026.00
23.91
181091.00
22.95
-0.828
Chenzhou
1346.38
460.52
2480990.00
539.23
0.40
550862.00
16991.13
19436.00
24.98
612686.00
29.85
-1.017
Yongzhou
945.39
521.25
4060190.00
954.03
0.46
587063.00
22601.58
8322.00
22.95
57400.00
25.36
-0.943
Huaihua
845.63
475.10
2157271.00
452.67
0.96
487785.00
19999.54
3880.08
23.91
183894.00
27.94
-0.884
Loudi
847.26
379.32
2139091.00
309.12
0.49
512531.00
14611.52
14476.34
23.89
337794.00
26.85
-1.821
West
Hunan
361.37
256.25
908715.40
263.73
0.42
176255.00
12259.29
3425.00
13.97
405130.00
17.81
-0.873 The KMO value is used to compare the simple correlation and partial correlation coefficient of items, which
values from 0 to 1. When it is greater than 0.9, the factor analysis is a very suitable method. When it is from 0.6
to 0.9, the factor analysis is a suitable method. When it is less than 0.6, the factor analysis is not a suitable
method. The result of Bartlett’s Test is to examine the significance of correlation coefficient of items. If the Sig is
less than 0.05, the factor analysis can be conducted. 4.3 The Calculation Process Because the dimension and the order of magnitudes of the different variables in table 1 are different, all the
indicators are standardized so as to make each variable comparable, and analyze the data in more equal condition. The new data after standardization are shown as table 2. Table 2. The data after standardization of factor analysis of agricultural products logistics in Hunan Province
Name
of
the city
X1
(100,000,
000 yuan)
X2
(10,000
person)
X3
(10,000
yuan)
X4
(10,000
ton)
X5
(10,000
person)
X6
(unit)
X7
(km)
X8
(10,000
ton)
X9
(100,000,
000 yuan)
X10
(10,000
yuan)
X11
(10,000
subscribers)
X12
(ton SCE/
10,000 Yuan
Changsha
3.21
1.36
0.82
1.06
2.37
2.70
-0.24
1.99
3.31
3.30
3.19
1.11
Zhuzhou
0.09
-0.47
-0.67
-0.44
-0.76
-0.01
-0.63
0.39
-0.05
-0.49
0.26
0.01
Xiangtan
-0.24
-1.11
-0.82
-0.86
-0.34
-0.53
-1.82
-0.57
-0.38
-0.37
-0.50
-1.14
Hengyang
0.22
1.40
1.48
1.08
-0.66
0.35
0.82
0.82
0.16
-0.24
0.36
0.27
Shaoyang
-0.41
1.37
0.29
0.37
1.67
0.07
1.04
0.52
-0.06
0.21
-0.06
0.11
Yueyang
0.35
0.44
0.76
0.71
0.85
-0.40
0.72
0.65
0.06
-0.53
0.20
-0.14
Changde
0.28
0.58
1.44
0.99
-0.66
0.89
1.15
-0.25
0.14
-0.12
0.22
0.81
Zhangjiajie
-0.88
-1.84
-1.66
-1.70
-0.71
-1.46
-1.62
-1.51
-0.89
-0.61
-0.95
0.91
Yiyang
-0.43
-0.23
0.08
0.16
-0.62
-0.27
-0.16
-0.45
-0.31
-0.41
-0.55
0.47
Chenzhou
-0.07
-0.06
-0.24
-0.13
-0.57
0.09
0.08
1.08
-0.26
0.41
-0.25
-0.17
Yongzhou
-0.38
0.29
0.96
1.53
-0.43
0.23
1.24
-0.55
-0.35
-0.65
-0.44
0.08
Huaihua
-0.46
0.02
-0.49
-0.48
0.74
-0.17
0.70
-1.20
-0.31
-0.41
-0.33
0.28
Loudi
-0.46
-0.52
-0.50
-1.05
-0.36
-0.07
-0.41
0.35
-0.31
-0.11
-0.38
-2.91
West
Hunan
-0.83
-1.23
-1.44
-1.24
-0.52
-1.44
-0.89
-1.27
-0.75
0.01
-0.76
0.32
The KMO value is used to compare the simple correlation and partial correlation coefficient of items, which
values from 0 to 1. When it is greater than 0.9, the factor analysis is a very suitable method. When it is from 0.6
to 0.9, the factor analysis is a suitable method. When it is less than 0.6, the factor analysis is not a suitable
method. The result of Bartlett’s Test is to examine the significance of correlation coefficient of items. If the Sig is
less than 0.05, the factor analysis can be conducted. 4.3 The Calculation Process The data after standardization of factor analysis of agricultural products logistics in Hunan Province The KMO value is used to compare the simple correlation and partial correlation coefficient of items, which
values from 0 to 1. When it is greater than 0.9, the factor analysis is a very suitable method. When it is from 0.6
to 0.9, the factor analysis is a suitable method. When it is less than 0.6, the factor analysis is not a suitable
method. The result of Bartlett’s Test is to examine the significance of correlation coefficient of items. If the Sig is
less than 0.05, the factor analysis can be conducted. The KMO value is used to compare the simple correlation and partial correlation coefficient of items, which
values from 0 to 1. When it is greater than 0.9, the factor analysis is a very suitable method. When it is from 0.6
to 0.9, the factor analysis is a suitable method. When it is less than 0.6, the factor analysis is not a suitable
method. The result of Bartlett’s Test is to examine the significance of correlation coefficient of items. If the Sig is
less than 0.05, the factor analysis can be conducted. 148 Journal of Management and Sustainability Vol. 5, No. 2; 2015 www.ccsenet.org/jms Table 3. The result of KMO and Bartlett's test
Kaiser-Meyer-Olkin Measure of Sampling Adequacy. .672
Bartlett's Test of Sphericity
Approx. Chi-Square
208.251
df
66
Sig. .000 As shown in table 3, the test value of KMO is 0.672(> 0.6), which indicates factor analysis is suitable for this
investigation. According to the result of Bartlett’s test of sphericity, the value of approx.chi-square is 208.251,
and the significance probability is 0.000, therefore the investigation data can be analyzed by using factor analysis
method. In the process of analyzing factor, the Principal Factor Analysis is utilized. The Varimax is used for factor
rotation, and the factors with eigenvalue greater than 1 are extracted. As shown in table 4, there are three factors
with eigenvalue greater than 1. The rate of total variance explained of these three factors is 89.774%, which
covers most of the variable information. Therefore, the first three factors are selected as the common factors. Table 4. 4.3 The Calculation Process Total variance explained
Component
Initial Eigenvalues
Extraction Sums of Squared Loadings
Rotation Sums of Squared Loadings
Total
% of Variance Cumulative % Total
% of Variance Cumulative % Total
% of Variance Cumulative %
1
7.397
61.643
61.643
7.397
61.643
61.643
5.773
48.111
48.111
2
2.316
19.298
80.941
2.316
19.298
80.941
3.886
32.380
80.491
3
1.060
8.834
89.774
1.060
8.834
89.774
1.114
9.283
89.774
4
.629
5.245
95.019
5
.244
2.031
97.050
6
.152
1.266
98.316
7
.074
.617
98.933
8
.061
.512
99.445
9
.041
.345
99.790
10
.021
.172
99.961
11
.003
.024
99.985
12
.002
.015
100.000
Extraction Method: Principal Component Analysis. Table 4. Total variance explained Table 4. Total variance explained By using Varimax rotation method，the factor loading of each indicator is shown as table 5. . Rotated component matrix
Component
1
2
3
X1(Regional GDP)
.947
.212
.106
X2(Population)
.469
.839
.015
X3(Gross output value of farming, forestry, animal husbandry and
fishery)
.266
.930
.025
X4(Total output of agricultural products)
.299
.905
.153
X5(Employees in traffic, transport, storage and post)
.711
.172
.112
X6(Number of civil vehicles owned)
.813
.489
.001 149 Journal of Management and Sustainability Vol. 5, No. 2; 2015 www.ccsenet.org/jms X7(Total length of highways)
-.052
.490
.085
X8(Freight traffic)
.741
.414
-.346
X9(Revenue from postal and telecommunication Service)
.965
.204
.114
X10(Investment in transport, storage and post)
.955
-.016
.103
X11(Number of internet users)
.950
.243
.106
X12(Opposite number of energy consumption of unit GDP)
.176
.161
.951
Extraction Method: Principal Component Analysis. The following conclusions can be drawn by analyzing the data of table 5. The first common factor F1 has greater
loading on the indicators X1, X5, X6, X8, X9, X10 andX11. These indicators are connected with the essential
element needed to ensure agricultural products logistics activities go smoothly, and can comprehensively reflect
the agricultural products logistics development in Hunan Province in the aspect of market supply condition,
which can be named as “supply factor” with the contribution rate of 48.111%. The second common factor F2 has
greater loading on the indicators X2, X3 and X4. These indicators are connected with the needs of consumers for
agricultural products logistics activities and can comprehensively reflect the agricultural products logistics
development in Hunan Province in the aspect of market demand condition, which can be named as “demand
factor” with the contribution rate of 32.380%. 4.3 The Calculation Process The ranking of Xiangtan, West Hunan and Zhangjiajie are the last three in this aspect. Zhangjiajie, Changde and
West Hunan do better job than other cities for energy consumption in agricultural products logistics activities. But Loudi has the worst performance in this aspect. The top 3 cities with comprehensive strength of the
agricultural products logistics are Changsha, Hengyang and Changde. Xiangtan, West Hunan and Zhangjiajie
rank the last three. According to table 6, Changsha, Chenzhou, Zhuzhou and Shaoyang are at the top of the list and West Hunan,
Zhangjiajie and Yongzhou are at the bottom of the list in terms of the supply ability of agricultural products
logistics. Hengyang, Yongzhou and Changde rank top 3 in terms of the agricultural products logistics demand. The ranking of Xiangtan, West Hunan and Zhangjiajie are the last three in this aspect. Zhangjiajie, Changde and
West Hunan do better job than other cities for energy consumption in agricultural products logistics activities. But Loudi has the worst performance in this aspect. The top 3 cities with comprehensive strength of the
agricultural products logistics are Changsha, Hengyang and Changde. Xiangtan, West Hunan and Zhangjiajie
rank the last three. 4.3 The Calculation Process The third common factor reflects the level of energy consumption
of agricultural products logistics activities in Hunan Province, which can be named as “energy consumption
factor” with contribution rate of 9.283% to table 4 and table 5, the score function of each common factor can be obtained as follows: According to table 4 and table 5, the score function of each common factor can be obtained as follows: F1=0.947X1+0.469X2+0.266X3+0.299X4+0.711X5+0.813X6-0.052X7+0.741X8+0.965X9+0.955X10+0.950X
11+0.176X12 F1=0.947X1+0.469X2+0.266X3+0.299X4+0.711X5+0.813X6-0.052X7+0.741X8+0.965X9+0.955X10+0.950X
11+0.176X12 F2=0.212X1+0.839X2+0.930X3+0.905X4+0.172X5+0.489X6+0.940X7+0.414X8+0.201X9-0.016X10+0.243X
11+0.161X12 F3=0.106X1+0.015X2+0.025X3+0.153X4+0.112X5+0.001X6+0.085X7-0.346X8+0.114X9+0.103X10+0.106X
11+0.951X12 And the factor evaluation model of agricultural products logistics in Hunan Province can consequently be
obtained: And the factor evaluation model of agricultural products logistics in Hunan Province can consequently be
obtained: F=(48.111×F1+32.380×F2+9.283×F3)/89.774 F=(48.111×F1+32.380×F2+9.283×F3)/89.774 F=(48.111×F1+32.380×F2+9.283×F3)/89.774 Taking the data into the model, the comprehensive evaluation score of the development of agricultural products
logistics in 14 cities of Hunan Province can be obtained. Sorted by the value of F1, F2, F3 and F from high to
low, the scores of 14 cities are shown as table 6. Table 6. The comprehensive score and rank of agricultural products logistics in Hunan
F1
F2
F3
F
Changsha
3.32
Hengyang
1.37
Zhangjiajie
1.28
Changsha
1.80
Chenzhou
0.07
Yongzhou
1.29
Changde
0.75
Hengyang
0.4
Zhuzhou
0.06
Changde
1.2
West Hunan
0.74
Changde
0.37
Shaoyang
0.05
Shaoyang
0.79
Huaihua
0.7
Shaoyang
0.31
Loudi
0
Yueyang
0.76
Changsha
0.67
Yueyang
0.22
Yueyang
-0.04
Yiyang
0.07
Yiyang
0.51
Yongzhou
0.06
Xiangtan
-0.07
Chenzhou
-0.01
Yongzhou
0.27
Chenzhou
-0.03
Hengyang
-0.15
Huaihua
-0.03
Shaoyang
-0.05
Zhuzhou
-0.17
Changde
-0.26
Changsha
-0.14
Hengyang
-0.15
Huaihua
-0.17
Huaihua
-0.43
Loudi
-0.5
Zhuzhou
-0.2
Yiyang
-0.2
Yiyang
-0.51
Zhuzhou
-0.51
Yueyang
-0.27
Loudi
-0.47
West Hunan
-0.56
Xiangtan
-1.21
Chenzhou
-0.6
Xiangtan
-0.56
Zhangjiajie
-0.69
West Hunan
-1.3
Xiangtan
-0.85
West Hunan
-0.69
Yongzhou
-0.8
Zhangjiajie
-1.77
Loudi
-2.79
Zhangjiajie
-0.88 Table 6. The comprehensive score and rank of agricultural products logistics in Hunan
F1
F2
F3 Table 6. The comprehensive score and rank of agricultural products logistics in Hunan 150 Journal of Management and Sustainability Vol. 5, No. 2; 2015 www.ccsenet.org/jms According to table 6, Changsha, Chenzhou, Zhuzhou and Shaoyang are at the top of the list and West Hunan,
Zhangjiajie and Yongzhou are at the bottom of the list in terms of the supply ability of agricultural products
logistics. Hengyang, Yongzhou and Changde rank top 3 in terms of the agricultural products logistics demand. 5. Conclusions and Recommendations The increasing living standard of consumer generates greater demand of agricultural products. Given this
background, the green development of agricultural products in Hunan Province means improving the capability
of providing the agricultural products logistics service from the aspects of processing, transport, storage and
distribution, guaranteeing the quality and safety of agricultural products in the processing of logistics, increasing
the efficiency of agricultural products logistics activities and reducing the level of energy consumption of
agricultural products logistics to the greatest extent. Based on the remarkable advantages of comprehensive capability of the whole province, the first category city
has the condition to develop advanced logistics mode. According to its current characteristics of the economy,
the development of transport and storage industry, information service industry should be given special attention
so as to provide strong support to the development of agricultural products logistics system of the whole
province. To take the agricultural products logistics mode lead by the chain supermarket, the third party logistics
as the main development pattern. To take the agricultural products logistics mode lead by production and
processing enterprises, wholesale market as the second development pattern. The third party cold chain logistics
needs to be enhanced by improving the research and promotion of cold chain logistics technology, increasing the
number of refrigeration car and refrigeration storage, establishing frozen products processing and distribution
center, and boosting the intensive distribution and joint distribution. Moreover, the control standard of
temperature and moisture of agricultural products in the process of transport, storage, processing, and
distribution also needs to be established so as to decrease the loss and waste of agricultural products. For the second category cities, the reasonable planning of agriculture logistics base is vital. The reconstruction
and expansion a batch of large-medium sized professional agricultural products wholesale markets with strong
distribution function and wide radiation scope in the main producing area of agricultural products are needed. To
speed up the construction of information management system of agricultural products logistics and form the
integrated information network of agricultural products market. The application of barcode technology in the
whole process of agricultural products logistics, the promotion of online transaction of agricultural products can
significantly increase the efficiency of agricultural products logistics. The continuous contact and good
communication among agricultural products logistics enterprises can assure the smooth connection of the
demand and supply of agricultural products. 4.4 The Cluster Analysis Based on the data of principal component indicators obtained by factor analysis, the sample matrix of cluster
analysis is established. The hierarchical clustering method is used to do the cluster analysis on agricultural
products logistics system of Hunan Province. The results of analysis are shown as figure 1. Figure 1. The dendrogram of agricultural products logistics of Hunan Province Figure 1. The dendrogram of agricultural products logistics of Hunan Province Based on figure 1 and table 6, the 14 cities of Hunan Province can be classified as five categories according to
the comprehensive capability of agricultural products logistics. The first category, Changsha, has the strongest
comprehensive capability of agricultural products logistics, especially the supply ability, and enjoys the relative
low energy consumption of agricultural products logistics. The second category, Yiyang, Huaihua, Shaoyang,
Yueyang, Hengyang, Changde and Yongzhou, have relatively good capability of agricultural products logistics,
and have large demand of agricultural products logistics due to the abundant resource of agricultural products
output. The third category, Zhuzhou and Chenzhou, have the medium level comprehensive capability of
agricultural products logistics and relatively good performance in supply ability. But the energy consumption of
agricultural products logistics activities of these cities is high. The fourth category, Xiangtan, Zhangjiajie and
West Hunan, have the weakest comprehensive capability of agricultural products logistics, both in supply and
demand. The energy consumption of agricultural products logistics activities in these cities is at a relative low
level. The fifth category, Loudi, has the weak comprehensive capability of agricultural products logistics, and the
poorest performance of energy consumption of agricultural products logistics activities. 151 Vol. 5, No. 2; 2015 Journal of Management and Sustainability www.ccsenet.org/jms 5. Conclusions and Recommendations The government can provide the assistance to help those
outstanding logistics enterprises to explore the market of rural areas and become the backbone of agricultural
products logistics system. The government also can encourage the enterprises dealing in the transport, storage
and freight forwarding service to expand the scope of their business and become the suppliers of agricultural
products logistics service. For the third category cities, the facilities construction of processing, storage and transport of agricultural
products is necessary, especially the research and development of temperature control equipment and damp
proof device of grain depot, cotton depot and refrigeration warehouse. To reduce the number of circulation
checkpoints and achieve the reasonable connection of various transportation means. To adopt the logistics plan
with little negative impact on the environment in planning the logistics system and decision making of logistics
activities. The trunk with small discharge capacity, short distance distribution, and night delivery are highly
recommended. For the fourth category cities, the construction of logistics infrastructure needs to be greatly reinforced. The
improvement of the accessibility of highway, the road grade can greatly assure the smooth delivery of
agricultural products. To cultivate and establish professional marketing organization of agricultural products, and
increase the consumer demand for agricultural products, thus increasing the demand for agricultural products
logistics. For the fifth category cities, the first task is to decrease the negative impact on environment during the process of
transport, storage, carrying, packing, and processing agricultural products. The energy consumption, pollutant
discharge, and noise making of means of conveyance need to be strictly controlled. The realization of energy
conservation and emissions reduction partially depends on the reasonable layout and planning of freight network
and distribution center, the route shortening and reduction of empty loading rate. HAN, S. (2009). Research on the construction of green logistics and supply chain system of agricultural
commodities in Henan Province. China Business and Trade, 8, 21-24. HUANG, F. H., & ZHOU, M. (2008). Analysis of the agricultural products logistics development in Hunan References 152 Vol. 5, No. 2; 2015 Journal of Management and Sustainability www.ccsenet.org/jms Province from the perspective of joint logistics. Issues in Agricultural Economy, 8, 90-93. Province from the perspective of joint logistics. Issues in Agricultural Economy, 8, 90-93. HUANG, F. H., & ZHOU, M. (2008). Analysis of the agricultural products logistics development in Hunan
Province from the perspective of joint logistics. Issues in Agricultural Economy, 8, 90-93. HUANG, F. H., & ZHOU, M. (2008). Analysis of the agricultural products logistics development in Hunan
Province from the perspective of joint logistics. Issues in Agricultural Economy, 8, 90-93. LI, C. X. (2009). Promoting the green logistics of agricultural products based on the construction of new rural. Jiangshu Commercial Forum, 8, 59-61. LI, C. X. (2009). The promotion strategy of green logistics of agricultural products. Hubei Social Science, 5,
94-97. LI, M. (2011). Analysis on the current situation of agricultural products logistics in Hunan Province. Jiangshu
Commercial Forum, 2, 75-76. LI, W. Q. (2010). The promotion strategy of green logistics of agricultural products in Guangxi Province based
on the ASEAN free trade area. Jiangshu Commercial Forum, 8, 87-89. LIU, C. J. (2011). Analysis on the security mechanism of the agricultural products logistics development in
Hunan Province. China Business & Trade, 10, 150-151. LU, L. Y. (2007). Discussion on the problems of green logistics of agricultural products in China. Anhui
Agricultural Sciences, 3, 853-854. LU, N. (2012). Discussion on the development situation of green logistics of agricultural products in China. Journal of Inner Mongolia agricultural university (social science edition), 3, 72-74. NIU, X. J. (2010). Discussion on the problem and countermeasures of the development of green logistics of
agricultural products in China. Logistics Technology, 7, 32-33, 43. PANG, Y., & WANG, Z. W. (2008). The research of the modern logistics system of agricultural products in
Hunan Province. China Logistics and Purchasing, 4, 74-75. SHENG, Y. (2009). The construction of modern logistics system of agricultural products in Hunan Province. Technological Development of Enterprise, 4, 118-120. WANG, W. W. (2010). The existing problem and countermeasures of green logistics of agricultural products in
China. Practice in Foreign Economic Relations and Trade, 10, 85-88. WANG, W., & LIU, X. T. (2010). Thoughts on improving the green logistics system of agricultural products in
Heilongjiang Province. Logistics Technology, 12, 8-10. WU, X. Y. (2008). Factor model analysis on the port logistics of both sides of Taiwan straits. Fujian Normal
University. References XU, H. L. (2011). Development and experience learning of the developed countries agricultural products green
logistics. Journal of Central University of Finance & Economics, 12, 70-74. XU, H. L. (2012). The discussion summary of the foreign agricultural products green logistics development. China Business and Market, 1, 28-32. YANG, L., & ZHANG, Y. Z. (2010). The construction of agricultural products logistics financial risk
management system based on factor analysis. Guangdong Agricultural Sciences, 4, 251-253. ZOU, X. M., & LI, Q. L. (2010). Research on agricultural products logistics of Hunan Province. Journal of
Yangtze University (natural science edition), 12, 78-81. Copyrights Copyright for this article is retained by the author(s), with first publication rights granted to the journal. Copyright for this article is retained by the author(s), with first publication rights granted to the journal. This is an open-access article distributed under the terms and conditions of the Creative Commons Attribution
license (http://creativecommons.org/licenses/by/3.0/). 153
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Evolution of Genome-Organizing Long Non-coding RNAs in Metazoans
|
Frontiers in genetics
| 2,020
|
cc-by
| 11,782
|
Evolution of Genome-Organizing
Long Non-coding RNAs in
Metazoans
América Ramírez-Colmenero 1, Katarzyna Oktaba 2 and Selene L. Fernandez-Valverde 1*
1 Unidad de Genómica Avanzada (Langebio), Centro de Investigación y de Estudios Avanzados del IPN, Irapuato, México,
2 Unidad Irapuato, Centro de Investigación y de Estudios Avanzados del IPN, Irapuato, México América Ramírez-Colmenero 1, Katarzyna Oktaba 2 and Selene L. Fernandez-Valverde 1*
1Unidad de Genómica Avanzada (Langebio) Centro de Investigación y de Estudios Avanzados del IPN Irapuato México Long non-coding RNAs (lncRNAs) have important regulatory functions across eukarya. It is now clear that many of these functions are related to gene expression regulation
through their capacity to recruit epigenetic modifiers and establish chromatin interactions. Several lncRNAs have been recently shown to participate in modulating chromatin within
the spatial organization of the genome in the three-dimensional space of the nucleus. The
identification of lncRNA candidates is challenging, as it is their functional characterization. Conservation signatures of lncRNAs are different from those of protein-coding genes,
making identifying lncRNAs under selection a difficult task, and the homology between
lncRNAs may not be readily apparent. Here, we review the evidence for these higher-order
genome organization functions of lncRNAs in animals and the evolutionary signatures
th
di
l Long non-coding RNAs (lncRNAs) have important regulatory functions across eukarya. It is now clear that many of these functions are related to gene expression regulation
through their capacity to recruit epigenetic modifiers and establish chromatin interactions. Several lncRNAs have been recently shown to participate in modulating chromatin within
the spatial organization of the genome in the three-dimensional space of the nucleus. The
identification of lncRNA candidates is challenging, as it is their functional characterization. Conservation signatures of lncRNAs are different from those of protein-coding genes,
making identifying lncRNAs under selection a difficult task, and the homology between
lncRNAs may not be readily apparent. Here, we review the evidence for these higher-order
genome organization functions of lncRNAs in animals and the evolutionary signatures
they display. Reviewed by: Reviewed by:
Daniel Vaiman,
Institut National de la Santé et de la
Recherche Médicale (INSERM), France
Sergey Razin,
Institute of Gene Biology (RAS), Russia Reviewed by:
Daniel Vaiman,
Institut National de la Santé et de la
Recherche Médicale (INSERM), France
Sergey Razin, Keywords: evolution, conservation, long-non-coding RNAs, chromatin conformation, three-dimensional
chromatin conformation, genome topology, gene expression regulation MINI REVIEW MINI REVIEW
published: 30 November 2020
doi: 10.3389/fgene.2020.589697 Keywords: evolution, conservation, long-non-coding RNAs, chromatin conformation, three-dimensional
chromatin conformation, genome topology, gene expression regulation INTRODUCTION *Correspondence:
Selene L. Fernandez-Valverde
selene.fernandez@cinvestav.mx The three-dimensional (3D) organization of DNA in the cell nucleus has become a significant
subject of study, particularly its influence on gene regulation. Recent advances in chromatin
conformation capture (3C) techniques, computational, and modeling approaches have made
its study feasible on a genome-wide scale, giving insight into the structure and the dynamics
of chromatin folding in space and time. Nuclear 3D organization has multiple levels and varies
between cell types and biological conditions. For instance, chromosomes are subdivided into
topologically associating domains (TADs) within which chromatin loops bring together regulatory
elements and target loci separated in the linear genome (Dixon et al., 2012). These chromatin
interactions are crucial for precise gene expression regulation (reviewed in Furlong and Levine,
2018; Schoenfelder and Fraser, 2019; Ibrahim and Mundlos, 2020). Importantly, changes in
transcriptional programs result in variation in chromatin interactions within TADs, while TAD
boundaries delimiting these domains are preserved (Dixon et al., 2015). TADs segregate in
the nuclear space into transcriptionally active (A) and inactive (B) compartments. A/B
compartments correlate well with histone modifications characteristic of euchromatin and
heterochromatin, respectively, and are described as cell type-specific, being able to undergo
switches during cell differentiation and lineage commitment (Lieberman-Aiden et al., 2009;
Rao et al., 2014; Dixon et al., 2015; Fortin and Hansen, 2015). Specialty section:
This article was submitted to
Epigenomics and Epigenetics,
a section of the journal
Frontiers in Genetics Specialty section:
This article was submitted to
Epigenomics and Epigenetics,
a section of the journal
Frontiers in Genetics
Received: 31 July 2020
Accepted: 09 November 2020
Published: 30 November 2020 Specialty section:
This article was submitted to
Epigenomics and Epigenetics,
a section of the journal
Frontiers in Genetics Received: 31 July 2020
Accepted: 09 November 2020
Published: 30 November 2020 Edited by:
Hehuang Xie,
Virginia Tech, United States Edited by:
Hehuang Xie,
Virginia Tech, United States Citation: Ramírez-Colmenero A, Oktaba K and
Fernandez-Valverde SL (2020)
Evolution of Genome-Organizing
Long Non-coding RNAs
in Metazoans. Front. Genet. 11:589697. doi: 10.3389/fgene.2020.589697 November 2020 | Volume 11 | Article 589697 1 Frontiers in Genetics | www.frontiersin.org Evolution of Genome-Organizing Animal lncRNAs Ramírez-Colmenero et al. These modes of conservation are not mutually exclusive and
may be present in a single lncRNA. In addition to DNA and histones, RNA is a major component
of the cell nucleus (Rinn and Chang, 2012). High-throughput
sequencing methods have revealed the pervasive transcription
of thousands of non-coding RNA (ncRNA) molecules in the
genome. Among the latter, long non-coding RNAs (lncRNAs)
have emerged as important gene regulators in eukaryotes. lncRNAs are broadly defined as transcripts longer than 200
nucleotides, with little to no protein-coding potential (Mercer
et al., 2009; Wang and Chang, 2011; Derrien et al., 2012). lncRNAs are more lowly expressed (Hezroni et al., 2015),
display more tissue-restricted expression patterns (Necsulea
et al., 2014), have fewer exons, and are shorter than protein-
coding genes (Hezroni et al., 2015). In animals, several lncRNAs
are essential to phenomena such as gene silencing, activation,
and
chromatin
remodeling,
with
significant
roles
in
development, immunity, and cancer (Guttman et al., 2011;
Schmitt and Chang, 2016; Delás et al., 2017). lncRNA functions
may predate the origin of metazoans, as several unicellular
holozans possess lncRNAs that are distinct in terms of their
histone marks as well as expression throughout their life
cycle (Gaiti et al., 2017). Beyond their apparent lack of conservation, many functionally
characterized lncRNAs modulate the organization of higher-
order chromatin structures in the nucleus (Saxena and Carninci,
2011; Marchese and Huarte, 2014). lncRNAs are involved in
the formation of DNA loops and domains (Wang and Chang,
2011; Zhang et al., 2014), interchromosomal structures
(Hacisuleyman et al., 2016), heterochromatic regions (Deng
et al., 2009; Engreitz et al., 2013), subnuclear bodies (Mao
et al., 2011), and the dynamic assembly of protein complexes
(Tsai et al., 2010; Lin et al., 2014; Marín-Béjar et al., 2017). Several novel experimental methods allow the identification
of lncRNAs binding to chromatin in vivo across the genome
(Li et al., 2017; Sridhar et al., 2017; Bell et al., 2018; Bonetti
et al., 2020; Gavrilov et al., 2020). Citation: Recruiting and binding to
effector molecules is a prevalent mode of action of lncRNAs
in both cis and trans activities.f Here, we summarize lncRNAs that affect, establish, or
maintain
three-dimensional
chromatin
organization
in
metazoans and the conservation signals that indicate they are
under selection. LNCRNAS THAT AFFECT TAD
CONFORMATION AND THEIR
CONSERVATION There has been a long debate on whether most lncRNAs are
functional or not (van Bakel et al., 2010; Clark et al., 2011;
Lindsay et al., 2013). This discussion was, in part, sparked by
the fact that the sequence of lncRNAs is generally poorly
conserved across species, suggesting that they are not under
purifying selection (Babak et al., 2005; Ponjavic et al., 2007;
Marques and Ponting, 2009). There are several examples of
orthologous RNAs that preserve their function, but whose
sequence is so divergent, they can no longer be identified as
orthologs by sequence similarity alone (Ponjavic et al., 2007;
Ulitsky et al., 2011; Ulitsky, 2016). Thus, the detection of
conservation beyond sequence is paramount to annotate candidate
lncRNAs for further functional characterization.hf Frontiers in Genetics | www.frontiersin.org Sequence Conservation Numerous ncRNAs are November 2020 | Volume 11 | Article 589697 2 Evolution of Genome-Organizing Animal lncRNAs Ramírez-Colmenero et al. A
B
C
D A
B
C
D
FIGURE 1 | Types of conservation and mechanism of action of example lncRNAs. Diagrams show exons (big filled boxes) and introns (colored links) of lncRNAs
genes. 5' and 3' UTRs are shown as light blue boxes in (C). (A) Sequence conservation: Some lncRNAs present high levels of sequence conservation (gray
shading). For example, the Metastasis Associated in Lung Adenocarcinoma Transcript 1 (MALAT1) lncRNA is highly conserved from human to zebrafish. Regions of
conservation are shown according to the “Vertebrate Multiz Alignment & Conservation” track of the UCSC genome browser. MALAT1 localizes to nuclear speckles,
nuclear bodies for co-transcriptional and post-transcriptional pre-mRNA processing. In humans, MALAT1 regulates the phosphorylation of serine/arginine splicing
factors, enriched at nuclear speckles. (B) Positional conservation: lncRNAs can have a conserved genomic position but very low sequence conservation. This is the
case for the roX lncRNAs in Drosophila, identified by a combination of synteny, microhomology, and secondary structure. roX1 (not shown) and roX2 spread to high-
affinity sites (HASs), landing regions of male-specific lethal (MSL) complex, in close spatial proximity, regulating local chromatin remodeling, leading to the increased
expression of genes for dosage compensation. (C) Structural conservation: lncRNAs can fold into a conserved secondary structure. The steroid receptor RNA
activator (SRA) gene produces both a protein and a lncRNA (ncSRA). A simplified representation of the structure of the human ncSRA, as determined by Novikova
et al. (2012), is depicted. ncSRA consists of four main domains, three of which are well-conserved at sequence across 36 vertebrate species and contain covariant
base pairs. Different segments of the structure have differences in sequence conservation, and specific helices are highly conserved. ncSRA binds to several
proteins including: trithorax group (TrxG), DEAD-box RNA helicase 5 (DDX5 or p68), and CCCTC-binding factor (CTCF), potentially acting as a scaffold for the
assembly of ribonucleoprotein complexes. (D) Functional convergence: lncRNAs with no common origin can have an equivalent function. The X-inactive specific
transcript (Xist) and RNA on the silent X (Rsx) lncRNAs act on the process of dosage compensation in different species. Both Xist and Rsx are expressed form the X
inactivation center (XIC) and are spread along the X chromosome to inactivate it. Sequence Conservation A
B A B B C C D FIGURE 1 | Types of conservation and mechanism of action of example lncRNAs. Diagrams show exons (big filled boxes) and introns (colored links) of lncRNAs
genes. 5' and 3' UTRs are shown as light blue boxes in (C). (A) Sequence conservation: Some lncRNAs present high levels of sequence conservation (gray
shading). For example, the Metastasis Associated in Lung Adenocarcinoma Transcript 1 (MALAT1) lncRNA is highly conserved from human to zebrafish. Regions of
conservation are shown according to the “Vertebrate Multiz Alignment & Conservation” track of the UCSC genome browser. MALAT1 localizes to nuclear speckles,
nuclear bodies for co-transcriptional and post-transcriptional pre-mRNA processing. In humans, MALAT1 regulates the phosphorylation of serine/arginine splicing
factors, enriched at nuclear speckles. (B) Positional conservation: lncRNAs can have a conserved genomic position but very low sequence conservation. This is the
case for the roX lncRNAs in Drosophila, identified by a combination of synteny, microhomology, and secondary structure. roX1 (not shown) and roX2 spread to high-
affinity sites (HASs), landing regions of male-specific lethal (MSL) complex, in close spatial proximity, regulating local chromatin remodeling, leading to the increased
expression of genes for dosage compensation. (C) Structural conservation: lncRNAs can fold into a conserved secondary structure. The steroid receptor RNA
activator (SRA) gene produces both a protein and a lncRNA (ncSRA). A simplified representation of the structure of the human ncSRA, as determined by Novikova
et al. (2012), is depicted. ncSRA consists of four main domains, three of which are well-conserved at sequence across 36 vertebrate species and contain covariant
base pairs. Different segments of the structure have differences in sequence conservation, and specific helices are highly conserved. ncSRA binds to several
proteins including: trithorax group (TrxG), DEAD-box RNA helicase 5 (DDX5 or p68), and CCCTC-binding factor (CTCF), potentially acting as a scaffold for the
assembly of ribonucleoprotein complexes. (D) Functional convergence: lncRNAs with no common origin can have an equivalent function. The X-inactive specific
transcript (Xist) and RNA on the silent X (Rsx) lncRNAs act on the process of dosage compensation in different species. Both Xist and Rsx are expressed form the X
inactivation center (XIC) and are spread along the X chromosome to inactivate it. Sequence Conservation Sequence conservation in lncRNAs can range from very high
to almost non-existent. Despite being generally presented as
poorly conserved, a subset of lncRNAs can present significant
sequence conservation across species (Necsulea et al., 2014;
Hezroni et al., 2015). However, sequence conservation does
not guarantee functional equivalence; a highly conserved lncRNA
can be fundamental in one species while dispensable in others. For example, the lncRNA Metastasis Associated in Lung
Adenocarcinoma Transcript 1 (MALAT1) is highly conserved
from human to zebrafish (Figure 1A; Hutchinson et al., 2007;
Lin et al., 2007). While the human MALAT1 functions in
nuclear speckles, regulating alternative splicing (Hutchinson
et al., 2007; Tripathi et al., 2010), cell-cycle associated genes
(Yang et al., 2011), and cancer progression (Gutschner et al., 2013),
the murine ortholog is neither essential for these functions nor
mouse development (Eißmann et al., 2012; Nakagawa et al., 2012;
Zhang et al., 2012). The conservation signals in lncRNAs can differ from those
typically found in protein-coding genes (Diederichs, 2014;
Ulitsky, 2016). For instance, conventional conservation analyses
applied to coding sequences, such as calculating the rate between
synonymous and non-synonymous mutations, are not suitable
for these elements. Nevertheless, lncRNAs display some sequence
conservation, generally in short sequence islands, potentially
due to selection constraints on sequences necessary for interacting
with other transcripts, proteins, or DNA (Kapusta and Feschotte,
2014; Quinn et al., 2016; Ulitsky, 2016). lncRNAs may also
display constraints on the post-transcriptional processing of
the transcript, leading to the conservation of splice sites across
different species (Nitsche et al., 2015; Ulitsky, 2016). lncRNAs
can also possess structural conservation – a constraint that
may not be readily detectable at the sequence level (Smith
et al., 2013; Tavares et al., 2019). Finally, lncRNAs can have
positional conservation, and be expressed from syntenic
loci despite having lost most or all sequence conservation. However, it is more common for lncRNAs to have short
conserved motifs or domains that are important for their
association with DNA or proteins that regulate chromatin
conformation. For example, lncRNAs that affect 3D genome
topology and arise from highly conserved syntenic loci, such
as the Hox clusters, display contrasting patterns of sequence
conservation compared to their protein counterparts in the
same cluster. Hox genes, organized in mammals in four clusters
(HoxA–HoxD), encode transcription factors crucial for patterning
along the anterior-posterior axis. Sequence Conservation Firre is also required
for the super-loop formation of the inactive X chromosome
(Xi), H3K27me3 deposition, and the localization of the Xi to
the perinuclear region (Yang et al., 2015; Barutcu et al., 2018).h The 3D architecture of TADs enables a group of multi-
exonic lncRNAs, termed immune gene-priming lncRNAs (IPLs),
to direct the active priming of the promoters of immune genes,
necessary for a rapid and robust pro-inflammatory response
as part of trained immunity (Fanucchi et al., 2019). Upon
induction of transcription of immune genes by the tumor
necrosis factor (TNF), chromatin contacts increase TNF-induced
genes and the lncRNAs loci. IPLs are somewhat conserved
between mouse and human; the majority possess an Alu element
in their first intron and share putative transcription-factor
binding motifs at their promoters. The region comprising an IPL, Upstream master lncRNA of
the inflammatory chemokine locus (UMLILO), engages in
chromosomal contacts with CXCL chemokine genes belonging
to the same TAD, but UMLILO does not have enhancer-RNA-
like characteristics. In contrast to other IPLs, UMLILO is not
conserved in mice and only partially conserved in pigs, suggesting
that IPLs are not essential across species, but have a
complementary role in ensuring robust gene expression. UMLILO
has short conserved sequence motifs and interacts with WDR5
through its conserved exon 3, directing WDR5/MLL1 to
chemokine gene promoters, mediating H3K4me3. Transcription
of chemokines in UMLILO knockdown cells was restored by
insertion of another WDR5-binding lncRNA, HOTTIP, under
the control of the UMLILO promoter (Fanucchi et al., 2019). The ability of HOTTIP to rescue the loss of UMLILO is an
example of convergent functional evolution, as they share
minimal sequence similarity. f
Another lncRNA expressed from HOX clusters is HOXA
transcript at the distal tip (HOTTIP), transcribed from the
5' end of the HOXA locus in mammals and conserved in
avians (Wang et al., 2011). Chromosomal looping brings
HOTTIP into spatial proximity to its target genes in cis,
allowing HOTTIP to activate transcription by binding the
WD repeat domain 5/mixed lineage leukemia (WDR5/MLL)
complex, driving H3K4me3 (Wang et al., 2011). HOTTIP and
its association with CCCTC-binding factor (CTCF), which
delineates active and inactive TADs within the HOXA cluster,
also influence the expression of HoxA genes (Narendra et al., 2015;
Wang et al., 2018). Another group of chromatin-modifying lncRNAs arises from
the syntenic estrogen receptor 1 (ESR1) locus. ESR1 is strongly
upregulated in cancerous cells undergoing estrogen deprivation. Sequence Conservation The HOX antisense intergenic RNA (HOTAIR) lncRNA is
transcribed from the boundary between domains with differential
chromatin marks at the HOXC locus but acts in trans repressing transcribed from the human HOX loci, and their expression
relates to differential histone marks and transcriptional
accessibility (Rinn et al., 2007). November 2020 | Volume 11 | Article 589697 Frontiers in Genetics | www.frontiersin.org 3 Evolution of Genome-Organizing Animal lncRNAs Ramírez-Colmenero et al. transcription of coding and non-coding genes on the HOXD
locus (Rinn et al., 2007). A chromatin loop established between
HOTAIR locus and the HOXC distal enhancer (HDE) located
downstream of HOTAIR promotes transcription of the lncRNA. This loop is disrupted by the recruitment of hepatocyte nuclear
factor 4-α (HNF4α), a master regulator of epithelial
differentiation, to the HDE (Battistelli et al., 2019). HOTAIR
exists across mammals, albeit poorly conserved in sequence;
it is only highly conserved in primates (He et al., 2011). Noteworthy, a highly conserved domain in exon 6, possibly
the backbone of HOTAIR, appeared first in kangaroos suggesting
the ab initio generation of HOTAIR in marsupials (He et al.,
2011). Despite its low sequence conservation across mammals,
key secondary structural elements of HOTAIR contain protein-
binding motifs and have significant conservation or covariation
(He et al., 2011; Somarowthu et al., 2015). However, studies
evaluating the functional conservation of murine HOTAIR
(mHotair) present contradictory results. On the one hand, the
deletion of the HoxC cluster, including mHotair, did not affect
HoxD silencing in vivo (Schorderet and Duboule, 2011). In
contrast, mice homozygous for mHotair KO presented homeotic
spine transformation and malformation of metacarpal bones,
and derived fibroblasts showed altered expression and levels
of epigenetic marks at hundreds of genes, including HoxD
genes (Li et al., 2013). Interestingly, human and mouse HOTAIR
differ in number, arrangement, and degree of sequence
conservation among their exons. The absence of exons with
protein-binding motifs in mHotair may partially explain
differences in their function. The RRD and other local repeats (LRs) are conserved to different
extents across Firre orthologs in mammals. Sequence Conservation A cluster of ncRNAs, ESR1 locus enhancing and activating
non-coding RNAs (Eleanors), are transcribed from introns in
a large chromatin cluster within a TAD that contains the
ESR1 locus (Tomita et al., 2015). These Eleanors form a
chromatin-associated RNA cloud that delineates the TAD and
cis-activate transcription. This TAD interacts with another
active TAD that contains the apoptotic transcription factor
forkhead Box O3 (FOXO3; Abdalla et al., 2019). Knockdown
of a promoter-associated Eleanor, pa-Eleanor(S), induced
repression of the rest of the Eleanors and the genes within
the TAD, including ESR1 (Abdalla et al., 2019). The abundant
and highly conserved Eleanor2 increases chromatin accessibility
in the ESR1 upstream region by destabilizing nucleosomes,
activating ESR1, and is required for the formation of the
RNA cloud (Fujita et al., 2020). Long non-coding RNAs also enable the establishment of
inter-chromosomal structures. The Functional intergenic
repeating RNA element (Firre) is a lncRNA involved in
pluripotency, hematopoiesis, and adipogenesis (Hacisuleyman
et al., 2014; Lewandowski et al., 2019). Firre accumulates
across a ~5 Mb domain around its transcription site on the
X chromosome (Hacisuleyman et al., 2014), located between
two TADs, and highly enriched in CTCF binding sites, required
for Firre transcription (Barutcu et al., 2018). This domain
colocalizes with five regions on different chromosomes that
contain genes with roles in adipogenesis. The formation of
this structure depends on the interaction of Firre with
Heterogeneous Nuclear Ribonucleoprotein U (HNRNPU),
through a 156-bp repeating RNA domain (RRD; Hacisuleyman
et al., 2014). This RRD is unique to Firre, and functions as
a lineage-specific nuclear retention signal in mice and humans. Frontiers in Genetics | www.frontiersin.org Positional Conservation Long non-coding RNAs may be expressed from syntenic loci,
suggesting a common origin, but may have lost the majority
of sequence conservation (Figure 1B). The functions of these
lncRNAs are thought to rely primarily on their transcription November 2020 | Volume 11 | Article 589697 4 Evolution of Genome-Organizing Animal lncRNAs Ramírez-Colmenero et al. (Diederichs, 2014; Ulitsky, 2016). Thus, the evolutionary signature
would be expected to reside outside the transcribed region
(Ulitsky, 2016). Indeed, many lncRNAs have a very conserved
promoter but little to no conservation in their transcribed
region (Guttman et al., 2009). A substantial difficulty in this
classification is defining when sequence conservation is entirely
lost. As outlined above, several lncRNAs only retain small
patches of conservation considered negligible by some authors
and meaningful by others. inactivation, interacts with proteins such as the polycomb
repressive complex 2 (PRC2; Zhao et al., 2008), ATRX chromatin
remodeler (Sarma et al., 2014), and SHARP repressor protein
(McHugh et al., 2015). RepA was experimentally shown to
have a complex structure that is preserved despite rapid
changes across mammalian evolution, strongly suggesting that
this structure is indispensable for Xist function (Liu et al.,
2017). lncRNAs involved in dosage compensation in
drosophilids, roX1 and roX2, have conserved boxes that
correspond precisely with stems that are necessary for binding
to the male-specific lethal (MSL) proteins. Domains outside
these interaction zones are not conserved and lack structure
(Ilik et al., 2013; Quinn et al., 2016). (Diederichs, 2014; Ulitsky, 2016). Thus, the evolutionary signature
would be expected to reside outside the transcribed region
(Ulitsky, 2016). Indeed, many lncRNAs have a very conserved
promoter but little to no conservation in their transcribed
region (Guttman et al., 2009). A substantial difficulty in this
classification is defining when sequence conservation is entirely
lost. As outlined above, several lncRNAs only retain small
patches of conservation considered negligible by some authors
and meaningful by others. Examples of this conundrum are dosage compensation lncRNAs
in Drosophila melanogaster (Figure 1B). Detailed syntenic analysis
of Drosophilid genomes revealed 47 new orthologs, where only
19 had been identified by sequence similarity (Quinn et al.,
2016). Importantly, it was shown that the roX RNA itself, only
its transcription, is necessary for dosage compensation (Quinn
et al., 2016). Furthermore, a distant roX RNA ortholog rescues
the loss of roX between two distant species (D. Positional Conservation melanogaster
and Drosophila busckii) despite almost no sequence conservation
outside an eight nucleotide-long conserved patch of microhomology
(Quinn et al., 2016). HOTAIR has also been shown to have a complex secondary
structure, with some evidence of conservation in mammals
acquired from computational methods (Somarowthu et al.,
2015). However, there is some debate as to whether there is
enough evidence to suggest that HOTAIR’s structure is conserved
in mammals (Rivas et al., 2017). Similarly, secondary-structure
predictions on Firre indicated that the RRD is a highly structured
domain (Nakagawa and Hirano, 2014), consistent with LRs
representing potential binding platforms for the specific targeting
of proteins to specific genomic regions by lncRNAs. A more traditional example of positional conservation is
the lncRNA antisense to Igf2r RNA non-coding (Airn), required
for paternal-specific silencing of imprinted genes in the
insulin-like growth factor 2 (Igf2r) cluster (Sleutels et al.,
2002). The function of Airn is conserved between human
and mouse despite them sharing little conserved sequence
(Yotova et al., 2008). The Igf2r silencing function of Airn
was shown to be dependent on transcriptional overlap and
not on the transcribed RNAs themselves (Latos et al., 2012). However, recent evidence shows that this is only the case
for nearby imprinted genes, as the murine Airn lncRNA
itself is necessary for the recruitment of chromatin-modifying
complexes to distant non-overlapping genes in the cluster
(Andergassen et al., 2019). Functional Convergence: The Case of
Dosage Compensation lncRNAsh g
p
The lncRNAs involved in the process of dosage compensation
are extraordinary examples of de novo emergence of novel
lncRNAs of unrelated evolutionary origins (Figure 1D). A
prominent example is the Xist lncRNA, required for dosage
compensation in the sex-chromosomes of eutherians (Penny
et al., 1996). Random X-chromosome inactivation in females
is necessary to balance the transcriptional output to that of
males. Xist localizes at the X inactivation center (XIC) and is
expressed exclusively from the inactivated X (Xi; Brown et al.,
1991). During the onset of X inactivation, Xist accumulates
at the XIC (Clemson et al., 1996), and then targets gene-rich
regions that are spatially close to its transcription site (Engreitz
et al., 2013; Simon et al., 2013), incorporating them into the
Xist silencing domain and spreading further to cover the
complete future Xi (Engreitz et al., 2013). Xist-mediated
inactivation involves the transcriptional silencing of most genes
on the Xi, and its compaction and recruitment to the nuclear
lamina (Zhao et al., 2008; Hasegawa et al., 2010; Chu et al.,
2015; McHugh et al., 2015; Minajigi et al., 2015). Frontiers in Genetics | www.frontiersin.org DISCUSSION lncRNAs are also the effectors of dosage compensation in
drosophilids, but they differ in both origin and mechanism to
those in mammals. Here, the roX1 and roX2 lncRNAs mediate
the upregulation of genes on the single male X chromosome
to equalize expression of the two X chromosomes in females. roX1 and roX2 associate to the MSL proteins, forming the MSL
complex that localizes to numerous specific sites along the male
X (Franke and Baker, 1999), mediating histone acetylation and
increasing transcription. The MSL complex does not alter the Distinctly, lncRNAs have emerged as an additional layer of
complexity
involved
in
shaping
the
three-dimensional
organization of the genome by interacting and modifying the
structure of chromatin. Several lncRNAs affect chromatin
conformation and display a combination of conservation signals
that may be difficult to identify solely by looking at traditional
genomic conservation metrics (summarized in Table 1). These
signatures could prove useful to identify and prioritize lncRNA TABLE 1 | Characterized long non-coding RNAs (lncRNAs) that are involved in nuclear genome topology. lncRNA
Function
Mode of
action
Interacting proteins
Association with chromatin
topology
Conservation
References
Xist
X chromosome
inactivation in mammals
In cis
PRC1, PRC2,
HNRNPU, RBM15,
SHARP, WTAP,
HNRNPK, LBR, and
many others
The organization of the XIC into two
topologically associating domains
(TADs) ensures the proper
interaction of Xist and its antisense
lncRNA Tsix with regulatory
elements
Present only in
eutherian mammals. Presence of common
core exonic
sequences, despite
species-specific
unique sequences,
and variation in length
and gene structure
Nesterova et al.,
2001; Plath et al.,
2003; Elisaphenko
et al., 2008; Zhao
et al., 2008;
Hasegawa et al.,
2010; Engreitz et al.,
2013; Chu et al.,
2015; McHugh et al.,
2015; Minajigi et al.,
2015; Moindrot et al.,
2015; Chen et al.,
2016; Pintacuda et al.,
2017; van Bemmel
et al., 2019; Galupa
et al., 2020
During X inactivation, Xist spreads
along the chromosome exploiting
the three-dimensional (3D)
organization, resulting in
compaction and recruitment to the
nuclear lamina
HOTTIP
Gene control of HOXA
locus for distal identity
In cis
WDR5/MLL and CTCF
A chromatin loop gets HOTTIP into
spatial proximity to HOXA genes. Structural Conservation Structural conservation is potentially the most telling signal
of conservation in lncRNAs, yet the most difficult to identify. The basic premise is that structural domains may be preserved
despite changes in the sequence, as long as complementary
base pairs are maintained.h The non-coding isoform of the steroid receptor RNA activator
(SRA), ncSRA, has a four-domain secondary structure with
varying levels of sequence conservation (Figure 1C). ncSRA
functions as a coactivator of several human hormone receptors
by modifying chromatin structure (Novikova et al., 2012). ncSRA associates with CTCF and the DEAD-BOX helicase 5
(DDX5), and this association is necessary for the insulator
activity of CTCF in vivo (Yao et al., 2010). The functional
RNA structure is conserved in all mammals, while its sequence
is not. Furthermore, several of the varying positions in other
species show changes predicted to help stabilize its structural
elements (Novikova et al., 2012). While exonic sequences of Xist are well-conserved among
eutherians, there are differences in the exon-intron structure,
length, and sequence between species (Nesterova et al., 2001;
Elisaphenko et al., 2008). This indicates that either Xist genes
present a high adaptation level or that their sequence and
structure are not essential (Elisaphenko et al., 2008). Xist is
not present in non-eutherian vertebrates, including marsupials,
despite common epigenetic features on the Xi, such as loss
of active histone marks and exclusion of RNA polymerase II
(Chaumeil et al., 2011). Homology of Xist with promoters and
exonic sequences of the protein-coding gene ligand of Dosage compensation lncRNAs (see next section) show
patches of structural conservation of biological importance. The Repeat A (RepA) region of X-inactive specific transcript
(Xist), essential to the establishment of X chromosome November 2020 | Volume 11 | Article 589697 5 Evolution of Genome-Organizing Animal lncRNAs Ramírez-Colmenero et al. numb-protein x 3 (Lnx3) found in marsupials, chicken, and
fish suggests that Xist emerged through pseudogenization of
Lnx3, possibly by the insertion of tandem repeats from
transposable elements (Duret et al., 2006; Elisaphenko et al., 2008). global architecture of the X chromosome, but it does spread
via spatial proximity from high-affinity sites – enriched at TAD
boundaries – to other regions (Ramírez et al., 2015). Structural Conservation Contrary
to Xist, whose activity is limited to the chromosome from which
it is expressed (Wutz and Jaenisch, 2000), roX transgenes target
the X chromosome in trans and rescue roX1 and roX2 mutant
males (Meller and Rattner, 2002).h Interestingly, in marsupials, X-chromosome inactivation is
imprinted, tissue-specific, and somewhat incomplete compared
to eutherians, and thought to be achieved by female-specific
expression of the lncRNA RNA on the silent X (Rsx), which
is transcribed from and coats the paternal chromosome (Grant
et al., 2012). The independent evolution of Xist and Rsx adds
to the notion of dosage systems rapidly evolving from ancient
silencing mechanisms common to all eukaryotes through the
use of lncRNAs (Gendrel and Heard, 2014; Graves, 2016). The
discoveries on the regulation of Xist by non-coding elements
located at its own and the neighboring TAD and the impact
of this 3D conformation on the regulatory landscape adds
another layer of complexity to the mechanisms for dosage
compensation (van Bemmel et al., 2019; Galupa et al., 2020). The independent origin of Xist in mammals, Rsx in marsupials,
and roX1 and roX2 in flies suggests that lncRNAs may be one
of the fastest mechanisms to evolve novel epigenetic controls. As these lncRNAs participate in dosage compensation but have
emerged
independently
in
several
lineages,
they
are
extraordinarily difficult to identify as functionally convergent. Additional examples of functionally equivalent lncRNAs with
no evolutionary relationship may likely have gone undetected. DISCUSSION Associates with CTCF to define
functional TADs at HOXA cluster
Portions conserved in
mammals and avians
Wang et al., 2011,
2018
Airn
Its transcription
prevents
overexpression of Igfr2
locus in a paternal-
specific matter
In cis
EHMT2
Forms an RNA cloud, creating a
repressive domain
Tandem direct
repeats at the CpG
island at 5' end are
conserved in human
and mouse at an
organizational level
but not by sequence
Lyle et al., 2000; Seidl
et al., 2006; Nagano
et al., 2008; Latos
et al., 2009, 2012;
Koerner et al., 2012;
Santoro et al., 2013
(Continued) TABLE 1 | Characterized long non-coding RNAs (lncRNAs) that are involved in nuclear genome topology. Frontiers in Genetics | www.frontiersin.org November 2020 | Volume 11 | Article 589697 6 Evolution of Genome-Organizing Animal lncRNAs Ramírez-Colmenero et al. DISCUSSION TABLE 1 | Continued
lncRNA
Function
Mode of
action
Interacting proteins
Association with chromatin
topology
Conservation
References
Kcnq1ot1
Silencing at imprinted
Kcnq1 locus in a
paternal-specific
manner
In cis
EHMT2, PRC2, PRC1,
and DNMT1
Formation of repressive chromatin
loop on the imprinting control region
of the locus
Well-conserved
motifs between
human and mouse
Pandey et al., 2004,
2008; Mancini-Dinardo
et al., 2006;
Mohammad et al.,
2008, 2010; Zhang
et al., 2014
pRNA
Mediates silencing by
CpG methylation of
rRNA genes at
nucleolus via DNA:RNA
triplex formation
In cis
NoRC and DNMT3b
Establishment of nucleolar
heterochromatin
Conserved across
eutharians, various
levels of sequence
conservation, and
highly conserved
secondary-structure
motifs
Mayer et al., 2006,
2008; Santoro et al.,
2010; Schmitz et al.,
2010;Guetg et al.,
2012; Jacob et al.,
2013; Savić et al.,
2014; Wehner et al.,
2014
LUNAR1
IGF1 signaling,
promotes cell
proliferation in cancers
In cis
None reported
A chromatin loop that brings into
contact the promoter of LUNAR1
and the enhancer of IFG1R is
necessary for the expression of
both genes, which reside in the
same TAD
Not reported
Trimarchi et al., 2014;
Peng and Feng, 2016
Khps1
Activates transcription
of its sense proto-
oncogene SPHK1, via
DNA:RNA triplex
formation at SPHK1
enhancer
In cis
EP300
Khps1 transcription leads
to a transcriptionally active
open chromatin state by
recruitment of EP300/CBP,
transcripton of KHPS1
enhancer and eviction
of CTCF
Conservation
between humans and
rodents
Imamura et al., 2004;
Postepska-Igielska
et al., 2015; Blank-
Giwojna et al., 2019
UMLILO
Trained immune
response on chemokine
genes
In cis
WDR5/MLL
In chemokine TAD, chromosomal
looping brings the super-enhancer
region harboring UMLILO into
contact with chemokine genes,
allowing UMLILO RNA to
guide WDR/MLL to the
promoters to facilitate
H3K4me3 epigenetic
priming
Partial conservation
between human,
chimpanzee, and pig,
absent in mouse
Fanucchi et al., 2019
Eleanors
Activation of ESR1
locus, apoptosis
resistance
In cis
None reported
Eleanors RNA cloud
delineate ESR1 TAD
and
activate transcription
Varying levels of
conservation for each
Eleanor
Tomita et al., 2015;
Abdalla et al., 2019;
Fujita et al., 2020
HOTAIR
Represses expression
in HOXD locus and
other genes, including
imprinted
In trans
PRC2, RCOR1, and
AR
HOTAIR transcripts demarcate silent
and active domains in HOXD locus. Partial conservation
between human,
chimpanzee, and pig,
absent in mouse
Fanucchi et al., 2019 Varying levels of
conservation for each
Eleanor Conservation
between humans and
rodents
Imamura et al., 2004;
Postepska-Igielska
et al., 2015; Blank-
Giwojna et al., 2019 DISCUSSION Poorly conserved by
sequence, secondary
structure motifs
conserved between
mouse and human
Rinn et al., 2007;
Gupta et al., 2010;
Tsai et al., 2010; Li
et al., 2013;
Somarowthu et al.,
2015; Zhang et al.,
2015; Portoso et al.,
2017
ncSRA
Activation of steroid
receptors (isoforms of
SRA code for protein)
In trans
SRC-1, PRC2, TrxG,
NANOG, CTCF,
SHARP, DDX5, and
others
Diverse, chromatin looping and
modification as scaffold for proteins
in both active and inactive domains
Significant sequence
conservation and
high structural
conservation
Lanz et al., 1999; Shi
et al., 2001; Zhao
et al., 2007; Yao et al.,
2010; Novikova et al.,
2012;
Wongtrakoongate
et al., 2015
roX1 and
roX2
Dosage compensation
in Drosophila
In trans
MSL Proteins
The MSL complex (rox + MSL
proteins) has high affinity sites on
There are roX
orthologs across
Franke and Baker,
1999; Park et al.,
2008; Ilik et al., 2013;
Maenner et al
2013; SRC-1, PRC2, TrxG,
NANOG, CTCF,
SHARP, DDX5, and
others Diverse, chromatin looping and
modification as scaffold for proteins
in both active and inactive domains The MSL complex (rox + MSL
proteins) has high affinity sites on
TAD borders The MSL complex (rox + MSL
proteins) has high affinity sites on
TAD borders November 2020 | Volume 11 | Article 589697 Frontiers in Genetics | www.frontiersin.org Evolution of Genome-Organizing Animal lncRNAs Ramírez-Colmenero et al. lncRNA
Function
Mode of
action
Interacting proteins
Association with chromatin
topology
Conservation
References
IPW
Repression of
maternally expressed
genes. Possibly
implicated in Prader-
Willi syndrome
In trans
EHMT2
Allele-specific formation of
heterochromatin at DLK1–DIO3
region. Poorly conserved by
sequence between
human and mouse
Wevrick et al., 1994;
Wevrick and Francke,
1997; Stelzer et al.,
2014
Firre
Role in adipogenesis,
nuclear architecture,
inflammatory response
(in vitro), and
hematopoiesis (in vivo)
In trans, but
occupies
domain in cis
HNRNPU
Firre acts as a scaffold for the
formation of an inter-chromosomal
structure. Locates at border of TAD
in a CTCF binding region. Required
for super loop formation of inactive
X
Conservation across
mammals, high
convergence of
repeating domain in
primates. FUNDING AR-C was funded by the Consejo Nacional de Ciencia y
Tecnología (CONACYT) M.Sc. fellowship. KO and SF-V were
funded by the Newton Advanced Fellowship (No. NAF\
R1\180303) awarded to SF-V. AR-C was funded by the Consejo Nacional de Ciencia y
Tecnología (CONACYT) M.Sc. fellowship. KO and SF-V were
funded by the Newton Advanced Fellowship (No. NAF\
R1\180303) awarded to SF-V. In the context of studying novel lncRNAs, its unique
conservation signatures, albeit more difficult to detect, are DISCUSSION Local
Repeats are conserved
between species of
the same order
Hacisuleyman et al.,
2014, 2016; Yang
et al., 2015; Lu et al.,
2017; Barutcu et al.,
2018; Lewandowski
et al., 2019
TERRA
Implicated in telomeric
and subtelomeric
heterochromatin
formation, stability and
maintenance
In cis (to
telomeres) and
in trans
Shelterin components
(TERF1 and TERF2),
ORC1, CBX5 NoRC,
ATRX, POT1, and
others
TERRA transcription depends on
chromosome looping
Telomere transcription
is conserved across
vertebrates and
Saccharomyces
cerevisiae
Azzalin et al., 2007;
Luke et al., 2008;
Schoeftner and
Blasco, 2008; Deng
et al., 2009;
Postepska-Igielska
et al., 2013; Beishline
et al., 2017
TABLE 1 | Continued candidates for experimental functional characterization. Sequence
conservation can be identified using traditional computational
sequence comparison methods. Recent examples have shown
that conserved sequence stretches can be much shorter in
lncRNAs than in protein-coding sequences, highlighting the
need to look for tiny stretches of sequence conservation
(microhomology; Quinn et al., 2016). Positional conservation
of lncRNAs can be identified using multiple genome alignments
complemented with transcriptomic data that support the existence
of non-coding transcripts in multiple taxa. The detection of
splice site conservation uses a similar approach but focuses
on identifying splice sites via modeling or direct RNA-seq
evidence, followed by comparison across taxa (Nitsche et al.,
2015). In the case of structural conservation, covariation
signatures in multiple sequence alignments may indicate the
conservation of a structure (Nawrocki et al., 2009; Gruber
et al., 2010; Will et al., 2012). One of the most significant
limitations is the difficult problem of distinguishing covariation
from sequence conservation. Thus, these methods can better
identify conserved structures in highly varying sequences in
diverse and multiple taxa (Rivas et al., 2017, 2020). excellent ways to identify potentially functional lncRNA
candidates and give a first insight on their possible mechanisms
of action. They can also help guide the search for homologous
mechanisms in other species. Complementing in silico studies
with experimental approaches in the context of spatiotemporal
gene expression programs is crucial to further assess the impact
of these ncRNAs on modulating genome architecture, including
their specific contribution to the complexity and evolution of
animal gene regulation. AUTHOR CONTRIBUTIONS All authors participated in writing and reviewing the manuscript
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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,
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Surface Plasmon Resonance Analysis Shows an IgG-Isotype-Specific Defect in ABO Blood Group Antibody Formation in Patients with Common Variable Immunodeficiency
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Frontiers in immunology
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Michael B. Fischer 1,2, Wendelin Wolfram 1, Christoph J. Binder 3, Georg A. Böhmig 4,
Markus Wahrmann 4, Martha M. Eibl 5 and Hermann M. Wolf 5* Michael B. Fischer 1,2, Wendelin Wolfram 1, Christoph J. Binder 3, Georg A. Böhmig 4,
Markus Wahrmann 4, Martha M. Eibl 5 and Hermann M. Wolf 5* 1 Department of Transfusion Medicine, Medical University of Vienna, Vienna, Austria, 2 Center of Biomedical Technology,
Danube University Krems, Krems an der Donau, Austria, 3 Department of Laboratory Medicine, Medical University of Vienna,
Vienna, Austria, 4 Division of Nephrology and Dialysis, Department of Medicine III, Medical University of Vienna, Vienna,
Austria, 5 Immunology Outpatient Clinic, Vienna, Austria Background: Common variable immunodeficiency (CVID) is the most common clinically
severe primary immunodeficiency and comprises a heterogeneous group of patients
with recurrent severe bacterial infections due to the failure to produce IgG antibodies
after exposure to infectious agents and immunization. Diagnostic recommendations for
antibody failure include assessment of isoagglutinins. We have readdressed this four
decades old but still accepted recommendation with up to date methodology. Keywords: blood groups, isoagglutinins, common variable immunodeficiency, B cell subsets, anti-ABO antibodies,
pneumococcal polysaccharide, natural antibodies Edited by:
Luigi Daniele Notarangelo,
Harvard Medical School, USA Edited by:
Luigi Daniele Notarangelo,
Harvard Medical School, USA Luigi Daniele Notarangelo,
Harvard Medical School, USA
Reviewed by:
Luigi Daniele Notarangelo,
Harvard Medical School, USA
Emily Mace,
Baylor College of Medicine, USA
*Correspondence:
Hermann M. Wolf,
Immunology Outpatient Clinic,
Schwarzspanierstrasse 15, Vienna
1090, Austria
hermann.wolf@itk.at Reviewed by:
Luigi Daniele Notarangelo,
Harvard Medical School, USA
Emily Mace,
Baylor College of Medicine, USA Methods: Anti-A/B IgM- and IgG-antibodies were measured by Diamed-ID Micro Typing,
surface plasmon resonance (SPR) using the Biacore® device and flow cytometry. *Correspondence:
Hermann M. Wolf,
Immunology Outpatient Clinic,
Schwarzspanierstrasse 15, Vienna
1090, Austria
hermann.wolf@itk.at Results: When Diamed-ID Micro Typing was used, CVID patients (n = 34) showed IgG-
and IgM-isoagglutinins that were comparable to healthy volunteers (n = 28), while all XLA
patients (n = 8) had none. Anti-A/B IgM-antibodies were present in more than 2/3 of the
CVID patients and showed binding kinetics comparable to anti-A/B IgM-antibodies from
healthy individuals. A correlation could be found in CVID patients between levels of anti-
A/B IgM-antibodies and levels of serum IgM and PnP–IgM-antibodies. In contrast in CVID
patients as a group ABO antibodies were significantly decreased when assessed by SPR,
which correlated with levels of switched memory, non-switched memory and naïve B
cells, but all CVID patients had low/undetectable anti-A/B IgG-antibodies. Specialty section:
This article was submitted to Primary
Immunodeficiencies, a section of the
journal Frontiers in Immunology Received: 29 January 2015
Accepted: 17 April 2015
Published: 06 May 2015 Conclusion: These results indicate that conventional isoagglutinin assessment and
assessment of anti-A/B IgM antibodies are not suited for the diagnosis of impaired
antibody production in CVID. Examination of anti-A/B IgG antibodies by SPR provides
a useful method for the diagnosis of IgG antibody failure in all CVID patients studied, thus
indicating an important additional rationale to start immunoglobulin replacement therapy
early in these patients, before post-infectious sequelae develop. Edited by: Edited by:
Luigi Daniele Notarangelo,
Harvard Medical School, USA ORIGINAL RESEARCH
published: 06 May 2015
doi: 10.3389/fimmu.2015.00211 CVID Patients and Healthy Controls y
Thirty-four patients with CVID diagnosed according to the cri-
teria established by the IUIS expert committee (5) were included
in the study after informed consent was obtained. The immuno-
logical characterization of the patients is depicted in Table 1. The study was conducted according to guidelines of the Medical
University of Vienna (MUW) and was approved by the ethic
commission of the MUW. The patients gave their informed
consent that anonymized data collected as part of the routine
medical attendance the patients received could be included in
a scientific publication. All results presented in this study were
obtained as part of the routine medical attendance the patients
received, no extra venipuncture was performed on the basis of this
study. Serum samples of CVID patients were drawn before regular
intravenous IVIG substitution therapy was started. Twenty-eight
healthy adult blood donors and eight X-linked primary antibody
deficiency (XLA) patients with no B cells and complete absence
of immunoglobulins of all isotypes due of a defect in bruton’s
tyrosine kinase (btk) served as positive and negative controls
(individuals with blood group AB were not included in the study). The distribution of ABO blood group among the controls (blood
group [N]: O [16], A [7], B [5], AB [0]) was not significantly
different as compared to the CVID patients (Table 1; P = 0.1, Chi-
squared analysis). All serum samples were immediately separated
from peripheral blood, dispensed into 500 µl aliquots and stored
until analysis at –30°C. IgG from plasma of eight healthy subjects
with known history of high anti-A/B antibodies was depleted
using a protein A column first and subsequently a Protein G
column (Enchant™IgG purification and depletion Kit, Pall Life
Science, Port Washington, NY, USA) in order to obtain IgM
antibodies only. The amount of IgM as well as IgG contamination
was determined by ELISA. Early IUIS, ESID/PAGID, and recent ESID diagnostic cri-
teria of CVID include poor antibody response to polypeptide
and polysaccharide vaccines and absence of anti-blood group
A (anti-A) and B (anti-B) antibodies as equivalent criteria (3–
5). However, studies addressing the question whether CVID
patients have defective production of isoagglutinin antibodies
examined in an isotype-specific manner are scarce. The immuno-
logically active epitopes of blood group ABO antigens share
carbohydrate moieties (13). Citation: Fischer MB, Wolfram W, Binder CJ,
Böhmig GA, Wahrmann M, Eibl MM
and Wolf HM (2015) Surface plasmon
resonance analysis shows an
IgG-isotype-specific defect in ABO
blood group antibody formation in
patients with common variable
immunodeficiency. Front. Immunol. 6:211. doi: 10.3389/fimmu.2015.00211 May 2015 | Volume 6 | Article 211 1 Frontiers in Immunology | www.frontiersin.org Anti-A/B antibodies in CVID Fischer et al. Introduction Common variable immunodeficiency (CVID) is a heterogeneous
disorder belonging to the group of primary antibody deficien-
cies, characterized by markedly reduced serum levels of IgG,
IgA, and often IgM, and a substantial failure to produce specific
IgG antibodies after vaccination or exposure to foreign antigens
(1–5). This renders patients susceptible to recurrent sinopul-
monary, ear, and gastrointestinal infections, the underlying reg-
ulatory immune defect can lead to autoimmune diseases, and
certain malignancies may occur more frequently. Several mono-
genetic defects including homo/heterozygous mutations in ICOS
(inducible costimulator of activated T cells), TACI (transmem-
brane activator and calcium-modulator and cyclophilin ligand
interactor), BAFF-R (B cell activating factor receptor), CD19,
CD81, and Msh5 were found in CVID patients, but genetics
of CVID are complicated by unaffected relatives that carry the
same mutations showing that the genotype–phenotype correla-
tion can be variable (6). For the majority of CVID patients, how-
ever, the underlying molecular defect has not yet been defined. Immunological abnormalities including defects in antigen pre-
sentation by monocytic cells and perturbed differentiation, matu-
ration, and function of DCs and invariant NK (iNK) cells were
described (7, 8). In addition to intrinsic B cell defects (9, 10)
functional abnormalities in T cells which can potentially lead
to defects in memory B cell compartment were described, and
included impaired early signaling events, production of cytokines
and expression of activation markers after antigenic stimula-
tion (11, 12). CVID Patients and Healthy Controls Blood group A/B specific antibod-
ies are produced by all individuals with a functional immune
system and are included under the broad classification of ‘nat-
ural antibodies’ in humans (14). They appear first in infants
at the age of 3–6 month when the developing immune system
can react with microorganisms and environmental antigens that
have carbohydrate epitopes similar to the blood group ABO
antigens (13, 14). The aim of the present study was to inves-
tigate whether freshly diagnosed CVID patients, who hitherto
had not received IVIG treatment, show a defect in anti-A/B
IgG or IgM antibodies. We used three different techniques, the
DiaMed-ID Micro Typing System, a blood group ABO flow
cytometry based assay (15), and the surface plasmon resonance
technology (SPR) using the Biacore® device and synthetic AB
trisaccharides (16–18) to study the blood group anti-A/B IgG
and IgM antibody titer in an isotype-specific assay and the
on/off rates of the binding of the respective antibodies to defined
blood group A/B trisaccharides in CVID patients and healthy
individuals. Determination of Anti-A/B Antibodies by the
DiaMed-ID Micro Typing System Anti-A/B antibodies were determined by the Diamed-ID Micro
Typing System (DiaMed AG, Cressier, Switzerland) according to
the manufacturer’s protocol. The ID-Card 50520 (NaCl) was used
to measure IgM, while the ID-Card 50531 (LISS/Coombs con-
taining polyspecific anti-human gammaglobulin [AHG] serum)
could determine IgG in addition to IgM. The serum samples were
serially diluted with 0.9% saline solution starting at 1:2, 1:4, until
1:265, and 25 µl of each dilution was pipetted into a single column
of the microtube gel card together with 50 µl of DiaMed ID-
DiaCell ABO/I–II test cell suspensions A1 or B (DiaMed AG). The
ID-Cards (NaCl) were subsequently incubated at room temper-
ature for 15 min, the ID-Cards (LISS/Coombs) at 37°C and then
centrifuged in a DiaMed ID-Centrifuge 24 s for 10 min at 910 rpm. Results were read immediately after centrifugation, the titer that
led to visible agglutination of erythrocytes dispersed in the gel was
selected positive. If an individual showed both anti-A and anti-
B antibodies then the higher titer of the two values was used for
statistical analysis. May 2015 | Volume 6 | Article 211 Frontiers in Immunology | www.frontiersin.org 2 Anti-A/B antibodies in CVID Fischer et al. Fischer et al. TABLE 1 | Immunological characterization of CVID patients. TABLE 1 | Immunological characterization of CVID patients. Patient
number
Sex
Agea
ABO blood
group
Serum immunoglobulins
(mg/dl)a
PnP antibodies
(reciprocal titer)b
CD19 +
(% ly)c
% of CD19-positive
lymphocytes
IgG
IgA
IgM
PnP–IgG
PnP–IgM
CD27+
IgD−
CD27+
IgD+
CD27−
IgD+
1
m
29
A
223
6
5
<20
<20
6
1,48
5,33
84,68
2
m
40
A
86
<6
12
<20
<20
6
4,75
6,27
83,17
3
m
47
A
160
6
4
21
<20
7
4,67
20,46
70,29
4
f
36
O
48
<7
<6
<20
<20
8
0,38
0,98
94,03
5
f
52
n.a. <7
<6
12
<20
<20
4
n.a. n.a. n.a. 6
f
48
n.a. 12
6
8
<20
<20
14
1,60
29,73
66,40
7
m
27
B
22
<6
<5
<20
<20
0
n.a. n.a. n.a. 8
f
49
O
94
6
23
<20
<20
5
0,66
4,80
91,95
9
f
29
O
169
<8
<7
63
<20
15
n.a. n.a. n.a. 10
f
54
O
199
<8
49
63
35
18
1,63
16,86
79,52
11
f
34
B
239
<8
47
<20
40
12
2,49
11,77
84,42
12
m
39
A
111
<7
<6
n.a. n.a. Determination of Anti-A/B Antibodies by the
DiaMed-ID Micro Typing System aAt time of diagnosis, before the initiation of immunoglobulin replacement therapy. bSerum IgG- and IgM-antibodies against 23-valent pneumococcal polysaccharides (PnP) were determined by ELISA before the initiation of immunoglobulin replacement therapy. cPercentage of peripheral blood CD19-positive lymphocytes as determined by flow cytometry. D t
t
il bl Determination of Anti-A/B Antibodies by the
DiaMed-ID Micro Typing System 9
n.a. n.a. n.a. 13
m
10
O
365
<5
17
<20
20
14
1,94
9,28
85,78
14
m
39
O
288
7
6
99
<20
13
1,60
41,62
49,28
15
m
50
O
<158
59
171
<20
49
18
n.a. n.a. n.a. 16
f
30
O
179
36
252
39
121
8
0,68
48,39
49,26
17
f
57
A
204
11
34
<20
30
20
14,52
17,86
62,60
18
m
33
A
84
<7
9
<20
<20
20
0,39
1,68
96,69
19
m
64
A
89
11
11
<20
36
3
n.a. n.a. n.a. 20
m
32
A
9
6
4
<20
<20
13
n.a. n.a. n.a. 21
f
44
A
219
44
26
<20
<20
5
n.a. n.a. n.a. 22
f
57
A
197
<8
<7
n.a. n.a. 6
n.a. n.a. n.a. 23
m
20
A
<148
<8
<6
<20
<20
5
1,80
6,10
87,02
24
m
42
O
60
<7
<4
<20
<20
15
8,91
29,57
59,65
25
m
13
A
76
<8
50
<20
<20
14
n.a. n.a. n.a. 26
m
27
A
<148
<8
41
39
<20
11
1,98
28,37
62,33
27
m
25
O
10
<6
8
<20
<20
13
0,77
25,32
73,26
28
f
10
O
383
356
76
<20
<20
5
n.a. n.a. n.a. 29
m
50
A
<7
<6
<5
<20
<20
3
2,75
9,89
84,07
30
m
19
O
210
<6
39
<20
<20
12
0,93
4,34
91,09
31
f
21
O
295
46
30
<20
117
10
1,35
7,72
89,37
32
m
22
A
154
13
31
n.a. n.a. 10
1,83
14,05
83,28
33
f
47
A
201
9
33
<20
50
13
1,90
15,81
80,98
34
m
17
O
<7
<6
<5
<20
<20
0
n.a. n.a. n.a. Normal
range
815–1784
93–287
108–237
>200
>100
7–23
9–36
6–32
37–77
aAt time of diagnosis, before the initiation of immunoglobulin replacement therapy. bSerum IgG- and IgM-antibodies against 23-valent pneumococcal polysaccharides (PnP) were determined by ELISA before the initiation of immunoglobulin replacement therapy. cPercentage of peripheral blood CD19-positive lymphocytes as determined by flow cytometry. n.a., Data not available. aAt time of diagnosis, before the initiation of immunoglobulin replacement therapy. bSerum IgG- and IgM-antibodies against 23-valent pneumococcal polysaccharides (PnP) were determined by ELISA before the initiation of immunoglobulin replacement therapy. cPercentage of peripheral blood CD19-positive lymphocytes as determined by flow cytometry. n.a., Data not available. Determination of Anti-A/B Antibodies by Surface
Plasmon Resonance Antibodies by Surface Plasmon Resonance
To measure anti-A/B IgG and IgM antibodies, crude human
serum was diluted by half with the HBS-EP and 100 µl of
the diluted plasma samples were passed through FC-1 and
FC-2 at a flow rate of 20 µl/min for 5 min. To discriminate
between anti-A/B IgM and IgG response, anti-human IgG Abs
(Coombs serum, Diamed) were injected first after the associ-
ation phase was finished, followed by injection of anti-human
IgM mAbs (Sigma Aldrich). The increase in RU following
the injection was indicative for the amount of IgG or IgM
bound to the synthetic trisaccharides. If an individual showed
both anti-A and anti-B IgG or IgM antibodies then the speci-
ficity with the higher titer (anti-A or anti-B) of the respec-
tive immunoglobulin isotype was used for statistical analysis. Before analysis was started, the binding of commercially avail-
able anti-blood group A/B antisera (anti-A murine monoclonal
Abs clone MH04 and A3D3, anti-B murine monoclonal Abs
clone NB1.19, NB10.5A5, and NB10.3B4, Ortho-Clinical Diag-
nostics, Neckargmünd, Germany) to the respective trisaccha-
rides was measured in order to examine chip performance. Serial dilutions of anti-blood group A mAbs (IgG clone 9A,
Abcam, Cambridge, MA, USA) and anti-blood group A/B anti-
sera (Ortho-Clinical Diagnostics) were performed to generate a
dose-dependent curve. The erythrocyte aggregation assay based on the Diamed-ID Micro
Typing System as well as the flow cytometric method to quantify
binding of anti-A/B IgG or IgM antibodies to fixed human A or
B red blood cells (ABO-FACS) are semi-quantitative assays that
lack sensitivity and specificity to precisely rank anti-A/B IgG and
IgM antibodies according to their strength of reaction with the
corresponding blood group A/B trisaccharide antigens. Therefore,
we used SPR to measure the association rate (ka) of the anti-
A/B IgG and IgM antibodies to the immobilized synthetic blood
group A or B trisaccharides and their dissociation rate (kd) on a
Biacore X device (16–18). We used the dextran hydrogel layer on
the CM-5 sensor chip to form a hydrophilic environment for the
attachment of blood group A or B trisaccharide amine derivative,
preserving the blood group antigens in a non-denatured state (18). This allows for measurement of antigen–antibody binding under
almost physiological conditions. Amine-labeled blood group A
and B trisaccharides (Dextra Laboratories Ltd., Reading, UK)
were immobilized on CM5 sensor chips (Biacore, GE Health-
care) according to standard protocols (16). Determination of Anti-A/B Antibodies by Surface
Plasmon Resonance In brief, the CM-5
sensor chip surface was activated with 100 µl of 0.05 mol/l N-
hydroxysuccinimide (Sigma Aldrich, St. Louis, MO, USA) and
0.2 mol/l N-ethyl-N’-dimethylamino propylcarbodiimide (Sigma
Aldrich) injected into the buffer stream of HBS-EP (0.01 mol/l
HEPES buffer, pH 7.4, containing 0.15 mol/l NaCl, 3 mmol/l
EDTA, and 0.005% surfactant P20). The buffer stream was passed
through FC-1 and FC-2 of the Biacore X instrument (GE Health
Care, Uppsala, Sweden) at a flow rate of 5 µl/min for 20 min. Subsequently, 15 µl of a blood group A/B trisaccharide amine
derivative solution at a concentration of 1 mg/ml, prepared with
borate (pH 8.5), was injected into the buffer stream and passed
through FC-1 at a flow rate of 5 µl/min and was halted after
injection of 7 µl for 2 h. Residual active ester groups on the sen-
sor surface were then deactivated by injecting 100 µl of 1 mol/l
ethanolamine-HCl (pH 8.5) into the buffer stream and passing it
through FC-1 and FC-2, and by passing the buffer stream through
the cell until a stable baseline was achieved. To measure anti-
A/B antibodies, crude human serum was diluted by half with the
HBS-EP and 100 µl of the diluted plasma samples were passed
through FC-1 and FC-2 at a flow rate of 20 µl/min for 5 min. When the association phase was finished, the flow cell was washed
for 20 s with the HBS-EP buffer and dissociation was recorded
in resonance units (RU) at a flow rate of 20 µl/min for 20 min. The chip was regenerated after each measurement in order to
reach the same baseline level as prior to measurement by remov-
ing bound antibody with 50 µl of 50 mmol/l NaOH buffer at a
flow rate of 60 µl/min. To establish a calibration curve for anti-
A/B antibody concentration, BIAevaluation software (General
Electric Healthcare) was used. The amount of anti-A or anti-B
antibody that associated with the corresponding blood group A
or B trisaccharide immobilized on the sensor chip surface was
obtained by subtracting the FC-II value (RU) from the FC-I value
(RU). If an individual showed both anti-A and anti-B antibodies Measurement of Serum Immunoglobulins and
Serum Antibodies against Pneumococcal
Polysaccharides Serum concentrations of IgG, IgA, and IgM were deter-
mined by laser nephelometry using reagents purchased from
Siemens–Behring Division (Siemens Healthcare Diagnostics
GmbH, Vienna, Austria). Serum levels of IgG- and IgM-antibodies
against 23-valent pneumococcal capsular polysaccharide were
determined using an in-house produced ELISA detecting all
serotypes contained in the 23-valent PnP vaccine as previously
described (19, 20). Frontiers in Immunology | www.frontiersin.org Flow Cytometric Analysis of Patients’ Anti-A/B
Antibodies and B Cell Populations All analysis were performed on a BD FACSCanto™(Becton Dick-
inson, Franklin Lakes, NJ, USA) interfaced to BD FACSDiva™
software. Mean fluorescence intensity (MFI) was calculated and
represented the titer of anti-A/B IgG or IgM antibodies present in
the sample. If an individual sample showed both anti-A and anti-
B IgG and IgM antibodies then the higher MFI-titer of the two
values was used for statistical analysis. Lymphocyte subpopula-
tions and B cell subsets were analyzed in peripheral whole blood
by flow cytometry using standard protocols with commercially
available directly conjugated monoclonal antibodies (anti-CD19
PerCP, Becton Dickinson Austria Ges.m.b.H., Schwechat, Austria;
anti-IgD FITC, Becton Dickinson Austria Ges.m.b.H., anti-CD27
PE, eBioscience, Vienna, Austria) and a FACSCalibur (Becton Undiluted serum samples of CVID patients and healthy control
subjects were incubated for 30 min at 4°C with 1% solution of
fixed RBCs (15). Thereafter, cells were washed two times in
FACS buffer (Hank’s buffered salt solution with 0.1% BSA) and
bound anti-A/B antibody to test erythrocytes was measured by
flow cytometry using either a DyLight 649 conjugated affinity
purified Fab fragment goat anti-human IgM (Fc5µ fragment spe-
cific, 15 µg/ml, Jackson ImmunoResearch Ltd, Newmarket, UK)
or a DyLight 649 conjugated affinity purified F(ab′)2 fragment
rabbit anti-human IgG (Fcγ fragment specific, 60 µg/ml, Jackson
ImmunoResearch). A minimum of 10, 000 events was acquired. May 2015 | Volume 6 | Article 211 Frontiers in Immunology | www.frontiersin.org 3 Anti-A/B antibodies in CVID Fischer et al. then the higher of the two RU values was used for statistical
analysis. Dickinson Austria Ges.m.b.H.). Data analysis was performed
using CellQuest software (Becton Dickinson Austria Ges.m.b.H.). Dickinson Austria Ges.m.b.H.). Data analysis was performed
using CellQuest software (Becton Dickinson Austria Ges.m.b.H.). Results IgG- in addition to IgM-isoagglutinins, the isoagglutinin titers
detected were higher as compared to the NaCl-system, but still
no difference was observed between CVID patients and controls
(Figure 1A, right panel). Blood Group Anti-A/B Antibodies did not Differ
Between CVID Patients and Healthy Individuals
When Determined by Diamed-ID Micro Typing
When the amount of anti-A/B antibodies in samples of 34 CVID
patients, diagnosed according to the criteria established by the
IUIS expert committee (5), was determined by the Diamed-ID
Micro Typing System using the ID-Card 50520 (NaCl) to mea-
sure IgM-isoagglutinins, no difference could be found between
CVID patients and healthy individuals (Figure 1A, left panel). X-linked agammaglobulinemia (XLA) patients with a defect in
btk showed no detectable isoagglutinin antibodies (Figure 1A, left
panel). When samples were further analyzed in the ID-Card 50531
(LISS/Coombs containing polyspecific AHG serum) to determine Surface Plasmon Resonance Analysis Shows
Defective ABO Blood Group Antibody Formation
in CVID Patients In the erythrocyte aggregation assay based on the Diamed-ID
Micro Typing System, 4/28 (14%, NaCl system) and 3/28 (11%,
LISS/Coombs system) of the healthy volunteers had low to unde-
tectable (serum titer ≤1:2) isoagglutinin antibodies (data not
shown). To better detect anti-A/B antibodies, SPR technology
using synthetic blood group A/B trisaccharides coupled to the
CM-5 sensor chip by amine binding was employed (16–18). 0
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
Healthy controls
(n=27)
CVID (n=31)
Agamma (n=3)
anti-A/B antibodies (resonance units)
p=0,000087
0
1
2
3
4
5
6
7
Healthy controls
(n=28)
CVID (n=34)
Agamma (n=8)
IgM-isoagglutinins
(DiaMed-ID microtyping, ID-Card 50520, NaCl,
reciprocal serum titer)
0
2
4
8
16
32
64
128
n.s. IgM
Healthy controls
(n=28)
CVID (n=34)
Agamma (n=8)
IgG- and IgM-isoagglutinins
(DiaMed-ID microtyping, ID-Card 50531, LISS/Coombs,
reciprocal serum titer)
0
4
16
64
256
1024
4096
n.s. IgG + IgM
B
A
FIGURE 1 | Examination of anti-A/B antibodies in CVID patients,
patients with XLA and healthy controls using the Diamed-ID Micro
Typing System with the ID-Card 50520 (NaCl) to measure IgM
isoagglutinins [(A), left panel] or with the ID-Card 50531
(LISS/Coombs containing polyspecific AHG serum) to determine IgG-
in addition to IgM-isoagglutinins [(A), right panel] and surface
plasmon resonance (SPR) technology without identification of the
antibody isotype using synthetic blood group A/B trisaccharides
coupled to the CM-5 Biacore® chip by amine binding (B). Reciprocal
serum isoagglutinin titers [(A), left panel, IgM isoagglutinins; (A), right panel,
IgG and IgM isoagglutinins] and SPR resonance units (B) are depicted using
box plot diagrams, the median of the study group is represented by a cross,
the interquartile range (IQR) is represented by the box, 5- and 95-percentile
values are represented by the whiskers, and minimum and maximum values
are represented by circles. The 95% confidence interval for the median
calculated according to McGill et al. (21) is indicated by horizontal bars. The
non-parametric Mann–Whitney U-test (two-sided P value) was used for
statistical comparison between patients and healthy controls. 0
1
2
3
4
5
6
7
Healthy controls
(n=28)
CVID (n=34)
Agamma (n=8)
IgM-isoagglutinins
(DiaMed-ID microtyping, ID-Card 50520, NaCl,
reciprocal serum titer)
0
2
4
8
16
32
64
128
n.s. IgM
Healthy controls
(n=28)
CVID (n=34)
Agamma (n=8)
IgG- and IgM-isoagglutinins
(DiaMed-ID microtyping, ID-Card 50531, LISS/Coombs,
reciprocal serum titer)
0
4
16
64
256
1024
4096
n.s. Surface Plasmon Resonance Analysis Shows
Defective ABO Blood Group Antibody Formation
in CVID Patients IgG + IgM
A 0
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
Healthy controls
(n=27)
CVID (n=31)
Agamma (n=3)
anti-A/B antibodies (resonance units)
p=0,000087
B Agamma (n=3) FIGURE 1 | Examination of anti-A/B antibodies in CVID patients,
patients with XLA and healthy controls using the Diamed-ID Micro
Typing System with the ID-Card 50520 (NaCl) to measure IgM
isoagglutinins [(A), left panel] or with the ID-Card 50531
(LISS/Coombs containing polyspecific AHG serum) to determine IgG-
in addition to IgM-isoagglutinins [(A), right panel] and surface
plasmon resonance (SPR) technology without identification of the
antibody isotype using synthetic blood group A/B trisaccharides
coupled to the CM-5 Biacore® chip by amine binding (B). Reciprocal FIGURE 1 | Examination of anti-A/B antibodies in CVID patients,
patients with XLA and healthy controls using the Diamed-ID Micro
Typing System with the ID-Card 50520 (NaCl) to measure IgM
isoagglutinins [(A), left panel] or with the ID-Card 50531
(LISS/Coombs containing polyspecific AHG serum) to determine IgG-
in addition to IgM-isoagglutinins [(A), right panel] and surface
plasmon resonance (SPR) technology without identification of the
antibody isotype using synthetic blood group A/B trisaccharides
coupled to the CM-5 Biacore® chip by amine binding (B). Reciprocal serum isoagglutinin titers [(A), left panel, IgM isoagglutinins; (A), right panel,
IgG and IgM isoagglutinins] and SPR resonance units (B) are depicted using
box plot diagrams, the median of the study group is represented by a cross,
the interquartile range (IQR) is represented by the box, 5- and 95-percentile
values are represented by the whiskers, and minimum and maximum values
are represented by circles. The 95% confidence interval for the median
calculated according to McGill et al. (21) is indicated by horizontal bars. The
non-parametric Mann–Whitney U-test (two-sided P value) was used for
statistical comparison between patients and healthy controls. serum isoagglutinin titers [(A), left panel, IgM isoagglutinins; (A), right panel,
IgG and IgM isoagglutinins] and SPR resonance units (B) are depicted using
box plot diagrams, the median of the study group is represented by a cross,
the interquartile range (IQR) is represented by the box, 5- and 95-percentile
values are represented by the whiskers, and minimum and maximum values
are represented by circles. The 95% confidence interval for the median
calculated according to McGill et al. (21) is indicated by horizontal bars. The
non-parametric Mann–Whitney U-test (two-sided P value) was used for
statistical comparison between patients and healthy controls. Statistical Analysis The non-parametric Mann–Whitney U-test (two-sided P value)
was used for unpaired comparison of results obtained from
patients and healthy control subjects. A P value of <0.05 was
assumed to be statistically significant. The Bravais–Pearson-
correlation coefficient was calculated to determine a statisti-
cally significant correlation between two parameters at a level
of P < 0.05. Where results are depicted using box plot dia-
grams, the median of the study group is represented by a
cross, the interquartile range (IQR) is represented by the box,
5- and 95-percentile values are represented by the whiskers,
and minimum and maximum values are represented by cir-
cles. The 95% confidence interval for the median calculated
according to McGill et al. (21) is indicated by horizon-
tal bars. May 2015 | Volume 6 | Article 211 Frontiers in Immunology | www.frontiersin.org 4 Anti-A/B antibodies in CVID Fischer et al. Surface Plasmon Resonance Analysis Shows
Defective ABO Blood Group Antibody Formation
in CVID Patients May 2015 | Volume 6 | Article 211 Frontiers in Immunology | www.frontiersin.org 5 Anti-A/B antibodies in CVID Fischer et al. This technology uses a molecularly defined carbohydrate anti-
gen and allows for real-time analysis of molecular interactions
of unlabeled antigen and antibody, thus leaving the functional
structures of both interaction partners intact. Furthermore, the
amine-labeling technology allows immobilization with a con-
trolled orientation for the carbohydrates (18). Thus the sensitivity
of detection of anti-A/B antibodies is enhanced by SPR tech-
nology (16). Accordingly, in those healthy individuals where no
or very low detectable anti-A/B titer could be observed in the
DiaMed-ID Micro Typing System and no binding of anti-A/B
specific antibodies to erythrocytes could be detected in the flow
cytometry based assay (data not shown), SPR showed slight but
detectable binding (100–130 RU) of anti-A/B antibodies to blood
group A/B tri-saccharides, and 27/27 (100%) of healthy individ-
uals had anti-AB antibody titers above 100 RU (Figure 1B). In
contrast to the isotype-specific SPR assay employed in later exper-
iments, the isotype of the anti-A/B antibodies was not identified
in these analyses. 0
4
8
12
16
20
switched memory B cells
(CD27+IgD-, % of CD19+)
p = 0,0320
0
10
20
30
40
50
60
70
IgM memory B cells
(CD27+IgD+, % of CD19+)
p = 0,0253
0
10
20
30
40
50
60
70
80
90
100
0
500
1000
1500
anti-A/B antibodies (RU)
naive B cells
(CD27-IgD+, % of CD19+)
p = 0,0109
FIGURE 2 | Correlation between levels of anti-A/B antibodies as
detected by surface plasmon resonance using the Biacore® device
and levels of B cell subsets in CVID patients. Anti-A/B antibodies were
determined using surface plasmon resonance (SPR) technology with synthetic
blood group A/B trisaccharides coupled to the CM-5 Biacore® chip by amine
binding, antibody titers are expressed as resonance units (RU). Peripheral B
cell subsets were determined by three-color-flow cytometry, results are
depicted as the percentage of total number of CD19+ B lymphocytes. y
In contrast to the findings obtained using the Diamed-ID Micro
Typing System, serum titers of anti-A/B antibodies as detected by
SPR were significantly decreased in CVID patients as compared to
healthy controls (Figure 1B). Surface Plasmon Resonance Analysis Shows
Defective ABO Blood Group Antibody Formation
in CVID Patients In 22/31 CVID patients (in whom
both assays could be performed in parallel) anti-A/B antibodies
were detectable in the erythrocyte aggregation assay, and these
patients also showed antibody binding to the A/B trisaccharides
immobilized on the surface of the chip as indicated by SPR-RU,
although with a significantly decreased titer. The remaining nine
patients showed no visible aggregation in the Diamed-ID Micro
System and also showed no or only very low antibody binding
to the trisaccharides (RU well below 100). There was a positive
correlation between the patients’ decreased levels of anti-A/B
antibodies as detected by SPR and decreased levels of switched
memory B cells and IgM memory B cells, and patients with low
anti-A/B IgM antibodies had higher levels of naïve B cells, thus
showing that SPR anti-A/B antibodies are decreased in parallel
with other parameters indicative of the defect in B cell function
present in CVID patients (Figure 2). CVID Patients Produce No Blood Group Anti-A/B
IgG as Examined by Flow Cytometry To further discriminate between blood group anti-A/B IgM-
and IgG-antibodies, patients’ samples were analyzed by a semi-
quantitative assay to detect ABO blood group antibodies by
flow cytometry in an isotype-specific manner (15). The anti-A/B
IgG and IgM MFI histograms of a representative CVID patient
and a representative healthy control are given in Figures 3A–D,
where the X-axis represents the MFI given on a logarithmic
scale and the Y-axis the cell number on a linear numeric scale. The filled histograms represent binding of an anti-A/B-negative
control sample (AB serum), the bold lines (CVID) and hatched
histograms (healthy control) represent the binding of IgM/IgG
antibodies present in the tested serum that react with human
red blood cells (RBCs). A statistically significant impairment
in anti-A/B IgG formation was detected in CVID patients as
compared to healthy controls (Figures 3A,B,E), while anti-A/B
IgM-antibody titers were comparable between CVID patients and
controls (Figures 3D,F). FIGURE 2 | Correlation between levels of anti-A/B antibodies as
detected by surface plasmon resonance using the Biacore® device
and levels of B cell subsets in CVID patients. Anti-A/B antibodies were FIGURE 2 | Correlation between levels of anti-A/B antibodies as
detected by surface plasmon resonance using the Biacore® device
and levels of B cell subsets in CVID patients. Anti-A/B antibodies were
determined using surface plasmon resonance (SPR) technology with synthetic
blood group A/B trisaccharides coupled to the CM-5 Biacore® chip by amine
binding, antibody titers are expressed as resonance units (RU). Peripheral B
cell subsets were determined by three-color-flow cytometry, results are
depicted as the percentage of total number of CD19+ B lymphocytes. Horizontal lines indicate the normal range of B cell subsets as determined in
134 healthy individuals (upper panel: 5th percentile; middle and lower panel:
upper line, 95th percentile, lower line, 5th percentile). The
Bravais–Pearson-correlation coefficient was calculated to determine a
statistically significant correlation between two parameters at a level of
P < 0.05. determined using surface plasmon resonance (SPR) technology with synthetic
blood group A/B trisaccharides coupled to the CM-5 Biacore® chip by amine
binding, antibody titers are expressed as resonance units (RU). Peripheral B
cell subsets were determined by three-color-flow cytometry, results are
depicted as the percentage of total number of CD19+ B lymphocytes. CVID Patients Produce No Blood Group Anti-A/B
IgG as Examined by Flow Cytometry Horizontal lines indicate the normal range of B cell subsets as determined in
134 healthy individuals (upper panel: 5th percentile; middle and lower panel:
upper line, 95th percentile, lower line, 5th percentile). The
Bravais–Pearson-correlation coefficient was calculated to determine a
statistically significant correlation between two parameters at a level of
P < 0.05. May 2015 | Volume 6 | Article 211 Frontiers in Immunology | www.frontiersin.org 6 Anti-A/B antibodies in CVID Fischer et al. 0
50000
100000
150000
200000
250000
AB-serum
(n=6)
healthy Co
(n=6)
CVID (n=5)
MFI
anti-A/B IgG
p=0.00538
0
20000
40000
60000
80000
100000
AB-serum
(n=6)
healthy Co
(n=6)
CVID (n=5)
MFI
p=0.27563
anti-A/B IgM
A
An-A IgG
101
102
103
104
105
250
200
150
100
50
0
Fluorescence intensity
An-A IgM
101
102
103
104
105
300
200
100
0
Fluorescence intensity
C
An-B IgG
101
102
103
104
105
200
150
100
50
0
Fluorescence intensity
B
An-B IgM
101
102
103
104
105
250
200
150
100
50
0
Fluorescence intensity
D
t
n
u
o
c
ll
e
C
t
n
u
o
c
ll
e
C
t
n
u
o
c
ll
e
C
t
n
u
o
c
ll
e
C
E
F
FIGURE 3 | Detection of anti-A/B IgG- and IgM-antibodies by flow
cytometry. The anti-A/B IgG and IgM mean fluorescence intensity (MFI)
histograms of a representative CVID patient (bold lines) with no anti-A/B IgG
A,B), no anti-A IgM but with anti-B IgM present (C,D), and a healthy individual
hatched histograms) with anti-A/B IgG (A B) and anti-A/B IgM present (C D)
are depicted. Background binding of fluorescence-labeled anti-IgG and anti-IgM
was assessed using AB serum [(A–D), filled histograms] containing no or only
low levels of anti-A/B IgG-(A,B) and IgM-antibodies (C,D). CVID Patients Produce No Blood Group Anti-A/B
IgG as Examined by Flow Cytometry The X-axis
represents the MFI given on a logarithmic scale and the Y-axis the cell number
on a linear numeric scale
(Continued) A
An-A IgG
101
102
103
104
105
250
200
150
100
50
0
Fluorescence intensity
t
n
u
o
c
ll
e
C An-B IgG
101
102
103
104
105
200
150
100
50
0
Fluorescence intensity
B
t
n
u
o
c
ll
e
C An-A IgM
101
102
103
104
105
300
200
100
0
Fl
i t
it
C
t
n
u
o
c
ll
e
C An-B IgM
101
102
103
104
105
250
200
150
100
50
0
Fluorescence intensity
D
t
n
u
o
c
ll
e
C C D D 0
50000
100000
150000
200000
250000
AB-serum
(n=6)
healthy Co
(n=6)
CVID (n=5)
MFI
anti-A/B IgG
p=0.00538
Fluorescence intensity
E 0
20000
40000
60000
80000
100000
AB-serum
(n=6)
healthy Co
(n=6)
CVID (n=5)
MFI
p=0.27563
anti-A/B IgM
Fluorescence intensity
F E F MFI MFI FIGURE 3 | Detection of anti-A/B IgG- and IgM-antibodies by flow
cytometry. The anti-A/B IgG and IgM mean fluorescence intensity (MFI)
histograms of a representative CVID patient (bold lines) with no anti-A/B IgG
(A,B), no anti-A IgM but with anti-B IgM present (C,D), and a healthy individual
(hatched histograms) with anti-A/B IgG (A,B) and anti-A/B IgM present (C,D) are depicted. Background binding of fluorescence-labeled anti-IgG and anti-IgM
was assessed using AB serum [(A–D), filled histograms] containing no or only
low levels of anti-A/B IgG-(A,B) and IgM-antibodies (C,D). The X-axis
represents the MFI given on a logarithmic scale and the Y-axis the cell number
on a linear numeric scale. (Continued) May 2015 | Volume 6 | Article 211 Frontiers in Immunology | www.frontiersin.org 7 Fischer et al. Anti-A/B antibodies in CVID antibodies in serum with the median of the study group represented by a cross,
the interquartile range (IQR) represented by the box, 5- and 95-percentile
values represented by the whiskers, and minimum and maximum values
depicted by circles. The non-parametric Mann–Whitney U-test (two-sided P
value) was used for statistical comparison between patients and healthy control
subjects. 0
200
400
600
800
1000
1200
1400
1600
healthy controls (n=10)
CVID (n=20)
Agamma (n=2)
anti-A/B IgG-antibodies (RU)
p=0,00000001
0
500
1000
1500
2000
2500
3000
healthy controls
(n=9)
CVID_M_normal
(n=14)
CVID_M_low (n=6)
Agamma (n=2)
anti-A/B IgM-antibodies (RU)
n.s. CVID Patients Produce No Blood Group Anti-A/B
IgG as Examined by Flow Cytometry p=0,0000012
p=0,000033
A
B
FIGURE 4 | Isotype-specific measurement of anti-A/B antibodies by
surface plasmon resonance using the Biacore® device in healthy
controls, patients with CVID and XLA patients (Agamma). The amount
of IgG- (upper panel) and IgM- (lower panel) anti-A/B antibodies present in the
serum sample are depicted as resonance units (RU) using box plot diagrams,
th
di
f th
t d
i
t d b
th i t
til antibodies in serum with the median of the study group represented by a cross,
the interquartile range (IQR) represented by the box, 5- and 95-percentile
values represented by the whiskers, and minimum and maximum values
depicted by circles. The non-parametric Mann–Whitney U-test (two-sided P
value) was used for statistical comparison between patients and healthy control
subjects. |
Defective anti-A/B IgG (E) but normal IgM (F) antibody response in patients with
CVID and controls (healthy Co) as examined by flow cytometry. Background
binding of fluorescence-labeled anti-IgG and anti-IgM to the erythrocytes was
assessed in the presence of an AB serum containing no or only low levels of
anti-A/B antibodies. Box plot diagrams show anti-A/B IgG (E) and IgM (F) Affinity of Blood Group Anti-A/B IgM Antibody
Binding to Blood Group A/B Trisaccharide
Antigens is Not Altered in CVID Patients The erythrocyte aggregation assay based on the Diamed-ID Micro
Typing System as well as the flow cytometric method to quantify
binding of anti-A/B antibodies to fixed human A or B red blood
cells (ABO-FACS) cannot rank anti-A/B antibodies according to
their strength of reaction with the corresponding blood group
A/B trisaccharide antigens. In order to compare affinity of the
anti-A/B IgM antibodies, we depleted serum IgG from healthy
individuals with high blood group anti-A or B isoagglutinins with
protein A- and protein G-affinity columns and measured the on
and off rates of the anti-AB IgM antibodies to the respective trisac-
charide immobilized on the CM-5 chip. For these experiments,
amine-coupling of the trisaccharides to the surface of the CM5
sensor chip was used in order to guarantee physiological condition
of binding (16–18). We found no difference in association (ka)
and dissociation rate (kd) between CVID patients and controls
depleted of IgG when sera were diluted to match with 700 RU (data
not shown). CVID Patients Produce No Blood Group Anti-A/B
IgG as Detected by Surface Plasmon Resonance
Using the Biacore® Device The amount
of IgG- (upper panel) and IgM- (lower panel) anti-A/B antibodies present in the
serum sample are depicted as resonance units (RU) using box plot diagrams,
the median of the study group is represented by a cross, the interquartile
range (IQR) is represented by the box, 5- and 95-percentile values are
represented by the whiskers, and minimum and maximum values are
represented by circles. The 95% confidence interval for the median calculated
according to McGill et al. (21) is indicated by horizontal bars. The
non-parametric Mann–Whitney U-test (two-sided P value) was used for
statistical comparison between patients and healthy controls. CVID_M_low,
CVID patients with very low to undetectable anti-A/B IgM-antibodies;
CVID_M_normal, CVID patients with anti-A/B IgM-antibody titers that were
comparable to the healthy control group. controls, patients with CVID and XLA patients (Agamma). The amount
of IgG- (upper panel) and IgM- (lower panel) anti-A/B antibodies present in the
serum sample are depicted as resonance units (RU) using box plot diagrams,
the median of the study group is represented by a cross, the interquartile
range (IQR) is represented by the box, 5- and 95-percentile values are
represented by the whiskers, and minimum and maximum values are
represented by circles. The 95% confidence interval for the median calculated
according to McGill et al. (21) is indicated by horizontal bars. The
non-parametric Mann–Whitney U-test (two-sided P value) was used for
statistical comparison between patients and healthy controls. CVID_M_low,
CVID patients with very low to undetectable anti-A/B IgM-antibodies;
CVID_M_normal, CVID patients with anti-A/B IgM-antibody titers that were
comparable to the healthy control group. Affinity of Blood Group Anti-A/B IgM Antibody
Binding to Blood Group A/B Trisaccharide
Antigens is Not Altered in CVID Patients CVID Patients Produce No Blood Group Anti-A/B
IgG as Detected by Surface Plasmon Resonance
Using the Biacore® Device 0
200
400
600
800
1000
1200
1400
1600
healthy controls (n=10)
CVID (n=20)
Agamma (n=2)
anti-A/B IgG-antibodies (RU)
p=0,00000001
A To further discriminate between IgM and IgG anti-A/B response
we added anti-IgM and anti-IgG antibodies to the SPR assay as
described in the Section “Materials and Methods.” An additional
binding of the anti-IgM and anti-IgG antibodies to the respective
anti-A/B antibodies present in the test sample caused a measurable
rise in the RU value that was proportional to the amount of
IgM/IgG-anti-A/B antibodies present in the sample. Commer-
cially available anti-blood group A/B mAbs in a concentration
of 50 µg of anti-A IgM mAbs gave 2500 RU in this assay, while
the limit of detection was observed at 300 ng of anti-A IgM, and
40 µg of anti-B IgM mAbs gave a maximum 1000 RU, while the
limit of detection in this case was observed at 640 ng. When the
assay was calibrated with 10 µg of anti-A IgG mAbs a maximum of
15,000 RU were measured, while the limit of detection for anti-A
IgG was observed at 100 ng. B B 0
500
1000
1500
2000
2500
3000
healthy controls
(n=9)
CVID_M_normal
(n=14)
CVID_M_low (n=6)
Agamma (n=2)
anti-A/B IgM-antibodies (RU)
n.s. p=0,0000012
p=0,000033
B While ≥90% of the healthy individuals presented with easily
detectable (≥100 RU) anti-A/B IgM and IgG antibodies, anti-
A/B IgG antibodies were undetectable or very low (<100 RU)
in all 20 CVID patients investigated (Figure 4, upper panel),
Six of these 20 patients also had very low to undetectable anti-
A/B IgM-antibodies (CVID_M_low in Figure 4, lower panel),
while levels of anti-A/B IgM-antibodies in the remaining patients
(CVID_M_normal in Figure 4, lower panel) were comparable to
the healthy control group. FIGURE 4 | Isotype-specific measurement of anti-A/B antibodies by
surface plasmon resonance using the Biacore® device in healthy
controls, patients with CVID and XLA patients (Agamma). The amount FIGURE 4 | Isotype-specific measurement of anti-A/B antibodies by
surface plasmon resonance using the Biacore® device in healthy FIGURE 4 | Isotype-specific measurement of anti-A/B antibodies by
surface plasmon resonance using the Biacore® device in healthy
controls, patients with CVID and XLA patients (Agamma). Impaired Blood Group Anti-A/B Antibody
Response in CVID Patients is Associated with
Defective IgM-Antibody Response to
Pneumococcal Polysaccharides Antibodies against 23-valent PnP were determined by ELISA
in 32 of the 34 CVID patients in order to find a correlation
between the immune response to PnPs and blood group ABO anti-
gens, because ABO- and PnP-antigens share similar carbohydrate May 2015 | Volume 6 | Article 211 Frontiers in Immunology | www.frontiersin.org 8 Anti-A/B antibodies in CVID Fischer et al. 10
30
50
70
90
110
130
0
200
400
600
800
1000
anti-A/B IgM-antibodies (RU)
PnP-IgM (reciprocal abtibody titer)
p = 0,054
0
50
100
150
200
250
0
200
400
600
800
1000
anti-A/B IgM-antibodies (RU)
serum IgM (mg/dl)
p = 0,0310
A
B
FIGURE 5 | Correlation between reduced blood group anti-A/B IgM
antibodies and defective IgM-antibody response to pneumococcal
polysaccharides (upper panel) or reduced serum IgM levels (lower
panel) in CVID patients. Serum IgM antibodies against 23-valent
pneumococcal polysaccharides (PnP–IgM) were determined by ELISA, serum
blood group anti-A/B IgM antibodies were assessed using isotype-specific
surface plasmon resonance technology, serum IgM levels were measured by
nephelometry. Results are depicted as reciprocal serum titer (for PnP–IgM),
resonance units (RU, for anti-A/B IgM-antibodies), and mg/dl (for serum IgM). The Bravais–Pearson-correlation coefficient was calculated to determine a
statistically significant correlation at a level of P < 0.05. moieties (22–24). Interestingly, among the 32 CVID patients in
whom both PnP antibodies and isoagglutinins could be tested,
nine patients showed no detectable isoagglutinins and no or only
very low blood group anti-A/B antibodies in the SPR assay and
also showed no PnP-specific IgM-antibody response. Among the
remaining 23 CVID patients with detectable blood group anti-
A/B isoagglutinins, only 15 showed no anti-PnP IgM-antibodies
(P = 0.042, Chi-squared analysis). In addition, there was a trend
toward a positive correlation between the levels of the patients’
anti-A/B IgM-response as measured using SPR and their serum
anti-PnP IgM, and low levels of total serum IgM were significantly
correlated with low levels of anti-A/B IgM-antibodies (Figure 5). Typical for all CVID patients was the incapability to produce PnP-
specific IgG (Table 1), which was in accordance with a defective
IgG-response to blood group ABO antigens. 10
30
50
70
90
110
130
0
200
400
600
800
1000
anti-A/B IgM-antibodies (RU)
PnP-IgM (reciprocal abtibody titer)
p = 0,054
A Frontiers in Immunology | www.frontiersin.org Discussion 0
50
100
150
200
250
0
200
400
600
800
1000
anti-A/B IgM-antibodies (RU)
serum IgM (mg/dl)
p = 0,0310
B Common variable immunodeficiency (CVID) comprises a het-
erogeneous group of patients with a multitude of immunological
abnormalities that have in common a severely impaired anti-
body production with altered isotype-switch and defective affinity
maturation of the antibodies produced (5). Dependent on the
underlying defect, B cell activation and development might be
compromised at different levels enabling B cells of certain patients
with CVID to mount an IgM antibody response, while B cells of
other CVID patients with more severe defects cannot produce any
antibodies at all (25). Due to the profound antibody deficiency,
the patients suffer from severe susceptibility to infections, and the
demonstration of defective IgG antibody production provides the
indication for immunoglobulin replacement therapy (26), as was
proposed by an IUIS expert committee already many years ago (5). The present study investigated the capability of CVID patients’
B cells to respond to blood group ABO antigens and shows
that 100% of the patients that receive long-term immunoglobulin
replacement therapy based on their inability to produce IgG anti-
bodies following vaccination or infection (26) also have a defect
in IgG antibody formation against ABO blood group antigens,
thus providing an additional diagnostic parameter of possible
clinical-therapeutic relevance. FIGURE 5 | Correlation between reduced blood group anti-A/B IgM
antibodies and defective IgM-antibody response to pneumococcal
polysaccharides (upper panel) or reduced serum IgM levels (lower FIGURE 5 | Correlation between reduced blood group anti-A/B IgM
antibodies and defective IgM-antibody response to pneumococcal
polysaccharides (upper panel) or reduced serum IgM levels (lower
panel) in CVID patients. Serum IgM antibodies against 23-valent
pneumococcal polysaccharides (PnP–IgM) were determined by ELISA, serum
blood group anti-A/B IgM antibodies were assessed using isotype-specific
surface plasmon resonance technology, serum IgM levels were measured by
nephelometry. Results are depicted as reciprocal serum titer (for PnP–IgM),
resonance units (RU, for anti-A/B IgM-antibodies), and mg/dl (for serum IgM). The Bravais–Pearson-correlation coefficient was calculated to determine a
statistically significant correlation at a level of P < 0.05. panel) in CVID patients. Serum IgM antibodies against 23-valent
pneumococcal polysaccharides (PnP–IgM) were determined by ELISA, serum
blood group anti-A/B IgM antibodies were assessed using isotype-specific
surface plasmon resonance technology, serum IgM levels were measured by
nephelometry. Discussion Results are depicted as reciprocal serum titer (for PnP–IgM),
resonance units (RU, for anti-A/B IgM-antibodies), and mg/dl (for serum IgM). The Bravais–Pearson-correlation coefficient was calculated to determine a
statistically significant correlation at a level of P < 0.05. Anti-ABO antibodies are thought to be natural antibodies
produced by all individuals with a functional immune system
(14). Their immunologically active epitopes share carbohydrate
moieties and appear first in infants at the age of 3–6 months when
the developing immune system can react with microorganisms
and environmental antigens that have similar carbohydrate epi-
topes as the blood group ABO antigens (22–24). Certain debate
exists, however, whether anti-AB antibodies completely fulfill
the criteria of natural antibodies (27). Natural antibodies were
described to be encoded by germline variable (V) genes, show
no somatic mutations and are produced predominantly by CD5+
B1 cells, at least in the mouse, although certain splenic CD5−B2
cells were also capable of producing natural antibodies (28, 29). Natural antibodies are mostly of the IgM isotype, are therefore
polyreactive and show a wide range of binding avidities (14, 30). High resolution crystallography has shown that anti-carbohydrate
antibodies in germline configuration can recognize a range of structurally related carbohydrate epitopes (22). A repertoire of
pre-existing ‘natural antibodies’ might exist in healthy individu-
als in the absence of immune stimulation which is encoded by
a relatively small number of germline progenitors recognizing
carbohydrate moiety with a define shape (31), although a second
stage of selection for antibodies directed against blood group ABO
antigens with higher affinity cannot be excluded at present. Natural antibody formation has up to now not been studied
in greater detail in patients diagnosed with CVID. This study
shows that a defective IgG antibody response to protein and
polysaccharide antigens that is characteristic for patients with May 2015 | Volume 6 | Article 211 Frontiers in Immunology | www.frontiersin.org 9 Anti-A/B antibodies in CVID Fischer et al. formation after vaccination or infection as equivalent criteria. SPR
analysis proved to be the most specific and sensitive method for
the detection of anti-blood group A/B IgG antibodies and applying
this method a pronounced defect in IgG antibody production
could be demonstrated and defined in all CVID patients tested. Discussion As can be seen in Figure 1, conventional methodology such as
the erythrocyte aggregation assay based on the Diamed-ID Micro
Typing System was unable to detect defective antibody production
in the majority of CVID patients, as a considerable proportion
(approximately 2/3) of these patients can produce normal levels
of anti-A/B IgM antibodies. Furthermore, SPR analysis of anti-
A/B antibodies can be applied to ascertain early diagnosis of IgG
antibody deficiency in CVID patients, while at present, antibody
failure has to be proven by the lack of response to vaccination. The
clinical application of SPR analysis for the diagnosis of antibody
deficiency certainly requires further development. Comparable
efforts have been undertaken for the application of this method in
the rapid quantitation of blood group antibodies (16), and more
recently a 96-well-plate format has been presented for clinical
applications of SPR in serum antibody quantification (37). In
contrast to microtyping, both flow cytometry and SPR can detect
anti-A/B antibodies in an isotype-specific manner. In addition
to a greater sensitivity in detecting anti-A/B antibodies, further
advantages of the SPR method include the use of molecularly
well defined, standardized blood group antigens as compared to
the interindividual variations in donor erythrocytes used as the
particulate antigen in flow cytometry, and the ability to detect
biologically relevant antibody specificities, as the carbohydrate
antigens are presented in a physiological manner. CVID (26) is accompanied by a defect in the formation of natural
IgG antibodies such as anti-A/B IgG-antibodies. Of the 34 patients
investigated, we found only nine CVID patients with no detectable
anti-A/B antibodies at all, similar to the eight patients with XLA
that had no detectable peripheral B cells due to btk-deficiency. The
remaining 25 patients showed detectable anti-A/B antibodies that
were of the IgM isotype. In contrast, healthy individuals showed
both anti-A/B IgM and IgG responses. These results are in accor-
dance with previous studies showing defects in class switching in
CVID patients (25, 26, 32). Interestingly, the association rate (ka)
and dissociation rate (kd) of blood group specific anti-A/B IgM
antibodies binding to defined blood group A/B trisaccharides was
normal in CVID patients, indicating that the majority of CVID
patients might produce blood group specific IgM antibodies that
are functional intact. The defective IgM response against A/B blood group antigens
observed in CVID patients was accompanied by defective IgM
antibody formation against pneumococcal capsular polysaccha-
rides as well. 2. Chapel H, Lucas M, Lee M, Bjorkander J, Webster D, Grimbacher B, et al.
Common variable immunodeficiency disorders: division into distinct clinical
phenotypes. Blood (2008) 112:277–86. doi:10.1182/blood-2007-11-124545
3. Conley ME, Notarangelo LD, Etzioni A. Diagnostic criteria for primary
immunodeficiencies.
Representing
PAGID
(Pan-American
Group
for Author Contributions Patients with predominantly antibody deficiency who have a
long history of clinical disease, in particular recurrent infections
of the lower respiratory tract, are well known to be prone to
developing organ damage such as chronic lung disease, which
determines their long-term prognosis (36). Increased awareness
for PID has made earlier diagnosis and initiation of adequate ther-
apy in PID patients feasible, so that more and more patients with
predominantly antibody deficiency lack a long history of clinical
disease, making it necessary to initiate immunoglobulin replace-
ment therapy based on laboratory findings rather than patient
history. Detection of impaired IgG antibody formation has been
suggested as a laboratory parameter to facilitate the decision on
early immunoglobulin replacement in hypogammaglobulinemic
patients (26). Currently used diagnostic criteria for CVID, e.g.,
the criteria proposed by an IUIS expert committee (5), the most
commonly used European Society for Immunodeficiencies/Pan
American Group for Immunodeficiency (ESID/PAGID) defini-
tion of CVID (3) or the 2014 diagnostic criteria for the ESID reg-
istry (4) propose defective isoagglutinins and impaired antibody HW and MF were the principal investigators, provided labora-
tory resources, analyzed clinical and immunological data, wrote
the first manuscript draft together, critically participated in all
revisions of the manuscript and take primary responsibilities for
the paper. WW performed all isoagglutinin and SPR analyses
and analyzed the results, MW and GB performed the ABO FACS
measurements and analyzed the results, ME provided clinical
patient data and laboratory resources, participated in data analysis
and interpretation and critically reviewed the initial draft and all
revisions of the manuscript, CB participated in data interpretation. Acknowledgments The authors would like to acknowledge Aysen Samstag for
performing the pneumococcal polysaccharide serum antibody
ELISAs, Slim David Bra and Ivan Divkovic for performing flow
cytometry analysis of B cell subsets, and our patients for their
consent to participate in a scientific study. Discussion One reason for the failure of CVID B cells to respond
to pneumococcal and blood group polysaccharides could be insuf-
ficient triggering of TLRs and their signaling pathways, as TLR-
activation is critical for T-independent antibody responses (33). CVID B cells were shown to have impaired responses to TLR7 and
TLR9 agonists including poor cell proliferation, loss of cytokine
production, and failure to produce and secrete IgG and IgA (34,
35). IFN-α could restore TLR7- and TLR9-triggered functional
responses in B cells of CVID subjects with >0.5% peripheral
isotype-switched memory B cells (34). An alternative explanation
for the observed association between anti-A/B and pneumococcal
IgM-antibodies could be a general decrease in IgM production
as is reflected by the low level of total serum IgM in most CVID
patients. 1. Wehr C, Kivioja T, Schmitt C, Ferry B, Witte T, Eren E, et al. The EUROclass
trial: defining subgroups in common variable immunodeficiency. Blood (2008)
111:77–85. doi:10.1182/blood-2007-06-091744 References 1. Wehr C, Kivioja T, Schmitt C, Ferry B, Witte T, Eren E, et al. The EUROclass
trial: defining subgroups in common variable immunodeficiency. Blood (2008)
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ducted in the absence of any commercial or financial relationships that could be
construed as a potential conflict of interest. Copyright © 2015 Fischer, Wolfram, Binder, Böhmig, Wahrmann, Eibl and Wolf. This is an open-access article distributed under the terms of the Creative Commons 20. Biagini RE, Schlottmann SA, Sammons DL, Smith JP, Snawder JC, Striley CAF,
et al. Method for simultaneous measurement of antibodies to 23 pneumococcal
capsular polysaccharides. Clin Diagn Lab Immunol (2003) 10:744–50. doi:10. 1128/CDLi.10.5.744-750.2003 Copyright © 2015 Fischer, Wolfram, Binder, Böhmig, Wahrmann, Eibl and Wolf. This is an open-access article distributed under the terms of the Creative Commons
Attribution License (CC BY). References The use, distribution or reproduction in other forums is
permitted, provided the original author(s) or licensor are credited and that the original
publication in this journal is cited, in accordance with accepted academic practice. No
use, distribution or reproduction is permitted which does not comply with these terms. Attribution License (CC BY). The use, distribution or reproduction in other forums is
permitted, provided the original author(s) or licensor are credited and that the original
publication in this journal is cited, in accordance with accepted academic practice. No
use, distribution or reproduction is permitted which does not comply with these terms. 21. McGill JW, Tukey W, Larsen A. Variations of box plots. Am Stat (1978) 32:12–6. doi:10.1080/00031305.1978.10479236 May 2015 | Volume 6 | Article 211 Frontiers in Immunology | www.frontiersin.org 11
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Mobile forms of carbon in trees: metabolism and transport
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Tree physiology
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© The Author(s) 2021. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/),
which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Pia Guadalupe Dominguez
1,3 and Totte Niittylä
2,3 1Instituto de Agrobiotecnología y Biología Molecular (IABIMO), Instituto Nacional de Tecnología Agropecuaria (INTA), Consejo Nacional de Investigaciones Científicas y
Técnicas (CONICET), Hurlingham, Buenos Aires B1686IGC, Argentina; 2Department of Forest Genetics and Plant Physiology, Umeå Plant Science Centre, Swedish University
of Agricultural Sciences, Umeå 90183, Sweden; 3Corresponding authors: P.G.Dominguez (piagdominguez@gmail.com); T. Niittylä (totte.niittyla@slu.se) Received February 18, 2021; accepted September 12, 2021; handling Editor Andrea Polle Plants constitute 80% of the biomass on earth, and almost two-thirds of this biomass is found in wood. Wood formation
is a carbon (C)-demanding process and relies on C transport from photosynthetic tissues. Thus, understanding the
transport process is of major interest for understanding terrestrial biomass formation. Here, we review the molecules
and mechanisms used to transport and allocate C in trees. Sucrose is the major form in which C is transported in plants,
and it is found in the phloem sap of all tree species investigated so far. However, in several tree species, sucrose is
accompanied by other molecules, notably polyols and the raffinose family of oligosaccharides. We describe the molecules
that constitute each of these transport groups, and their distribution across different tree species. Furthermore, we detail
the metabolic reactions for their synthesis, the mechanisms by which trees load and unload these compounds in and
out of the vascular system, and how they are radially transported in the trunk and finally catabolized during wood
formation. We also address a particular C recirculation process between phloem and xylem that occurs in trees during
the annual cycle of growth and dormancy. A search of possible evolutionary drivers behind the diversity of C-carrying
molecules in trees reveals no consistent differences in C transport mechanisms between angiosperm and gymnosperm
trees. Furthermore, the distribution of C forms across species suggests that climate-related environmental factors will
not explain the diversity of C transport forms. However, the consideration of C-transport mechanisms in relation to
tree–rhizosphere coevolution deserves further attention. To conclude the review, we identify possible future lines of
research in this field. Keywords: C transport, C metabolism, dormancy, mobile forms of C, sucrose, polyols, RFO, phloem loading, phloem unloading,
radial transport, trees. Keywords: C transport, C metabolism, dormancy, mobile forms of C, sucrose, polyols, RFO, phloem loading, phloem unloading,
di l Tree Physiology 42, 458–487
https://doi.org/10.1093/treephys/tpab123
Arboreal review Tree Physiology 42, 458–487
https://doi.org/10.1093/treephys/tpab123
Arboreal review Tree Physiology 42, 458–487 Introduction between mature leaves (source tissues) and the heterotrophic
tissues (sink tissues) (Yu et al. 2015, Smith et al. 2018). The C transport and metabolism steps allow coordination of C
assimilation and export, and incorporation into sink tissues in
relation to the availability of nutrients and environmental cues. The ∼3 trillion trees on the planet constitute an estimated
sink of 2.4 petagrams of carbon (C) per year globally (Pan
et al. 2011, Crowther et al. 2015). This makes C assimilation
by trees a central part of the global C cycle and signifies
forest’s potential in the mitigation of climate change. Most of
the biomass in trees resides in above and below ground woody
tissues and roots, which account for ∼70% of the terrestrial
plant biomass (Bar-on et al. 2018). Woody biomass is derived
from the photosynthetically fixed C imported primarily from
leaves and to a lesser extent from photosynthetic bark tissues. The process of C allocation in trees involves communication Following photosynthetic C assimilation in the leaf mesophyll
cells, fixed C (mostly in the form of sugars and/or sugar
alcohols collectively called assimilates) are loaded into the
phloem system in the minor veins of the leaves (Lalonde
et al. 2003). Phloem loading is considered to include the
transport of assimilates from their synthesis or storage sites to
the conduits comprised of the sieve element/companion cell Mobile forms of carbon in trees 459 complexes (SE/CCCs), which form the long-distance transport
pathway. Phloem unloading occurs when assimilates reach the
sink tissue and move across the SE/CCC boundary to their
utilization or storage sites. There are two main mechanisms by
which molecules move cell-to-cell toward or from the phloem to
achieve either phloem loading or unloading: the symplasmic and
the apoplasmic routes (Lalonde et al. 2003, Braun et al. 2014). In the symplasmic route, assimilates move passively through
interconnecting plasmodesmata between cells, i.e., through the
symplasm. This process requires a gradient driving assimilate
diffusion from high to low concentration. In the apoplasmic
route, assimilates are exported into the apoplast of the tissue
by facilitated diffusion through plasma membrane transporters
and then actively imported by cells against a concentration gra-
dient. Sometimes, both apoplasmic and symplasmic pathways
are combined to achieve the movement of assimilates. The
cell-to-cell movement of assimilates is called lateral or radial
transport and it can be bidirectional: phloem-to-xylem or xylem-
to-phloem (Aubry et al. 2019). Introduction The connections and transport
processes between phloem and xylem vascular systems allow
the distribution of assimilates at the whole tree level. Upon
reaching the destination sink tissues/organs, assimilates are
catabolized to produce energy or used as building blocks to
synthetize macromolecules and other compounds. which form sucrose-6-phosphate in a reaction catalyzed by
sucrose-phosphate synthase (SPS; EC 2.4.1.14). In trees, SPS
activity has been characterized from leaves of hybrid poplar
(Populus alba L. × Populus grandidentata Michx.) and Prosopis
juliflora (Sw.) DC. (Table 1) (Sinha et al. 1997, Park et al. 2009). After SPS, sucrose-6-phosphate is dephosphorylated
by the sucrose-phosphate phosphatase (SPP; EC 3.1.3.24) to
produce sucrose. Interestingly, 35S promoter-driven expression
of a chimeric fusion construct between SPS and SPP increased
the growth rate of hybrid poplars, suggesting that metabolic
channeling between these two enzymes can alleviate a bottle-
neck in C allocation to wood (Table 1) (Maloney et al. 2015). After synthesis, sucrose can be stored locally in the vacuole or
loaded into the phloem, either symplasmically or apoplasmically
depending on the species (Fu et al. 2011). The presence of
sucrose in phloem sap has been demonstrated in several tree
species (Table 1). In addition to sucrose, phloem sap analyses of several plant
species including trees have shown that other molecules, such
as sugar alcohols and oligosaccharides, can also contribute to
long-distance C transport (Figure 3, Tables 2 and 3) (Rennie
and Turgeon 2009, Fu et al. 2011). The presence of these
molecules in the phloem sap along with evidence collected from
other type of experiments such as the existence of synthetizing
enzymes in source tissues and degrading enzymes in sink
tissues, and labeling studies to follow the localization of the
molecules, have contributed to define two main molecule groups
that in addition to sucrose account as mobile forms of C in
the phloem: the sugar alcohols and the raffinose family of
oligosaccharides (RFOs) (Figure 1B and C, Tables 2 and 3)
(Bieleski 1982, Moing 2000, Madore 2001). Sucrose, sugar
alcohols and RFOs share some chemical features such as being
highly hydrophilic and reduced, weakly charged and exhibiting
relatively low molecular weight (Peters et al. 2007, Merchant
and Richter 2011, Dumschott et al. 2017). This makes them
well suited for transport in the aqueous phloem sap and less
likely to react chemically with other cellular components such
as proteins. Introduction During the growth of trees, developing wood (i.e., the living
region of woody tissue undergoing mitosis and cell differentia-
tion) constitutes one of the strongest C sinks. Thus, how trees
distribute C and how C is incorporated into developing wood
are of major interest for understanding terrestrial biomass for-
mation. The phloem unloading process and the radial transport
of assimilates in stems are key steps in the incorporation and
distribution of C into developing wood. In this article, we review
the literature on metabolism, long-distance transport and radial
transport of the main molecules in which C is transported in
trees. The sugar alcohols The mobile forms of C are molecules used to transport and allo-
cate C, incorporated in their backbones, between distant organs
of the plant. Sucrose (Figure 1A) is the main form of transported
C in most plant species (Ruan 2014), including trees (Table 1). It is synthetized as a product of the photosynthesis in source
tissues, where triose phosphate from the Calvin–Benson cycle in
the chloroplasts is exported to the cytosol by triose-phosphate
transporters. In the cytosol, triose phosphates contribute to
a pool of interconvertible hexose-phosphates and nucleotide
sugars that serve as substrates for sucrose synthesis as well
as for other primary metabolic reactions (Figure 2A). Sucrose
is synthesized from UDP-glucose and fructose-6-phosphate, Sugar alcohols, also called polyols, polyalcohols or polyhydric
alcohols, are a diverse family of molecules distributed widely in
plants. In total, sugar alcohols are estimated to compose ∼30%
of the photosynthetically fixed C on the planet (Bieleski 1982). They are common in trees and represent the main form of C in
the phloem of several members of the Rosaceae family such as
the genera Malus (apple), Pyrus (pear) and Prunus (stone fruits
such as plum). The synthesis of sugar alcohols involves the action of reduc-
tases or reductases combined with phosphatases on hexoses or
hexose-phosphates (Moing 2000). Thus, sugar alcohols are the
result of the reduction of the aldo or keto group of a sugar to Tree Physiology Online at http://www.treephys.oxfordjournals.org 460 Dominguez and Niittylä Figure 1. Molecular structures of phloem mobile carbon forms. (A) Sucrose. (B) Sugar alcohols. Left panel: glycitols. Right panel: cyclitols. (C)Raffinose
family of oligosaccharides. Downloaded from https://academic.oup.com/treephys/article/42/3/458/6372535 by Sveriges Lantbruksuniversitet user on 06 October 2022 Downloaded from https://academic.oup.com/treephys/article/42/3/458/6372535 by Sveriges Lantbruksuniversitet user on 06 October 2022 Figure 1. Molecular structures of phloem mobile carbon forms. (A) Sucrose. (B) Sugar alcohols. Left panel: glycitols. Right panel: cyclitols. (C)Raffinose
family of oligosaccharides. 2014, Gallinger and Gross 2018). The role of D-pinitol in C
transport is still debated and more solid biochemical data are
needed, especially in trees (Dumschott et al. 2017). However,
evidence is accumulating that pinitol is a form of C transport
in the phloem: it is synthetized in source tissues (Dittrich and
Kandler 1972, Dittrich and Brandl 1987), and it can be found
in phloem and xylem sap of both angiosperm and gymnosperm
plants (Figure 3, Table 2) (Richter and Popp 1992, Gallinger
and Gross 2018, Shi et al. 2020). The sugar alcohols While Gallinger and Gross
(2018) reported that pinitol is the major mobile C form in the
phloem sap of Scots pine and other gymnosperm trees, Devaux
et al. (2009) found that pinitol was in lower levels in comparison
with sucrose in Pinus pinaster Aiton (Figure 3). This could be 2014, Gallinger and Gross 2018). The role of D-pinitol in C
transport is still debated and more solid biochemical data are
needed, especially in trees (Dumschott et al. 2017). However,
evidence is accumulating that pinitol is a form of C transport
in the phloem: it is synthetized in source tissues (Dittrich and
Kandler 1972, Dittrich and Brandl 1987), and it can be found
in phloem and xylem sap of both angiosperm and gymnosperm
plants (Figure 3, Table 2) (Richter and Popp 1992, Gallinger
and Gross 2018, Shi et al. 2020). While Gallinger and Gross
(2018) reported that pinitol is the major mobile C form in the
phloem sap of Scots pine and other gymnosperm trees, Devaux
et al. (2009) found that pinitol was in lower levels in comparison
with sucrose in Pinus pinaster Aiton (Figure 3). This could be a hydroxy group, rendering their physical chemical properties
close to those of carbohydrates. Structurally, they can be a
linear chain or acyclic (called glycitols) or cyclic (called cyclitols)
(Figure 1B). Mannitol, sorbitol (glucitol) and galactitol (dulcitol)
are considered the most frequent linear polyols that act as
mobile C forms in several tree species including olive trees (Olea
europaea L.) and Rosaceae (Table 2) (Bieleski 1982, Noiraud
et al. 2001, Reidel et al. 2009). D-pinitol (3-O-methyl-D-chiro-
inositol) is found in eucalyptus (Eucaliptus sp.), acacias (Acacia
sp.), European larch (Larix decidua Mill.), black spruce (Picea
mariana (Mill.) Britton, Sterns & Poggenb.), Norway spruce
(Picea abies (L.) Karst.) and Scots pine (Pinus sylvestris L.)
(Merchant et al. 2006, Simard et al. 2013, Deslauriers et al. Tree Physiology Volume 42, 2022 Mobile forms of carbon in trees
461 461 Table 1. List of species in which evidence of sucrose metabolism and transport has been reported. Angiosperm trees/shrubs
Gymnosperm
trees
Herbaceous plants
Presence of sucrose in
phloem sap
P. persica (Moing et al. 1997, Nadwodnik
and Lohaus 2008); E. globulus (Pate et al. 1998, Merchant et al. 2012); F. sylvatica
(Gessler et al. 2004, Fink et al. 2018);
Cocos nucifera Cocos nucifera L. cv. Namhom (Nakamura et al. 2004);
P. abies, Abies
alba, P. sylvestris,
L. Populus sp. (Coleman et al. 2009,
Dominguez et al. 2021, Gerber et al.
2014, Li et al. 2020, Rende et al. 2017) The sugar alcohols deciduas
(Gallinger and
Gross 2018)
Arabidopsis thaliana (Tetyuk et al. 2013); Helianthus
annuus, Solanum lycopersicum, Nicotiana rustica,
Phaselus vulgaris, Solanum tuberosum, C. sativus, A. graveolens, P. major (Fu et al. 2011). Others: Rennie
and Turgeon (2009), Fu et al. (2011) Table 1. List of species in which evidence of sucrose metabolism and transport has been reported. Angiosperm trees/shrubs
Gymnosperm
trees
Herbaceous plants
Presence of sucrose in
phloem sap
P. persica (Moing et al. 1997, Nadwodnik
and Lohaus 2008); E. globulus (Pate et al. 1998, Merchant et al. 2012); F. sylvatica
(Gessler et al. 2004, Fink et al. 2018);
Cocos nucifera Cocos nucifera L. cv. Namhom (Nakamura et al. 2004);
Pseudotsuga menziesii (Mirb.) Franco
(Woodruff 2014); Querus robus, F. excelsior (Öner-Sieber and Lohaus 2014);
Magnolia kobus, Gnetum gnemon (Fink
et al. 2018); Citrus sinensis, Murraya
paniculata, Bergera koenegii (Killiny
2016); Prunus domestica (Gallinger and
Gross 2018); P. trichocarpa × Populus
deltoides (Dafoe et al. 2009). Others:
Rennie and Turgeon (2009), Fu et al. (2011). P. abies, Abies
alba, P. sylvestris,
L. deciduas
(Gallinger and
Gross 2018)
Arabidopsis thaliana (Tetyuk et al. 2013); Helianthus
annuus, Solanum lycopersicum, Nicotiana rustica,
Phaselus vulgaris, Solanum tuberosum, C. sativus, A. graveolens, P. major (Fu et al. 2011). Others: Rennie
and Turgeon (2009), Fu et al. (2011)
S
h
i
i
P
l
(M l
l
P k
A
h li
(S
d
l
P k
l Table 1. List of species in which evidence of sucrose metabolism and transport has been reported. Presence of sucrose in
phloem sap Synthesis reactions
(biochemical or
physiological experiments
in source tissues) A. thaliana (Strand et al. 2000, Park et al. 2008,
Volkert et al. 2014, Bahaji et al. 2015, Albi et al. 2016), Solanum lycopersicum L., Spinacia oleracea
L. (Huber and Huber 1992), Oryza sativa L. (Hashida et al. 2016), Spinacia oleracea (Guy et al. 1992), Zea mays (Bilska-Kos et al. 2020),
Saccharum spp. (Partida et al. 2021). Populus sp. (Maloney et al. 2015, Park et
al. 2009), P. juliflora (Sinha et al. 1997) A. thaliana (Barratt et al. 2009), S. lycopersicum
(D’Aoust et al. 1999, Zanor et al. 2009), S. tuberosum (Zrenner et al. 1995, Hajirezaei et al. 2000), Z. mays (Li et al. 2013), O. sativa (Morey et
al. 2018), Solanum chmielewskii (Sun et al. 1992),
P. sativum (Lunn and Rees 1990). Populus sp. (Payyavula et al. 2011,
Mahboubi et al. 2013, Zhang et al. 2021) Populus sp. Presence of sucrose in
phloem sap The sugar alcohols (Payyavula et al. 2011,
Mahboubi et al. 2013, Zhang et al. 2021) Transport evidence
(transporters in
sink/source tissues or
tracer experiments) A. thaliana (Baud et al. 2005, Chen et al. 2012, Kim
et al. 2021, Le Hir et al. 2015), S. lycopersicum
(Shammai et al. 2018), Nicotiana tabacum (Bürkle
et al. 1998), Z. mays (Bezrutczyk et al. 2018,
Slewinski et al. 2009, Sosso et al. 2015), O. sativa
(Eom et al. 2012, Ma et al. 2017, Sosso et al. 2015, Yang et al. 2018), S. tuberosum (Hackel et al. 2006, Riesmeier et al. 1993), Sorghum bicolor
(Milne et al. 2017) due to different seasonal levels or to methodological differences. Pinitol has also been found in the phloem sap of herbaceous
plants (Table 2) and in small amounts in the Broussonetia
papyrifera tree (Figure 3, Table 2) (Shi et al. 2020). There are
other sugar alcohols that are found in high levels in trees such
as quercitol, polygalatol, bornitol and quebrachitol (Bieleski and
Briggs 2005, Merchant et al. 2006, 2007, Arndt et al. 2008). Myo-inositol, also a cyclitol, is ubiquitous in plants but found only
in low levels in phloem sap, so other roles related to signaling
have been suggested for this compound (Figure 3) (Noiraud et
al. 2001). However, it participates in the production of D-pinitol
and of galactinol, which is part of the raffinose pathway and, as such, involved in the RFO synthesis (Nadwodnik and Lohaus
2008, Dumschott et al. 2017). Evidence of sugar alcohol
metabolic reactions in trees is largely based on enzymatic
activity measurements of purified or partially purified proteins
from tissue extracts. Thus, in order to provide a metabolic
framework for sugar alcohol synthesis in trees, we have overlaid
the evidence of individual reaction steps found in gymnosperm
or angiosperm trees on the pathways described for herbaceous
plants (Figure 2). Mannitol is found in the Oleaceae family, which encompasses
trees such as olive and ash (Fraxinus excelsior L.) (Table 2)
(Reidel et al. 2009). Its synthesis starts by the isomerization of Tree Physiology Online at http://www.treephys.oxfordjournals.org 462 Dominguez and Niittylä 2. Synthesis of phloem mobile carbon forms. (A) Sucrose (orange). (B) Sugar alcohols (greens and blues). (C) Raffinose family of
ccharides (RFOs) (gray). The colors of the circles indicate the type of plant for which there is evidence for each enzyme. Mobile forms of carbon in trees
463 Mobile forms of carbon in trees
463 463 Mobile forms of carbon in trees
463
Figure 3. Metabolite content in the phloem sap of different tree species. The colors and the sizes of the dots represent the relative values of the
metabolite contents in comparison with sucrose, whose value is equivalent to 1. Collection data (material-collection method): 1. phloem sap-aphid; 2. phloem sap-bleeding; 3. phloem sap-exudate; 4. total phloem tissue; 5. phloem sap-centrifugation method; 6. phloem and xylem sap-centrifugation
method. RFOs: raffinose family of oligosaccharides. The plot was generated with the ggplot2 library of R using the data published in the references. Figure 3. Metabolite content in the phloem sap of different tree species. The colors and the sizes of the dots represent the relative values of the
metabolite contents in comparison with sucrose, whose value is equivalent to 1. Collection data (material-collection method): 1. phloem sap-aphid; 2. phloem sap-bleeding; 3. phloem sap-exudate; 4. total phloem tissue; 5. phloem sap-centrifugation method; 6. phloem and xylem sap-centrifugation
method. RFOs: raffinose family of oligosaccharides. The plot was generated with the ggplot2 library of R using the data published in the references. galactitol-synthetizing aldose reductase (aldehyde reductase)
(EC:1.1.1.21) (Figure 2B, Table 2) (Bliss et al. 1972, Negm
1986). myo-inositol), which is further epimerized to D-pinitol. The
first step is catalyzed by myo-inositol-O-methyltransferase
(IMT) (EC 2.1.1.129), which transfers a methyl group from
S-adenosylmethionine to myo-inositol (Vernon and Bohnert
1992, Chiera et al. 2006). The second step is catalyzed
by a D-ononitol epimerase (OEP) (Ahn et al. 2018). This
is the case for the jojoba (Simmondsia chinensis (Link) C. K. Schneid.) shrub (Dittrich and Korak 1984) and some
herbaceous species (Dittrich and Kandler 1972) as studied by
tracer experiments. In some species such as Medicago truncatula
Gaertn., the epimerization is a two-step reaction, which includes
the formation of an intermediate compound (Pupel et al. 2019). This pathway involves the conversion of D-ononitol to
4-O-methyl-D-myo-inosose by D-ononitol dehydrogenase (EC
1.1.1). The intermediate product 4-O-methyl-D-myo-inosose is
converted to D-pinitol by D-pinitol dehydrogenase (EC 1.1.1). In some gymnosperm trees (Juniperus communis L., Taxus
baccata), myo-inositol is converted into sequoyitol (5-O-Methyl-
myo-inositol) by an uncharacterized methyltransferase (EC
2.1.1). Sequoyitol is then epimerized to give D-pinitol (Dittrich
and Kandler 1972). The sugar alcohols Downloaded from https://academic.oup.com/treephys/article/42/3/458/6372535 by Sveriges Lantbruksuniversitet user on 06 October 202 Downloaded from https://academic.oup.com/treephys/article/42/3/458/6372535 by Sverige 2. Synthesis of phloem mobile carbon forms. (A) Sucrose (orange). (B) Sugar alcohols (greens and blues). (C) Raffinose family of
oup.com/treephys/article/42/3/458/6372535 by Sveriges Lantbruksuniversitet user on 06 October Figure 2. Synthesis of phloem mobile carbon forms. (A) Sucrose (orange). (B) Sugar alcohols (greens and blues). (C) Raffinose family of
oligosaccharides (RFOs) (gray). The colors of the circles indicate the type of plant for which there is evidence for each enzyme. Figure 2. Synthesis of phloem mobile carbon forms. (A) Sucrose (orange). (B) Sugar alcohols (greens an
oligosaccharides (RFOs) (gray). The colors of the circles indicate the type of plant for which there is evidence fo dephosphorylated
by
M1PP
(mannitol-1-phosphate
phos-
phatase) (EC 3.1.3.22) into mannitol (Rumpho et al. 1983). dephosphorylated
by
M1PP
(mannitol-1-phosphate
phos-
phatase) (EC 3.1.3.22) into mannitol (Rumpho et al. 1983). Galactitol is abundantly found in the phloem sap of the
Celastraceae family, which includes the spindle tree (Euonymus
europaeus L.) (Bieleski 1982). It is considered to be syn-
thetized from Dfructose-6-P-galactose by a NADPH-dependent fructose-6-phosphate (P) into mannose-6-P by M6PI (mannose-
6-phosphate isomerase) (EC 5.3.1.8) (Figure 2B) (Rumpho
et al. 1983, Merchant and Richter 2011). Mannose-6-P is
converted to mannitol-1-P by the action of M6PR (NADPH-
dependent mannose-6-P reductase) (EC 1.1.1.224) (Loescher
et al. 1992, Everard et al. 1997). Mannitol-1-P is subsequently Galactitol is abundantly found in the phloem sap of the
Celastraceae family, which includes the spindle tree (Euonymus
europaeus L.) (Bieleski 1982). It is considered to be syn-
thetized from Dfructose-6-P-galactose by a NADPH-dependent Tree Physiology Volume 42, 2022 Mobile forms of carbon in trees
463 1982,
Negm and Loescher 1979, Nosarszewski
et al. 2004, Yamaguchi et al. 1994,
Yamaguchi et al. 1996); Vitis vinifera,
Citrus sinensis (Jia et al. 2015); Prunus sp. (Walker et al. 2020), P. persica (Lo Bianco
and Rieger 2002, Morandi et al. 2008)
A. thaliana (Aguayo et al. 2013,
Nosarzewski et al. 2012)
Transport evidence
P. cerasus (Gao et al. 2003), M. domestica
(Li et al. 2018, Watari et al. 2004)
P. major (Fu et al. 2011,
Ramsperger-Gleixner et al. 2004), A. thaliana (Klepek et al. 2005), gramineae
crops (Kong et al. 2020)
Pinitol
Presence in tissues
Eucaliptus sp., Acacia sp. (Merchant et al. 2006)
L. decidua, P. mariana, P. abies, P. sylvestris
(Deslauriers et al. 2014,
Gallinger and Gross
2018, Merchant et al. 2006, Simard et al. 2013)
Glycine max (Streeter et al. 2001)
Presence in phloem sap
B. papyrifera (Shi et al. 2020)
P. abies, Abies alba, P. Medicago sativa (Campbell et al. 1984), . List of species in which evidence of polyol metabolism and transport has been reported. P. sylvestris (Gallinger and
Gross 2018)
A. graveolens (Nadwodnik and Lohaus
2008) A. graveolens (Everard et al. 1997,
Loescher et al. 1992, Rumpho et al. 1983)
A. graveolens (Rumpho et al. 1983, Stoop
and Pharr 1992, Stoop and Pharr 1994,
Williamson et al. 1995, Stoop et al. 1996,
Zamski et al. 2001); Arabidopsis thaliana
(Maruta et al. 2008)
A. graveolens (Fu et al. 2011, Noiraud et
al. 2001)
P. major, Plantago maritima (Pommerrenig
et al. 2007, Nadwodnik and Lohaus
2008)
Hordeum vulgare (Bartels et al. 1991),
Zea mays (Yang et al. 2020)
A. thaliana (Aguayo et al. 2013,
Nosarzewski et al. 2012)
P. major (Fu et al. 2011,
Ramsperger-Gleixner et al. 2004), A. thaliana (Klepek et al. 2005), gramineae
crops (Kong et al. 2020)
P. 14,
Glycine max (Streeter et al. 2001)
Medicago sativa (Campbell et al. 1984),
P. sativum (Blicharz et al. 2021), Lupinus
angustifolius (Merchant 2012)
Downloaded from https://academic.oup.com/treephys/article/42/3/458/6372535 by Sveriges Lantbruksuniversitet user on 06 October 2022 A. graveolens (Everard et al. 1997,
Loescher et al. 1992, Rumpho et al. 1983)
A. graveolens (Rumpho et al. 1983, Stoop
and Pharr 1992, Stoop and Pharr 1994,
Williamson et al. 1995, Stoop et al. 1996,
Zamski et al. 2001); Arabidopsis thaliana
(Maruta et al. 2008)
A. graveolens (Fu et al. 2011, Noiraud et
al. 2001) Euonymus japonica (Bliss et al. 1972,
Negm 1986) Tree Physiology Volume 42, 2022 Mobile forms of carbon in trees
463 persica (Moing et al. 1997, Nadwodnik
and Lohaus 2008), Cocos nucifera
(Nakamura et al. 2004), Citrus sinensis
(Killiny 2016), Prunus domestica
(Gallinger and Gross 2018)
Synthesis reactions
M. domestica (Cheng et al. 2005, Negm
and Loescher 1981, Tao et al. 1995,
Zhou et al. 2003), Eriobotrya japonica
(Hirai 1981), P. persica (Hartman et al. 2017)
Catabolism reactions
M. domestica (Loescher et al. 1982,
Negm and Loescher 1979, Nosarszewski
et al. 2004, Yamaguchi et al. 1994,
Yamaguchi et al. 1996); Vitis vinifera,
Citrus sinensis (Jia et al. 2015); Prunus sp. (Walker et al. 2020), P. persica (Lo Bianco
and Rieger 2002, Morandi et al. 2008)
Transport evidence
P. cerasus (Gao et al. 2003), M. domestica
(Li et al. 2018, Watari et al. 2004)
Pinitol
Presence in tissues
Eucaliptus sp., Acacia sp. (Merchant et al. 2006)
Presence in phloem sap
B. papyrifera (Shi et al. 2020) Table 2. List of species in which evidence of polyol metabolism and transport has been reported. Evidence in angiosperm trees/shrubs
Evidence in gymnosperm
trees
Evidence in herbaceous plants
Mannitol
Presence in phloem sap
F. excelsior (Öner-Sieben and Lohaus
2014); Citrus sinensis, Murraya
paniculata, Bergera koenegii (Killiny
2016); Prunus domestica (Gallinger and
Gross 2018)
P. sylvestris (Gallinger and
Gross 2018)
A. graveolens (Nadwodnik and Lohaus
2008)
Synthesis reactions
Ligustrum vulgare (Loescher et al. 1992)
A. graveolens (Everard et al. 1997,
Loescher et al. 1992, Rumpho et al. 1983)
Catabolism reactions
O. europaea (Conde et al. 2008, 2011)
A. graveolens (Rumpho et al. 1983, Stoop
and Pharr 1992, Stoop and Pharr 1994,
Williamson et al. 1995, Stoop et al. 1996
Zamski et al. 2001); Arabidopsis thaliana
(Maruta et al. 2008)
Transport evidence
O. europaea (Conde et al. 2007, 2008)
A. graveolens (Fu et al. 2011, Noiraud et
al. 2001)
Galactitol/dulcitol
Synthesis reactions
Euonymus japonica (Bliss et al. 1972,
Negm 1986)
Sorbitol/glucitol
Presence in phloem sap
P. persica (Moing et al. 1997, Nadwodnik
and Lohaus 2008), Cocos nucifera
(Nakamura et al. 2004), Citrus sinensis
(Killiny 2016), Prunus domestica
(Gallinger and Gross 2018)
P. major, Plantago maritima (Pommerrenig
et al. 2007, Nadwodnik and Lohaus
2008)
Synthesis reactions
M. domestica (Cheng et al. 2005, Negm
and Loescher 1981, Tao et al. 1995,
Zhou et al. 2003), Eriobotrya japonica
(Hirai 1981), P. persica (Hartman et al. 2017)
Hordeum vulgare (Bartels et al. 1991),
Zea mays (Yang et al. 2020)
Catabolism reactions
M. domestica (Loescher et al. Mobile forms of carbon in trees
463 The epimerization transformation is a two-
reaction step: first, sequoyitol is transformed into a keto interme-
diate, D-5-O-methyl-2,3,5/4,6-pentahydroxycyclohexanone, by
a NAD-dependent sequoyitol dehydrogenase (EC 1.1.1.143);
afterward, the keto intermediate is converted into D-pinitol by
a NADP-dependent D-pinitol dehydrogenase (EC 1.1.1.142). It is tempting to suggest that angiosperms and gymnosperm
trees synthetize pinitol through different pathways (Figure 2B),
but systematic analyses need to be performed for irrefutable
conclusions. Sorbitol is the most abundant C form in the phloem
of the Rosaceae (Bieleski 1982), which serves as model
species for sorbitol metabolism studies. It is synthesized from
glucose-6-P, which is converted to sorbitol-6-P by a NADPH-
dependent aldose-6-P-reductase (ALD-6-PRase; EC 1.1.1.200)
(Figure 2B, Table 2) (Hirai 1981, Negm and Loescher 1981,
Tao et al. 1995, Cheng et al. 2005, Hartman et al. 2017). Sorbitol-6-P is dephosphorylated by a sorbitol-6-P phosphatase
(S6PP; EC 3.1.3.50) into sorbitol (Zhou et al. 2003). The synthesis of myo-inositol starts with the conversion of
glucose-6-P into myo-inositol-1-P by INPS (inositol-3-P syn-
thase) (also called myo-inositol-1-P synthase) (EC 5.5.1.4)
(Figure 2B) (Gumber et al. 1984, Loewus et al. 1984, Johnson
and Sussex 1995, Zhang et al. 2018, Hu et al. 2020). This
is the first step in the synthesis of all the inositols found
in plants. Myo-inositol-1-P is then converted into myo-inositol
by the action of IMPase (inositol mono phosphatase) (EC
3.1.3.25) (Laing et al. 2004, Torabinejad et al. 2009, Nour-
bakhsh et al. 2015, Ruszkowski and Dauter 2016, Yadav et al. 2020). As mentioned above, pinitol may be used for C transport
in gymnosperm trees and angiosperm plants (Table 2). The
synthesis of D-pinitol has been mainly studied in herbaceous
plants where it occurs through the formation of D-ononitol
or of sequoyitol (Figure 2B, Table 2). In most of the studied
angiosperms, the synthesis occurs through D-ononitol. Myo-
inositol is methylated producing D-ononitol (1D-4-O-methyl Tree Physiology Online at http://www.treephys.oxfordjournals.org Dominguez and Niittylä 464 Table 2. List of species in which evidence of polyol metabolism and tra
Evidence in angiosperm trees/shrubs
Mannitol
Presence in phloem sap
F. excelsior (Öner-Sieben and Lohaus
2014); Citrus sinensis, Murraya
paniculata, Bergera koenegii (Killiny
2016); Prunus domestica (Gallinger and
Gross 2018)
Synthesis reactions
Ligustrum vulgare (Loescher et al. 1992)
Catabolism reactions
O. europaea (Conde et al. 2008, 2011)
Transport evidence
O. europaea (Conde et al. 2007, 2008)
Galactitol/dulcitol
Synthesis reactions
Euonymus japonica (Bliss et al. 1972,
Negm 1986)
Sorbitol/glucitol
Presence in phloem sap
P. Euonymus japonica (Bliss et al. 1972,
Negm 1986) Catabolism reactions: biochemical or physiological experiments in
sink tissues. Transport evidence: transporters in sink/source tissues or tracer experiments. Table 3. List of species in which evidence of RFOs metabolism and transport has been reported. Evidence in angiosperm trees
Evidence in
gymnosperm trees
Evidence in herbaceous plants
Presence in tissues
P. trichocarpa × deltoides (Philippe et al. 2010), E. globulus (Merchant et al. 2012),
Populus sp. (Zhou et al. 2014), B. pendula
(Riikonen et al. 2013), E. speciosa (Hell et al. 2019)
P. halepensis (Ben
Youssef et al. 2016),
P. taeda (Pullman and
Buchanan 2008),
Pinus sp.,
Cupressus × leylandii
(Fischer and Höll 1991,
Hinesley et al. 1992)
Cucurbita sp., Cucumis sp., Phaseolus sp.,
Coleus blumei Benth, Vicia sp. (Madore
2001)
Presence in phloem
sap
O. europaea (Flora and Madore 1993), E. globulus (Pate et al. 1998, Merchant et al. 2010, Merchant et al. 2012), Cocos nucifera
(Nakamura et al. 2004), F. excelsior
(Öner-Sieben and Lohaus 2014)
Madore (2001)
Synthesis
(Biochemical or
physiological
experiments in
source tissues)
Populus sp. (Unda et al. 2012, Zhou et al. 2014)
Cucurbita pepo, C. sativus, Phaseolus
vulgaris, Coleus blumei Benth, Vicia faba, C. melo, Lens culinaris, Vigna anularis (Madore
2001); A. reptans (Haab and Keller 2002);
P. sativum (Peterbauer et al. 2002). Catabolism
(Biochemical or
physiological
experiments in sink
tissues)
C. melo (Carmi et al. 2003, Gao and
Schaffer 1999, Hubbard et al. 1989), C. sativus (Hu et al. 2009), Cucurbita pepo
(Gaudreault and Webb 1983), Arabidopsis
thaliana (Peters et al. 2010)
Transport
(transporters in
sink/source tissues
or tracer
experiments)
O. europaea (Flora and Madore 1993); C. speciosa, F. americana (Fu et al. 2011); S. meyeri (Rennie and Turgeon 2009); S. reticulata (Fu et al. 2011, Rennie and Turgeon
2009); F. excelsior (Öner-Sieben and Lohaus
2014)
Putative transporter in Arabidopsis thaliana
and A. reptans (Schneider and Keller 2009)
Downloaded from https://academic.oup.com/treephys/article/42/3/458/6372535 by Sveriges Lantbruksuniversitet user on 06 October 2022 Mobile forms of carbon in trees
465 465 Synthesis reactions: biochemical or physiological experiments in source tissues. Catabolism reactions: biochemical or physiological experiments in
sink tissues. Transport evidence: transporters in sink/source tissues or tracer experiments. Synthesis reactions: biochemical or physiological experiments in source tissues. Catabolism reactions: biochemical or physiological experiments in
sink tissues. Transport evidence: transporters in sink/source tissues or tracer experiments. Table 3. List of species in which evidence of RFOs metabolism and transport has been reported. Table 3. Euonymus japonica (Bliss et al. 1972,
Negm 1986) List of species in which evidence of RFOs metabolism and transport has been reported. Evidence in angiosperm trees
Evidence in
gymnosperm trees
Evidence in herbaceous plants
Presence in tissues
P. trichocarpa × deltoides (Philippe et al. 2010), E. globulus (Merchant et al. 2012),
Populus sp. (Zhou et al. 2014), B. pendula
(Riikonen et al. 2013), E. speciosa (Hell et al. 2019)
P. halepensis (Ben
Youssef et al. 2016),
P. taeda (Pullman and
Buchanan 2008),
Pinus sp.,
Cupressus × leylandii
(Fischer and Höll 1991,
Hinesley et al. 1992)
Cucurbita sp., Cucumis sp., Phaseolus sp.,
Coleus blumei Benth, Vicia sp. (Madore
2001)
Presence in phloem
sap
O. europaea (Flora and Madore 1993), E. globulus (Pate et al. 1998, Merchant et al. 2010, Merchant et al. 2012), Cocos nucifera
(Nakamura et al. 2004), F. excelsior
(Öner-Sieben and Lohaus 2014)
Madore (2001)
Synthesis
(Biochemical or
physiological
experiments in
source tissues)
Populus sp. (Unda et al. 2012, Zhou et al. 2014)
Cucurbita pepo, C. sativus, Phaseolus
vulgaris, Coleus blumei Benth, Vicia faba, C. melo, Lens culinaris, Vigna anularis (Madore
2001); A. reptans (Haab and Keller 2002);
P. sativum (Peterbauer et al. 2002). Catabolism
(Biochemical or
physiological
experiments in sink
tissues)
C. melo (Carmi et al. 2003, Gao and
Schaffer 1999, Hubbard et al. 1989), C. sativus (Hu et al. 2009), Cucurbita pepo
(Gaudreault and Webb 1983), Arabidopsis
thaliana (Peters et al. 2010)
Transport
(transporters in
sink/source tissues
or tracer
experiments)
O. europaea (Flora and Madore 1993); C. speciosa, F. americana (Fu et al. 2011); S. meyeri (Rennie and Turgeon 2009); S. reticulata (Fu et al. 2011, Rennie and Turgeon
2009); F. excelsior (Öner-Sieben and Lohaus
2014)
Putative transporter in Arabidopsis thaliana
and A. reptans (Schneider and Keller 2009)
ic.oup.com/treephys/article/42/3/458/6372535 by Sveriges Lantbruksuniversitet user on 06 October 2022 Cucurbita pepo, C. sativus, Phaseolus
vulgaris, Coleus blumei Benth, Vicia faba, C. melo, Lens culinaris, Vigna anularis (Madore
2001); A. reptans (Haab and Keller 2002);
P. sativum (Peterbauer et al. 2002). C. melo (Carmi et al. 2003, Gao and
Schaffer 1999, Hubbard et al. 1989), C. sativus (Hu et al. 2009), Cucurbita pepo
(Gaudreault and Webb 1983), Arabidopsis
thaliana (Peters et al. 2010) O. europaea (Flora and Madore 1993); C. speciosa, F. americana (Fu et al. 2011); S. meyeri (Rennie and Turgeon 2009); S. reticulata (Fu et al. 2011, Rennie and Turgeon
2009); F. excelsior (Öner-Sieben and Lohaus
2014) O. europaea (Flora and Madore 1993); C. speciosa, F. Euonymus japonica (Bliss et al. 1972,
Negm 1986) P. persica (Moing et al. 1997, Nadwodnik
and Lohaus 2008), Cocos nucifera
(Nakamura et al. 2004), Citrus sinensis
(Killiny 2016), Prunus domestica
(Gallinger and Gross 2018)
M. domestica (Cheng et al. 2005, Negm
and Loescher 1981, Tao et al. 1995,
Zhou et al. 2003), Eriobotrya japonica
(Hirai 1981), P. persica (Hartman et al. 2017) P. persica (Moing et al. 1997, Nadwodnik
and Lohaus 2008), Cocos nucifera
(Nakamura et al. 2004), Citrus sinensis
(Killiny 2016), Prunus domestica
(Gallinger and Gross 2018)
M domestica (Cheng et al 2005 Negm Synthesis reactions Catabolism reactions M. domestica (Loescher et al. 1982,
Negm and Loescher 1979, Nosarszewski
et al. 2004, Yamaguchi et al. 1994,
Yamaguchi et al. 1996); Vitis vinifera,
Citrus sinensis (Jia et al. 2015); Prunus sp. (Walker et al. 2020), P. persica (Lo Bianco
and Rieger 2002, Morandi et al. 2008)
P. cerasus (Gao et al. 2003), M. domestica
(Li et al. 2018, Watari et al. 2004) Continued Mobile forms of carbon in trees
465
Table 2. Continued. Evidence in angiosperm trees/shrubs
Evidence in gymnosperm
trees
Evidence in herbaceous plants
Synthesis reactions
S. chinensis (Dittrich and Korak 1984)
J. communis, T. baccata
(Dittrich and Kandler
1972)
M. sativa, Ononis spinosa, Trifolium
incarnatum (Dittrich and Brandl 1987);
Mesembryanthemum crystallinum (Chiera
et al. 2006, Vernon and Bohnert 1992);
A. thaliana (Ahn et al. 2018); M. truncatula (Pupel et al. 2019)
Quebrachitol
Presence in tissues
Heterodendrum oleifolium (Merchant et al. 2006)
Presence in phloem sap
A. platanoides (Schill et al. 1996); L. chinensis (Wu et al. 2018)
Synthesis reactions
L. chinensis (Wu et al. 2018)
Synthesis reactions: biochemical or physiological experiments in source tissues. Catabolism reactions: biochemical or physiological experiments in
sink tissues. Transport evidence: transporters in sink/source tissues or tracer experiments. Mobile forms of carbon in trees
465
Table 2. Continued. Evidence in angiosperm trees/shrubs
Evidence in gymnosperm
trees
Evidence in herbaceous plants
Synthesis reactions
S. chinensis (Dittrich and Korak 1984)
J. communis, T. baccata
(Dittrich and Kandler
1972)
M. sativa, Ononis spinosa, Trifolium
incarnatum (Dittrich and Brandl 1987);
Mesembryanthemum crystallinum (Chiera
et al. 2006, Vernon and Bohnert 1992);
A. thaliana (Ahn et al. 2018); M. truncatula (Pupel et al. 2019)
Quebrachitol
Presence in tissues
Heterodendrum oleifolium (Merchant et al. 2006)
Presence in phloem sap
A. platanoides (Schill et al. 1996); L. chinensis (Wu et al. 2018)
Synthesis reactions
L. chinensis (Wu et al. 2018)
Synthesis reactions: biochemical or physiological experiments in source tissues. Euonymus japonica (Bliss et al. 1972,
Negm 1986) americana (Fu et al. 2011); S. meyeri (Rennie and Turgeon 2009); S. reticulata (Fu et al. 2011, Rennie and Turgeon
2009); F. excelsior (Öner-Sieben and Lohaus
2014) Putative transporter in Arabidopsis thaliana
and A. reptans (Schneider and Keller 2009) from the genus Quercus, which presents high levels of this
compound. There are some indications suggesting that it could
be transported in phloem based on phloem sap measurements
in eucalyptus, although the data are not clear (Arndt et al. Quercitol (cyclohexanepentol) is a cyclitol present in several
trees including Mimusops hexandra Roxb., Eucalyptus sp. and
oak (Quercus sp.) (Misra and Mitra 1968, Merchant et al. 2006, 2007, Rodríguez-Sánchez et al. 2010). Its name derives Tree Physiology Online at http://www.treephys.oxfordjournals.org Tree Physiology Online at http://www.treephys.oxfordjournals.org Dominguez and Niittylä 466 species suggests a correlation to environmental adaptation
(Merchant et al. 2007); similar studies related to other trees
remain to be done. There are also other hypotheses that could
explain the reason of plants evolving to transport sugar alcohols. In this regard, Li et al. (2018) offered two interesting hypothe-
ses for sorbitol transport in apple trees (Malus domestica
Borkh.). On one hand, they observed that reducing the synthesis
of sorbitol in apple tree leaves did not alter the phenotype of the
fruits, but rather increased sucrose transport and metabolism. This suggests that being able to transport two different types
of molecules increases the metabolic flexibility of the Rosaceae
trees. On the other hand, sorbitol is converted to fructose by
a sorbitol dehydrogenase (SDH, EC 1.1.1.14) upon phloem
unloading in fruits. Fructose is sweater than any other sugars
present in fruits, which could make them more attractive for
seed-dispersing animals. 2008). The synthesis pathway of quercitols in plants has not yet
been elucidated. In bacteria, it has been proposed that it could
proceed from glucose and from myo-inositol (Itoh 2018). The
presence and role of quercitols in trees have also been related
to different types of environmental stresses (Arndt et al. 2008,
Sardans et al. 2014). Bornesitol
(O-methyl-myo-inositol),
quebrachitol
(2-O-
methyl-chiro-inositol)
and
polygalatol
(1,5-anhydrosorbitol)
were systematically analyzed in the Proteaceae family (Bieleski
and Briggs 2005). These sugar alcohols were found in high
amounts in several tree genera including Carnarvonia, Leuca-
dendron and Faurea, among others. Quebrachitol represents
40% of sugars in the phloem exudate of the fruit tree Litchi
chinensis (Wu et al. The raffinose family of oligosaccharides Galactose-containing oligosaccharides are involved in several
physiological processes such as preventing seed desiccation,
transient C storage in seeds and C phloem transport (Madore
2001). These oligosaccharides contain short galactan chains
usually linked to a sucrose molecule. They form a large chemical
family that includes umbelliferose (present in Apiaceae),
planteose and sesamose [present in the seeds of plantain
(Musa × paradisiaca), sesame (Sesamum indicum) and ash],
galactosylcyclitols (they have a cyclic sugar alcohol backbone
instead of a sucrose backbone and are present in legumes) and
galactosyloligosaccharides based on raffinose. The latter group
is composed of molecules that have a raffinose backbone and
includes the RFOs, the lychnose and the isolychnose series;
the last two being produced in the Caryophyllaceae family. Of
all these oligosaccharides, RFOs are the most abundant in the
plant kingdom and are recognized to participate in phloem C
transport. A common feature of the phloem mobile C biosynthesis
pathways summarized in Figure 2 is the phosphatase catalyzed
hydrolysis reaction. sucrose-phosphate phosphatase (SPP) cat-
alyzes essentially an irreversible reaction in vivo pulling the SPS
reaction toward net sucrose synthesis (Lunn and Rees 1990). M1PP, S6PP and IMPase have not been characterized in detail,
but an irreversible phosphatase reaction could facilitate the
generation of the concentration gradient driving symplasmic C
transport. In addition to C transport, sugar alcohols have other functions
including C storage (Moing 2000), involvement in the response
to abiotic and biotic stresses (Arndt et al. 2008, Kanayama
2009, Conde et al. 2011, Wu et al. 2015, Pupel et al. 2019),
boron transport (Liakopoulos et al. 2005), and stamen develop-
ment and pollen tube growth (Meng et al. 2018). Their role as
osmoprotectants during stress may be particularly relevant for
trees. For example, mannitol accumulates in olive trees under
salt stress and drought (Conde et al. 2011) and pinitol in black
spruce (P. mariana) under mild water deficit (Deslauriers et al. 2014). The prevalence of sugar alcohols as a C transport form
is believed to be related to its chemical inertness (Dumschott
et al. 2017) and to the fact that sugar alcohols are not part
of the core primary metabolism, which may prevent them
from being metabolized quickly or from inhibiting photosynthe-
sis (Merchant and Richter 2011). Euonymus japonica (Bliss et al. 1972,
Negm 1986) 2018), and it can also be found in
the sugar maple (Acer saccharum) xylem sap and the syrup
produced from it (Stinson et al. 1967), and in the phloem
sap of Acer platanoides (Table 2) (Schill et al. 1996). The
synthesis of quebrachitol starts when myo-inositol is methylated
by an inositol-1-O-methyltransferase gene (IMT) (EC 2.1.1.40)
to form bornesitol (Figure 2B, Table 2) (Wu et al. 2018). Hypothetically, bornesitol is epimerized to quebrachitol by an
unidentified epimerase (EC 5.1.3) (Wu et al. 2018). For its part,
the synthesis of polygalatol has not been described in plants,
but it is probably synthetized through a different pathway since
it is not a structural derivative of inositol (Bieleski and Briggs
2005). The raffinose family of oligosaccharides In eucalyptus, it has been
observed that the distribution of sugar alcohols among the The RFOs are the main form of C transport in the Cucur-
bitaceae and Scrophulariaceae (Ma et al. 2019), and in trees,
they can be found in olive, Eucalyptus globulus Labill. (eucalyp-
tus), Catalpa speciosa (Northern catalpa), Pinus halepensis Mill. (Aleppo pine) and ash (Flora and Madore 1993, Rennie and
Turgeon 2009, Merchant et al. 2012, Öner-Sieben and Lohaus
2014, Ben Youssef et al. 2016) (an extended list can be found
in Table 3). Despite this, the study of RFOs transport in trees at
the molecular level has not received much attention, and most
of the research has been focused on classic plant models such
as cucumber (Cucumis sativus L.). In trees, RFOs have been
mainly studied related to their role in stress responses. Raffinose
family of oligosaccharides (RFOs) (and also polyols) can act
during stresses as compatible solutes, which are highly soluble
non-toxic compounds that protect cells during stress. Several
different protective mechanisms of compatible solutes have
been proposed including reactive oxygen species scavenging Tree Physiology Volume 42, 2022 Mobile forms of carbon in trees 467 (by reacting with them), and stabilization of proteins and
membranes (by replacing the hydroxyl groups of water that
help to maintain their structural integrity) (Chen and Murata
2002, Van den Ende 2013, Elsayed et al. 2014, Sengupta et
al. 2015). Zhou et al. (2014) found that all GolS (galactinol
synthase) genes (except for GolS9), which are involved in RFO
synthesis, changed following salt and water-deficit stresses in
aspen trees. Raffinose family of oligosaccharides (RFO) levels
can also be increased under herbivorous stress in hybrid poplar
(Populus trichocarpa Torr. & A. Gray ex. Hook × deltoides W. Bartram ex Marshall) (Philippe et al. 2010) or under elevated
temperatures in silver birch (Betula pendula) buds (Riikonen et
al. 2013). Raffinose has also been linked to seed desiccation
tolerance in the tree Erythrina speciosa Andrews (Hell et al. 2019). The accumulation of RFOs in gymnosperms trees has
been described in seeds of P. halepensis (Ben Youssef et al. 2016), in embryos of Pinus taeda L. (Pullman and Buchanan
2008) and in the needles of Pinus sp. and Leyland cypress
(Cupressus × leylandii) (Fischer and Höll 1991, Hinesley et
al. 1992). Although Saranpää and Höll (1989) detected RFOs
in the xylem sap of P. sylvestris, their role in gymnosperm C
transport remains unexplored. The raffinose family of oligosaccharides degree of polymerization can also be formed in a galactinol-
dependent pathway. In pea (Pisum sativum L.), the donor of
the galactosyl group to form verbascose from stachyose is
galactinol instead of an RFO via a reaction also mediated by
a galactosyltransferase (Peterbauer et al. 2002). So far Unda et
al. (2012) and Zhou et al. (2014) are the only studies in which
galactinol synthases of a tree species (Populus sp.) have been
characterized (Table 3). Overexpression of AtGolS3 in hybrid
poplar (P. alba × grandidentata) altered stem secondary cell
walls and caused starch accumulation in ray cells (Unda et al. 2017), indicating that these compounds may have a role in C
partitioning during wood formation. Phloem loading in trees All three current phloem loading models have been reported
to exist in trees: symplasmic, apoplasmic and symplasmic poly-
mer trapping loading. The evidence for these different loading
models is mainly based on the presence or absence of sym-
plasmic connections between cells, and in the case of polymer
trapping the presence of RFOs in the phloem tissue. Except for
the RFO polymer trapping, the loading mechanism seems to
be species specific rather than molecule-type specific (Moing
2000). A summary of the loading mechanisms found in trees
and shrubs is presented in Table 4. Approximately half of the
plant families that include several tree species are characterized
as symplasmic loaders; however, more systematic analyses
are needed to establish whether symplasmic loading is the
predominant form of transport in trees (Liesche 2017). Strong
evidence for sucrose symplasmic loading in poplars (Populus
sp.) comes from the lack of C transport-related defects in
transgenics expressing a yeast invertase in the apoplasm, which
would interfere with apoplasmic sucrose loading (Zhang et al. 2014). In addition, symplasmic loading is thought to dominate
in some species that predominantly transport sugar alcohols
such as willow (Salix babylonica L.), apple trees and peach
trees (Prunus persica) (Turgeon and Medville 1998, Reidel et al. 2009, Fu et al. 2011), among others (Table 4). Phloem loading
strategies in gymnosperm trees have been only tested in three
species: P. sylvestris (Liesche and Schulz 2012), Pinus mugo
Turra and Ginkgo biloba L. (Liesche 2017). The tested species
are symplasmic loaders, but more studies are needed to estab-
lish whether this is a common trait of gymnosperms (Table 4)
(Liesche 2017). Pinus sylvestris is known to load pinitol and
mannitol together with sucrose (Figure 3) (Gallinger and Gross
2018), but similar measurements on P. mugo and G. biloba have
not been performed so far. hys/article/42/3/458/6372535 by Sveriges Lantbruksuniversitet user on 06 October 2022 Downloaded from https://academic.oup.com/treephys/article/42/3/458/6372535 by Sveriges Table 4. Phloem loading mechanisms in trees and shrubs. Sucrose loading
Sucrose and polyol loading
RFO loading1
Symplasmic loaders
Populus sp. (Zhang et al. 2014), S. babylonica (Turgeon and Medville 1998);
Cercidiphyllum japonicum, Corylus colurna,
F. sylvatica, Juglans ailanthifolia, Platanus
acerifolia, P. alba, Q. coccinea (Fu et al. 2011); Corylus colurna, J. ailantifolia, F. sylvatica, Pterocarya illinoisensis, Tilia
americana, Platanus occidentalis,
Rhododendron sc lippenbachii, A. saccharum, Aesculus pavia (Rennie and
Turgeon 2009); P. mugo and G. biloba
(Liesche 2017)
M. domestica (Fu et al. 2011,
Reidel et al. 2009); Amelanchier
laevis, Prunus armeniaca, Prunus
avium, P. cerasus, Prunus
domestica, P. persica, Pyrus
communis, Spiraea japonica (Fu
et al. 2011); Prunus laurocerasus,
Sorbus hybrida (Rennie and
Turgeon 2009); P. sylvestris
(Liesche and Schulz 2012)
Apoplasmic loaders
Q. robur (Öner-Sieben and Lohaus 2014);
Alnus glutinosa, Liquidambar styraciflua,
Liriodendron tulipifera, Phellodendron
lavallei (Fu et al. 2011); Ilex meservae,
Cercis candensis (Rennie and Turgeon
2009); Halesia tetraptera (Fu et al. 2011,
Rennie and Turgeon 2009)
Combined symplasmic
and apoplasmic loaders
F. excelsior (Öner-Sieben and Lohaus
2014)
Polymer trap loaders
Any species transporting
ROFs including C
Downloaded from https://academic.oup.com/treephys/a Table 4. Phloem loading mechanisms in trees and shrubs. Table 4. Phloem loading mechanisms in trees and shrubs. Downloaded from https://academic.oup.com/treephys/article/42/3/458/6372535 by Sveriges Lantbruksuniversitet user on 06 October 2022 1RFO loading is accompanied by sucrose and/or polyol loading by means of one of the other mechanisms. herbaceous species are known to use this pathway, e.g., in the
mannitol transporting celery (Apium graveolens L.) (Nadwodnik
and Lohaus 2008, Fu et al. 2011). Apoplasmic phloem
loading requires the presence of sugar/polyol exporters and
importers in leaves. In herbaceous species, SWEET (sugar will
be eventually exported) and SUT (sucrose/proton symport)
transporters have been shown to be involved in the sucrose
export and import, respectively, driving sucrose movement
toward the SE/CCC (sieve element-companion cell complexes)
in leaves (Table 1) (Riesmeier et al. 1993, Bürkle et al. 1998,
Slewinski et al. 2009, Chen et al. 2012, Bezrutczyk et al. 2018, Kim et al. 2021). Most of them remain to be functionally
characterized in trees. Some SUT expression and localization
studies have been performed in hybrid poplar (Populus tremula
L. × alba L.) leaves (Payyavula et al. 2011). The silencing
of the most abundant leaf SUT, which is tonoplast SUT4, led
to increased leaf-to-stem biomass ratio, which suggests a role
in C partitioning. hys/article/42/3/458/6372535 by Sveriges Lantbruksuniversitet user on 06 October 2022 mugo and G. biloba
(Liesche 2017)
M. domestica (Fu et al. 2011,
Reidel et al. 2009); Amelanchier
laevis, Prunus armeniaca, Prunus
avium, P. cerasus, Prunus
domestica, P. persica, Pyrus
communis, Spiraea japonica (Fu
et al. 2011); Prunus laurocerasus,
Sorbus hybrida (Rennie and
Turgeon 2009); P. sylvestris
(Liesche and Schulz 2012)
Apoplasmic loaders
Q. robur (Öner-Sieben and Lohaus 2014);
Alnus glutinosa, Liquidambar styraciflua,
Liriodendron tulipifera, Phellodendron
lavallei (Fu et al. 2011); Ilex meservae,
Cercis candensis (Rennie and Turgeon
2009); Halesia tetraptera (Fu et al. 2011,
Rennie and Turgeon 2009)
Combined symplasmic
and apoplasmic loaders
F. excelsior (Öner-Sieben and Lohaus
2014)
Polymer trap loaders
Any species transporting
ROFs, including C. speciosa (Fu et al. 2011), F. americana (Fu
et al. 2011), S. meyeri
(Rennie and Turgeon
2009), S. reticulata (Fu
et al. 2011, Rennie and
Turgeon 2009), F. excelsior (Öner-Sieben
and Lohaus 2014)
1RFO loading is accompanied by sucrose and/or polyol loading by means of one of the other mechanisms. Downloaded from https://academic.oup.com/treephys/article/42/3/458/6372535 by Sverige Table 4. Phloem loading mechanisms in trees and shrubs. Sucrose loading
Sucrose and polyol loading
RFO loading1
Symplasmic loaders
Populus sp. (Zhang et al. 2014), S. babylonica (Turgeon and Medville 1998);
Cercidiphyllum japonicum, Corylus colurna,
F. sylvatica, Juglans ailanthifolia, Platanus
acerifolia, P. alba, Q. coccinea (Fu et al. 2011); Corylus colurna, J. ailantifolia, F. sylvatica, Pterocarya illinoisensis, Tilia
americana, Platanus occidentalis,
Rhododendron sc lippenbachii, A. saccharum, Aesculus pavia (Rennie and
Turgeon 2009); P. mugo and G. biloba
(Liesche 2017)
M. domestica (Fu et al. 2011,
Reidel et al. 2009); Amelanchier
laevis, Prunus armeniaca, Prunus
avium, P. cerasus, Prunus
domestica, P. persica, Pyrus
communis, Spiraea japonica (Fu
et al. 2011); Prunus laurocerasus,
Sorbus hybrida (Rennie and
Turgeon 2009); P. sylvestris
(Liesche and Schulz 2012)
Apoplasmic loaders
Q. robur (Öner-Sieben and Lohaus 2014);
Alnus glutinosa, Liquidambar styraciflua,
Liriodendron tulipifera, Phellodendron
lavallei (Fu et al. 2011); Ilex meservae,
Cercis candensis (Rennie and Turgeon
2009); Halesia tetraptera (Fu et al. 2011,
Rennie and Turgeon 2009)
Combined symplasmic
and apoplasmic loaders
F. excelsior (Öner-Sieben and Lohaus
2014)
Polymer trap loaders
Any species transporting
ROFs, including C. speciosa (Fu et al. 2011), F. americana (Fu
et al. 2011), S. meyeri
(Rennie and Turgeon
2009), S. reticulata (Fu
et al. 2011, Rennie and
Turgeon 2009), F. excelsior (Öner-Sieben
and Lohaus 2014)
1RFO loading is accompanied by sucrose and/or polyol loading by means of one of the other mechanisms. Combined symplasmic
and apoplasmic loaders
Polymer trap loaders hys/article/42/3/458/6372535 by Sveriges Lantbruksuniversitet user on 06 October 2022 The RFOs have one or more α-D-galactopyranosyl (galactose
in the pyranose form) groups in their structures linked to one
molecule of sucrose by means of α-(1→6) glycosidic linkages
(Figure 1C) (Peterbauer et al. 2001). Like sucrose, they are
non-reducing sugars, but unlike sucrose, they can accumulate
as storage compounds without being directly involved in the
core reactions of primary metabolism (Peters et al. 2007). An additional advantage can be that RFOs deliver at least 1.5
times the amount of C compared with sucrose, but without
increasing the osmotic potential (Madore 2001). The most
usual RFOs are raffinose, stachyose and verbascose (Peterbauer
et al. 2001, Sengupta et al. 2015, Vinson et al. 2020). The
synthesis of these compounds has been mainly studied in
herbaceous plants (Table 3). As a first step in their synthesis,
galactinol is formed by the reaction between UDP-galactose
(uridine diphosphate-galactose) and myo-inositol, catalyzed by
the galactinol synthase (EC 2.4.1.123) (GolS) (Figure 2C). The galactinol serves as the donor of the galactosyl moiety
to one molecule of sucrose to form raffinose via a reaction
catalyzed by the raffinose synthase (EC 2.4.1.82) (RafS). The
thus formed raffinose can be added another galactosyl moiety by
the stachyose synthase (StaS) (EC 2.4.1.67) to form stachyose. Verbascose and larger polymers are formed by the transfer of
galactosyl moiety between different raffinose family oligosac-
charides in reactions catalyzed by galactan:galactan galactosyl-
transferases (EC 2.4.1) (Madore 2001, Haab and Keller 2002). For example, four molecules of stachyose can originate two
molecules of verbascose and two of raffinose, or one molecule
of verbascose reacting with one molecule of stachyose can orig-
inate one ajugose and one raffinose. These polymers with higher Apoplasmic phloem loading has been documented in
several tree species including common oak (Quercus robur L.)
transporting mainly sucrose (Table 4) (Öner-Sieben and Lohaus
2014). More detailed analyses are needed to establish whether
there are apoplast sugar alcohol loading trees, although several Tree Physiology Online at http://www.treephys.oxfordjournals.org Dominguez and Niittylä 468 Table 4. Phloem loading mechanisms in trees and shrubs. Sucrose loading
Sucrose and polyol loading
RFO loading1
Symplasmic loaders
Populus sp. (Zhang et al. 2014), S. babylonica (Turgeon and Medville 1998);
Cercidiphyllum japonicum, Corylus colurna,
F. sylvatica, Juglans ailanthifolia, Platanus
acerifolia, P. alba, Q. coccinea (Fu et al. 2011); Corylus colurna, J. ailantifolia, F. sylvatica, Pterocarya illinoisensis, Tilia
americana, Platanus occidentalis,
Rhododendron sc lippenbachii, A. saccharum, Aesculus pavia (Rennie and
Turgeon 2009); P. Any species transporting
ROFs, including C.
speciosa (Fu et al.
2011), F. americana (Fu
et al. 2011), S. meyeri
(Rennie and Turgeon
2009), S. reticulata (Fu
et al. 2011, Rennie and
Turgeon 2009), F.
excelsior (Öner-Sieben
and Lohaus 2014) hys/article/42/3/458/6372535 by Sveriges Lantbruksuniversitet user on 06 October 2022 Polyol transporters have been identified in leaves of celery (Noiraud et al. 2001), Plantago major L. (Ramsperger-Gleixner et al. 2004), Arabidopsis (Klepek et al. 2005) and gramineae crops (Table 2) (Kong et al. 2020). In
trees, sorbitol transporters (SOTs) have been identified in apple
leaves (Table 2) (Watari et al. 2004), but functional studies
related to their role have not been performed yet. Öner-Sieben
and Lohaus (2014) have shown evidence of the existence
of a complex combined apoplasmic and symplasmic phloem
loading mechanism in ash (Table 4). Discrepancies in the
loading mechanisms of the Quercus genera, in which Quercus
coccinea Münchh. is described as a symplasmic loader (Fu et al. 2011) and Q. robur as an apoplasmic loader (Öner-Sieben and
Lohaus 2014), could be due to mixed loading types (Table 3)
(Liesche 2017). Hence, the emerging picture is that some tree
species may even combine different types of phloem loading
mechanisms (Öner-Sieben and Lohaus 2014, Liesche 2017). The phloem loading of RFOs is thought to occur through the
polymer trap mechanism (Turgeon 1996, Zhang and Turgeon Tree Physiology Volume 42, 2022 Mobile forms of carbon in trees 469 2018). In species that transport RFOs, leaf minor veins have
a specialized type of companion cells called intermediary cells
(ICs). Sucrose is transported from the mesophyll cells to the
bundle sheath where it diffuses to the ICs through specialized
plasmodesmata, characterized by being narrower than regular
plasmodesmata. Once in the ICs, RFOs are synthetized from
sucrose. Because of their size, RFOs cannot diffuse back to
the bundle sheath through the specialized plasmodesmata,
but they can move toward the sieve elements from where
they are transported to sink tissues. In trees, evidence for
this mechanism has been observed in C. speciosa, Fraxinus
americana L., Syringa meyeri C.K.Schneid. and Syringa reticulata
(Blume) H.Hara (Table 4) (Rennie and Turgeon 2009, Fu et
al. 2011). Since this mechanism is specific for RFO transport,
it is found together with symplasmic or apoplasmic loading
mechanisms that transport sucrose and/or polyols (Rennie and
Turgeon 2009). composed; of the bark, the outer layer that provides protection
(Bdeir et al. 2017); the phloem, the living cell conducting
system that transports C and other metabolites from the source
tissues to the sink tissues (Furze et al. hys/article/42/3/458/6372535 by Sveriges Lantbruksuniversitet user on 06 October 2022 2018); and the vascular
cambium, which by repeated division produces phloem cells
to the outside and xylem cells to the inside forming the bulk
of the trunk biomass (Mellerowicz et al. 2001, Campbell and
Turner 2017). Each of these tissues is comprised of a variety
of different cell types organized into an axial and a radial system
(Ek et al. 2009). The axial cell system is orientated in the longi-
tudinal direction of the trunk and its main function is to provide
mechanical support, storage and transport water and nutrients. It
is mainly composed of vessels, longitudinal (axial) parenchyma
(called paratracheal parenchyma if associated to vessels and
apotracheal parenchyma if not in contact with vessels) and
fibers. The radial system is oriented perpendicularly to the tree
and is mostly composed of ray cells that are arranged in lines (or
rays) from the bark to the pith (Figure 4A). All ray cells in one
radial ray cell file are originated from the same ray initial located
in the cambium (Spicer 2014). Thus, the rays stretch from the
cambium toward the phloem and toward the xylem, providing
a radial transport pathway for phloem unloading and C import
to wood. This means that radial transport of C occurs across
the three tissues, the phloem, the cambium and the xylem. In addition to transport and C distribution, ray cells are also
used for C storage (Sauter and Van Cleve 1994, Larisch et al. 2012). Downloaded from https://academic.oup.com/treephys/article/42/3/458/6372535 by Sveriges Lantbruksuniversitet user on 06 October 2022 Since phloem loading processes in plants have been reviewed
and discussed in detail elsewhere (Lalonde et al. 2003, De
Schepper et al. 2013, Lemoine et al. 2013, Slewinski et al. 2013, Liesche 2017, Zhang and Turgeon 2018), the focus in
the subsequent section is on phloem unloading. Trunk tissues and wood formation The development of wood begins with the differentiation of
the lateral meristem, the vascular cambium, which forms a
continuous cylinder extending from shoot to root. Vascular cam-
bium contains secondary xylem mother cells/fusiform initials,
which after periclinal division undergo cell expansion, secondary
wall deposition, programmed cell death and maturation, and
finally heartwood/duramen formation (Ye and Zhong 2015). This process, called xylogenesis, is highly dependent on sugar
supply, which supports cell divisions in the cambium and cell
wall synthesis in the xylem creating a strong C sink. Several
factors influence the C partitioning between sink and source
tissues. However, phloem unloading is the first step that makes C
available for sink tissues and is therefore a key factor influencing
sink strength and, consequently, C partitioning to wood. Phloem unloading and metabolism of the mobile
forms of carbon Trunk tissues and wood formation The phloem unloading and radial transport of carbon in
hardwood xylem during growth ther species such as Arabidopsis and sorghum (Sorghum
SUT and SWEET transporters have been suggested to be
ved in the process based on expression and localization
and Pfanz 2003, Vandegehuchte et al. 2015) might enter the
phloem ray cells directly or through the dilatation parenchyma
cells (a group of enlarged parenchyma cells that form part of
the phloem) or via phloem axial cells (Figure 4B) (Chaffey and
Downloaded from https://academic.oup.com/treephys/article/42/3/458/6372535 by Sveriges Lantbruksuniversitet user on 06 October 2022 Figure 4. Stem anatomy and carbon flow. (A) Cross-section of stems. Upper panel: light microscope image of aspen stem tissues dyed with Safranin–
Alcian blue. Lower panel: representation of a stem. (B and C) Stem carbon flow during growth (B) and during the activation period after dormancy
(C). Arrows in B and C indicate the direction of the carbon flow. Figure 4. Stem anatomy and carbon flow. (A) Cross-section of stems. Upper panel: light microscope image of aspen stem tissues dyed with Safranin–
Alcian blue. Lower panel: representation of a stem. (B and C) Stem carbon flow during growth (B) and during the activation period after dormancy
(C). Arrows in B and C indicate the direction of the carbon flow. and Pfanz 2003, Vandegehuchte et al. 2015) might enter the
phloem ray cells directly or through the dilatation parenchyma
cells (a group of enlarged parenchyma cells that form part of
the phloem) or via phloem axial cells (Figure 4B) (Chaffey and
Barlow 2001). In conifers, the SE/CCCs can directly unload
sugars into the ray cells or via the Strassburger cells follow-
ing a symplasmic route (Sauter 1976, 1980, Pfautsch et al. 2015a). transporters involved in this step are largely unknown in trees. In other species such as Arabidopsis and sorghum (Sorghum
sp.), SUT and SWEET transporters have been suggested to be
involved in the process based on expression and localization
experiments (Liesche and Patrick 2017, Milne et al. 2017,
Falchi et al. 2020). Silencing of SWEET11 and -12, which
are expressed in the phloem and the xylem of Arabidopsis
inflorescence stems, alters vascular development and could
be involved in the phloem unloading (Le Hir et al. 2015). Carbohydrates from the phloem SE/CCCs (or produced by the
photosynthetic phloem cells; Chaffey and Barlow 2001, Aschan As mentioned, C moves across the ray cells following a
radial direction crossing the phloem, the cambium and the
xylem (Figure 4B). The phloem unloading and radial transport of carbon in
hardwood xylem during growth The phloem unloading and radial transport of carbon in
hardwood xylem during growth All cell types that make up the radial and axial cell systems
of wood need to be connected and interacting. The com-
plexity of the stem anatomy and analytical limitations mean
that the knowledge of radial C transport is still fragmented. However, the basic steps in the lateral solute transport dur-
ing growth in trees have been proposed: primary unloading
from the SE/CCC of the phloem, uptake by the other phloem
elements, transport through the phloem ray cells, transport
through the cambium ray cells, transport through the xylem
ray cells and delivery into developing xylem cells or axial
parenchyma cells (Figure 4B) (van Bel 1990, Spicer 2014,
Pfautsch et al. 2015a). In some species, transport between
paratracheal parenchyma and phloem through ray cells was
detected employing radioactive tracers (Van Bel 1990). This
highlights the role of ray cells as a connector between phloem
and xylem. Paratracheal parenchyma and mature vessels can
exchange solutes and water most likely through pits (Plavcová
and Jansen 2015). Angiosperms such as birch (Betula sp.), beech (Fraxinus
sp.), oak and poplar are commonly classified as hardwood,
as opposed to gymnosperms trees (Picea sp., Pinus sp.)
that produce softwood. Hardwoods are anatomically and
physiologically different from softwoods, having a greater variety
in cell morphology (Ek et al. 2009). Below, we will mainly
focus on angiosperm wood, in which the mechanism of phloem
unloading, lateral C transport and C metabolism have been
studied more extensively at the molecular level. There is agreement that SE/CCCs unload sugars apoplasmi-
cally in most angiosperms, including trees such as Salix alba
L. and Eucalyptus saligna Sm. (van Bel 1990, 1996, van Bel
and Kempers 1991, Spicer 2014, Pfautsch et al. 2015b). The Phloem unloading in trees is governed by the arrangement of
the different tissues that form the stem (Figure 4A). The trunk is Tree Physiology Online at http://www.treephys.oxfordjournals.org 470 Dominguez and Niittylä re 4. Stem anatomy and carbon flow. (A) Cross-section of stems. Upper panel: light microscope image of aspen stem tissues dyed with Safranin–
n blue. Lower panel: representation of a stem. (B and C) Stem carbon flow during growth (B) and during the activation period after dormancy
Arrows in B and C indicate the direction of the carbon flow. sporters involved in this step are largely unknown in trees. The phloem unloading and radial transport of carbon in
hardwood xylem during growth In the phloem, axial parenchyma could be Tree Physiology Volume 42, 2022 Mobile forms of carbon in trees 471 SWEET2, -11 and -12 in poplar xylem (Mahboubi and Niittylä
2018), makes them good candidates for being involved in the
sucrose efflux from xylem ray cells. In support of this model,
overexpression of SWEET7 in aspen increased the xylem area
and the stem height and diameter, suggesting that increased
SWEET levels increases C allocation to xylem (Zhang et al. 2021). On the other hand, the sucrose/proton symports SUTs
are involved in sucrose uptake in fibers. Some of them have
been studied in herbaceous plants (Table 1) (Baud et al. 2005,
Hackel et al. 2006, Eom et al. 2012, Milne et al. 2017). In
hybrid aspen (P. tremula × tremuloides) fibers, the silencing
of the plasma membrane sucrose transporter SUT3 generated
alterations in the secondary cell wall chemical composition
and reduced secondary cell wall formation due to impaired C
transport, suggesting that this mechanism is important for the
transport of sucrose into fiber cells (Table 1) (Mahboubi et al. 2013). connected to rays symplasmically since pits are found in their
contact zones. However, information about these cells is limited
due to the difficulties in their identification (Chaffey and Barlow
2001, Spicer 2014). In black poplar (Populus nigra L.), cambial cells are connected
by plasmodesmata, whose number varies according to the
differentiation stage and the season (Figure 4B) (Fuchs et al. 2010). However, other authors have suggested that cambial ray
and fusiform cells are isolated in sycamore (Acer pseudoplatanus
L.) and field elm (Ulmus minor Mill.) during growth (Sokołowska
and Zagórska-Marek 2007). Assimilates may move symplasmically across xylem ray cells
(Figure 4B) (van Bel 1990, Blokhina et al. 2019). The tangen-
tial walls of the ray parenchyma are perforated by numerous
plasmodesmata, aggregated in pit fields in several species
(Chattaway 1951). Sauter and Kloth (1986) suggested that
according to the observed plasmodesmatal frequencies and the
sugar translocation rates, the xylem ray parenchyma cells in
poplar trees were comparable to cells specialized in short dis-
tance translocation such as symplasmic transport. However, the
presence of plasmodesmata is insufficient to assume symplas-
mic movement given that it does not indicate active transport. A
further confirmation of the presence of symplasmic transport
came from dye tracing studies that showed the continuum
formed by the xylem rays (Sokolowska and Zagórska-Marek
2012, Pfautsch et al. 2015b). The phloem unloading and radial transport of carbon in
hardwood xylem during growth Moreover, it has been suggested
that the microtubule, microfilament and myosin components
of the cytoskeleton of the ray and axial parenchyma cells of
horse chestnut (Aesculus hippocastanum L.) and hybrid aspen
(P. tremula × tremuloides) have a role in the delivery of the
photosynthates to the vascular cambium and to the sites of C
storage (Chaffey and Barlow 2001). However, the regulation
and specificity of the symplasmic transport across rays is
practically unknown. Analysis of radial sucrose and hexose
levels support the hypothesis that there is a passive transport
mechanism down a steep concentration gradient from phloem
to developing wood in both Scots pine and Populus sp. (Uggla
et al. 2001, Roach et al. 2017). Although studies on phloem unloading processes of polyols
in trees are limited, work on mannitol and sorbitol transport
shed light on the possible mechanisms. Olive trees accumulate
mannitol in fruits (also a strong sink tissue) via a mannitol/proton
transporter (MaT1) (Conde et al. 2007), suggesting that the
phloem unloading is apoplasmic in this tissue and species
(Conde et al. 2008). Other polyols, such as dulcitol, sorbitol and
xylitol, can compete with mannose for the uptake through this
transporter (Conde et al. 2007). Two SOTs, whose expression
is associated to sorbitol accumulation in sink tissue, have been
cloned from fruits of sour cherry (Prunus cerasus) (Gao et
al. 2003). Besides, SOT1 expression levels are reduced in
apple when leaf sorbitol synthesis is reduced (Li et al. 2018),
suggesting that they are involved in the incorporation of sorbitol
into the fruits. Transporters of RFOs have not been identified in plants so far. However, the presence of a raffinose transporter was postulated
to exist in the chloroplast membrane of Arabidopsis and Ajuga
reptans L. leaves based on raffinose uptake into isolated chloro-
plasts (Schneider and Keller 2009). This transporter would be
involved in carrying the raffinose from its synthesis site in the
cytosol to the chloroplasts to scavenge reactive oxygen species. Based on this work, other authors have proposed that such RFO
transporters would be involved in stress responses (Valluru and
Van den Ende 2011) and in apoplasmic phloem unloading (Hu
et al. 2011). Xylem ray cells and developing wood fibers may not be sym-
plasmically connected, suggesting that photosynthates exit the
ray cells toward the fibers apoplasmically (Figure 4B) (Chaffey
and Barlow 2001). This implies the presence of sucrose trans-
porters in both ray cells and fibers. The phloem unloading and radial transport of carbon in
hardwood xylem during growth Several SWEET transporters
have been described in herbaceous plants as involved in sucrose
transport in sink tissues (Table 1) (Le Hir et al. 2015, Sosso
et al. 2015, Ma et al. 2017, Shammai et al. 2018, Yang et
al. 2018, Kim et al. 2021). The identification of SWEETs in
several tree species, such as rubber tree (Hevea brasiliensis
(Willd. ex A.Juss.) Müll.Arg.) (Sui et al. 2017), L. chinensis (Xie
et al. 2019), tung tree (Vernicia fordii (Hemsl.) Airy Shaw)
(Cao et al. 2019) and poplar (P. trichocarpa) (Zhang et al. 2021) together with the differential expression of the orthologs The catabolism of different carbon forms in wood fibers and
other sink tissues After carbohydrates move across the ray cells, they can be
stored in the axial parenchyma cells or they can be delivered to
the developing xylem cells such as fibers (Figure 4B). Sucrose
can be degraded in these sink cells by sucrose synthase (SUS)
and/or invertases (INVs) (Figure 5A). Sucrose synthase (SUS)
is a cytosolic enzyme that degrades sucrose into UDP-glucose Tree Physiology Online at http://www.treephys.oxfordjournals.org 472
Dominguez and Niittylä
igure 5. Catabolism of phloem mobile carbon forms. (A) Sucrose (orange). (B) Sugar alcohols (greens and blues). (C) Raffinose family
ligosaccharides (gray). The colors of the circles indicate the type of plant for which there is evidence for each enzyme. and fructose (Ruan 2014). While there are indices in several
rees that SUS is involved in wood C metabolism, including
Pinus radiata D. Don (Li et al. 2012) and Catalpa fargesii
B
(L
t
l 2018) f
ti
l
t di
h
l
b
Alteration of aspen SUS1 and SUS2 levels affects the tot
amount of secondary cell wall polymers and wood biomas
characteristic of a key C allocation determinant (Colema
t
l 2009 G
b
t
l 2014 Li
t
l 2020 D
i 472 Dominguez and Niittylä Downloaded from https://academic.oup.com/treephys/article/42/3/ Figure 5. Catabolism of phloem mobile carbon forms. (A) Sucrose (orange). (B) Sugar alcohols (greens and blues). (C) Raffinose family of
oligosaccharides (gray). The colors of the circles indicate the type of plant for which there is evidence for each enzyme. Figure 5. Catabolism of phloem mobile carbon forms. (A) Sucrose (orange). (B) Sugar alcohols (greens a
oligosaccharides (gray). The colors of the circles indicate the type of plant for which there is evidence for each e mobile carbon forms. (A) Sucrose (orange). (B) Sugar alcohols (greens and blues). (C) Raffinose family o
rs of the circles indicate the type of plant for which there is evidence for each enzyme. Alteration of aspen SUS1 and SUS2 levels affects the total
amount of secondary cell wall polymers and wood biomass,
characteristic of a key C allocation determinant (Coleman
et al. 2009, Gerber et al. 2014, Li et al. 2020, Dominguez
et al. 2021). While SUS catalyzes a reversible reaction, inver-
tases degrade sucrose into glucose and fructose irreversibly Alteration of aspen SUS1 and SUS2 levels affects the total
amount of secondary cell wall polymers and wood biomass,
characteristic of a key C allocation determinant (Coleman
et al. The catabolism of different carbon forms in wood fibers and
other sink tissues This is clearly observed in cucumber, in which stachyose is
found in high levels in leaves but in extremely low levels in
the fruits, which suggests that they are converted into simpler
sugars in the SEs of the fruits (Hu et al. 2009, Ma et al. 2019). Reverse reactions of the RFO synthesis pathway shown
in Figure 2C can lead to the catabolism of RFOs (Figure 5C)
(Madore 2001). Thereby, stachyose can be converted into
raffinose and galactinol by the action of stachyose synthase. The reaction product raffinose can be converted to sucrose and
galactinol by a raffinose synthase. These reactions are believed
to control the flux of RFOs in the tissues (Sengupta et al. 2015). An alternative pathway can be led by α-galactosidases
(EC 3.2.1.22), which sequentially remove galactose groups
from RFOs (Madore 2001). They can be either neutral/basic
or acid and have been described in squash (Cucurbita sp.),
melon (Cucumis melo L.), Arabidopsis and Coleus blumei Benth
(Plectranthus scutellarioides (L.) Benth.) (Table 3) (Gaudreault
and Webb 1983, Gao and Schaffer 1999, Madore 2001, Carmi
et al. 2003, Peters et al. 2010). These reactions are believed
to be involved in the phloem unloading of RFOs in sink tissues
(Gaudreault and Webb 1983, Pharr and Sox 1984, Gao and
Schaffer 1999). The general pathway of mannitol catabolism in plants has
been elucidated in herbaceous plants (Stoop et al. 1996,
Noiraud et al. 2001), and it is believed to be conserved in
olive trees (Figure 5B, Table 2) (Conde et al. 2008). In sink
tissues, mannitol is first converted to mannose via a mannitol
dehydrogenase (MTD) (EC 1.1.1.255) (Stoop and Pharr 1992,
Williamson et al. 1995, Conde et al. 2011). This enzyme is
a pivotal control point for the regulation of mannitol levels in
celery (Stoop et al. 1996, Zamski et al. 2001). Mannose is next
phosphorylated into mannose-6-P by the action of a hexokinase
(HK) (EC 2.7.1.1) (Stoop and Pharr 1994). In general, plants
do not have specific mannose kinases, so mannose is assumed
to be phosphorylated by hexokinases (Renz and Stitt 1993). Hexokinases are ubiquitous in plants (Aguilera-Alvarado and
Sánchez-Nieto 2017), and hexokinase activity is high in the
cambium and secondary cell wall formation zone in aspen
(Roach et al. 2017). The phosphorylation product mannose-
6-P is converted into fructose-6-P by a phospho-mannose
isomerase (EC 5.3.1.8) (Maruta et al. 2008). The catabolism of different carbon forms in wood fibers and
other sink tissues 2017), while one Cell wall invertases is specifically
expressed in growing tissues, including xylem, in hybrid aspen
(Canam et al. 2008). These data suggest that CWINV could
also have a role in sucrose degradation in sink tissues. Cell
wall invertase activity is coupled to hexose transporter activity
in recipient cells; thus, future studies on the roles of CWINVs
and hexose transporters should increase the knowledge on the
fine tuning of sucrose catabolism in wood. Moreover, two VINs
(Potri.015G127100 and Potri.003G112600) are differentially
expressed in the expansion zone of aspen wood, suggesting
that they are involved in the cell expansion process providing C
and energy (see Figure S1 available as Supplementary data at
Tree Physiology Online) (Sundell et al. 2017). Yamaguchi et al. 1996, Nosarszewski et al. 2004). The product
fructose is further phosphorylated by a fructokinase (FK; EC
2.7.1.4) or a hexokinase (Stein and Granot 2019). In aspen
trees, albeit not being sorbitol loaders, reduction in fructokinase
activity leads to the reduction of C flux to developing wood
(Roach et al. 2012). This suggests that fructokinase could be
a link between sorbitol metabolism and C allocation to wood
in sorbitol loaders. An alternative sorbitol catabolic pathway
is through the sorbitol oxidase (SOX; Walker et al. 2020),
turning it into glucose, which can be further phosphorylated by
a hexokinase. This mechanism has been shown to exist in peach
fruits (Lo Bianco and Rieger 2002) and seeds (Morandi et al. 2008). The catabolism of other polyols has not been studied in detail
in plants (Table 2). It is worth noting that polyol catabolism in
wood has received little attention, even in species in which a
significant amount of C is transported as polyols and wood is the
main sink tissue, e.g., pinitol in Scots pine and Norway spruce
(Figure 3). The catabolism of RFOs in trees has been scarcely stud-
ied, but studies in other species can give clues about the
pathways that may exist in trees (Table 3). The RFOs are
rapidly metabolized upon arrival to the sink tissue, showing
low levels in sink tissues when they are used with the aim
of transporting C (Hubbard et al. 1989, Carmi et al. 2003). The catabolism of different carbon forms in wood fibers and
other sink tissues There are two main sorbitol catabolic pathways (Figure 5B,
Table 2) (Walker et al. 2020). Sorbitol can be converted into
fructose by the action of the Sorbitol dehydrogenase (SDH)
(EC 1.1.1.14) in sink tissues (Negm and Loescher 1979,
Yamaguchi et al. 1994, Aguayo et al. 2013, Hartman et al. 2014). SDH has its highest activity on sorbitol but it can oxidize
other polyols such as xylitol and ribitol as well (Jia et al. 2015). It is a key step in the regulation of sorbitol content in plants
(Nosarzewski et al. 2012, Jia et al. 2015) and it contributes
to the accumulation of sorbitol in sink tissues such as fruits
and developing leaves of Rosaceae trees (Loescher et al. 1982, The catabolism of different carbon forms in wood fibers and
other sink tissues 2009, Gerber et al. 2014, Li et al. 2020, Dominguez
et al. 2021). While SUS catalyzes a reversible reaction, inver-
tases degrade sucrose into glucose and fructose irreversibly and fructose (Ruan 2014). While there are indices in several
trees that SUS is involved in wood C metabolism, including
Pinus radiata D. Don (Li et al. 2012) and Catalpa fargesii
Bureau (Lu et al. 2018), functional studies have only been
performed in aspen (Populus sp.) (Table 1). This species has
seven SUS genes, called SUS1–SUS7 (Zhang et al. 2011). Tree Physiology Volume 42, 2022 Mobile forms of carbon in trees 473 (Ruan 2014). They can be cytoplasm basic/neutral invertases
(CINs) or cell wall and vacuole acidic invertases (CWINVs and
VINs, respectively) (Bocock et al. 2008, Ruan 2014, Chen
et al. 2015). The reduction of CIN1 and CIN2 expression in
hybrid aspen reduces cellulose levels, suggesting that they
contribute to sucrose degradation and cellulose synthesis in
developing wood (Rende et al. 2017). Acidic invertase activity
is also detected in wood (Chen et al. 2015). CWINVs are
expressed in the xylem of Populus (Chen et al. 2015, Sundell
et al. 2017), while one Cell wall invertases is specifically
expressed in growing tissues, including xylem, in hybrid aspen
(Canam et al. 2008). These data suggest that CWINV could
also have a role in sucrose degradation in sink tissues. Cell
wall invertase activity is coupled to hexose transporter activity
in recipient cells; thus, future studies on the roles of CWINVs
and hexose transporters should increase the knowledge on the
fine tuning of sucrose catabolism in wood. Moreover, two VINs
(Potri.015G127100 and Potri.003G112600) are differentially
expressed in the expansion zone of aspen wood, suggesting
that they are involved in the cell expansion process providing C
and energy (see Figure S1 available as Supplementary data at
Tree Physiology Online) (Sundell et al. 2017). (Ruan 2014). They can be cytoplasm basic/neutral invertases
(CINs) or cell wall and vacuole acidic invertases (CWINVs and
VINs, respectively) (Bocock et al. 2008, Ruan 2014, Chen
et al. 2015). The reduction of CIN1 and CIN2 expression in
hybrid aspen reduces cellulose levels, suggesting that they
contribute to sucrose degradation and cellulose synthesis in
developing wood (Rende et al. 2017). Acidic invertase activity
is also detected in wood (Chen et al. 2015). CWINVs are
expressed in the xylem of Populus (Chen et al. 2015, Sundell
et al. Interaction between phloem and xylem The ray and axial parenchyma in sycamore maple (A. pseudo-
platanus), poplar and eucalyptus were shown to form a 3D Tree Physiology Online at http://www.treephys.oxfordjournals.org 474 Dominguez and Niittylä symplasmic transport pathway through plasmodesmata by
applying fluorescent tracers (Chaffey and Barlow 2001, Ehlers
and van Bel 2010, Sokolowska and Zagórska-Marek 2012,
Pfautsch et al. 2015b). Wood cells are also connected by
different types of pits, which are channels that facilitate the
solute exchange between different cell types, including simple
pits between parenchyma cells as well as between vessels
and parenchyma cells, bordered pits between vessels and
half-bordered pits between fiber cells and parenchyma cells
(Chattaway 1951, Van Bel 1990, Ek et al. 2009, Spicer 2014,
Pfautsch et al. 2015a, Slupianek et al. 2019). Vessel-associated
cells (VACs) and paratracheal parenchyma, which are cell types
in contact with vessels, can modify the content of xylem sap
(Plavcová and Jansen 2015, Morris et al. 2018). These cells
form a continuum with vessel-distant ray and axial parenchyma,
which stretches to the phloem. Besides, sugar transport in
xylem sap during growth has also been described (Mayrhofer
et al. 2004, Diacci et al. 2021). Combined, these anatomical
and transport results suggest that xylem functions as a sink-
to-source translocation system during growth in trees. These
data also emphasize that the vascular system is interconnected,
which supports its role as an integrator of the root-to-shoot
and shoot-to-root signaling networks (Lucas et al. 2013). For
example, changes in xylem sap metabolome and proteome
composition induced by variations in the nitrogen nutrition
are associated with large alterations in the systemic defense
transcripts in leaves of poplar (Populus × canescens) (Kasper
et al. 2021). In this root-to-shoot and shoot-to-root signaling
networks, cells associated to the vessels (such as VACs and
paratracheal parenchyma) could have a determinant role given
their strategic connection with vessels and, in the case of VACs,
their functional specialization in solute transport. leaf C assimilation could be expected in trees. However, more
research addressing sink tissue activity, sucrose xylem content
and leaf physiology is needed to corroborate these links. In
fact, many other metabolites have been detected in the phloem
(Figure 3) (Dinant et al. 2010, references therein) and xylem
sap of trees (Saranpää and Höll 1989, Schill et al. 1996,
Deslauriers et al. 2014, Diacci et al. 2021, Kasper et al. treephys/article/42/3/458/6372535 by Sveriges Lantbruksuniversitet user on 06 October 2022 p
p
Communication between source and sink organs is essential
for coordinating C assimilation and partitioning during growth
(Yu et al. 2015). If the sink tissue strength is altered, then
the source tissue performance will also be affected through
feedback mechanisms, possibly through xylem transmitted sig-
nals (Chang and Zhu 2017). Typical examples of reduction of
the sink strength are root area reduction and impaired phloem
transport, which are known to produce C assimilation alterations
in trees (Campany et al. 2017, Jing et al. 2018, Rainer-Lethaus
and Oberhuber 2018). This regulation can happen at other
levels as well. Coleman et al. (2008) found that reduction in
lignin synthesis in stems by silencing the enzyme coumaroyl 3′-
hydroxylase reduced photosynthesis due to the accumulation
of photosynthates in sources tissues. Sucrose is known to be a
xylem signal that controls assimilate partitioning in herbaceous
species (Chiou and Bush 1998, Griffiths et al. 2016). Given that
sucrose is the main form of C transport in trees and that sucrose
and/or its turn over inhibits photosynthesis in the leaves of some
trees (Layne and Flore 1995, Zhou and Quebedeaux 2003,
Nebauer et al. 2011), a similar role as a xylem signal regulating Interaction between phloem and xylem 2021), but whether these metabolites have other roles beyond
C mobilization is not very well known. Sorbitol, for example, also
increases together with sucrose when sink activity is limited
in fruit trees (Layne and Flore 1995, Zhou and Quebedeaux
2003), suggesting that it could potentially function as a xylem
signal. Other type of molecules such as peptides could also
be acting as signal molecules in both the phloem and the
xylem (Notaguchi and Okamoto 2015). Thus, studying the
relation between metabolites (and other type of molecules)
transported in the vessels and different environmental cues
could help to understand the long-distance coordination of
the functioning of organs and tissues. Cytokinins, gibberellins,
trehalose- 6-P, SnRK1.11 and hexokinase are thought to be
involved in the source–sink signaling in herbaceous species
(Rolland et al. 2006, Chang and Zhu 2017). The molecular
aspects of the source–sink regulatory mechanisms are scarcely
known in trees, including the signaling steps, the sensing of the
signaling and the identity of the signals. A combination of tracer
experiments, metabolomics and proteomics measurements on
vessel exudates, and grafting experiments could be used to
move the field forward. Furthermore, sometimes, low rates of
sink tissue growth coincide with high availability of photoas-
similates (Körner 2015), or C storage exceeds the balance of
C supply and demand under stress (Sala et al. 2012). Hence,
the evaluation of different environmental effects (e.g., nutritional
status, exposure to stresses) could help to define the long-
distance signals that produce C allocation trade-offs during
stress/nutrient deficit. Carbon transport during growth-dormancy
transitions in trees These pits contain an amorphous layer, possibly to enlarge the
exchange surface; this layer is enriched in arabinogalactan-rich
glycoproteins and extensins, which may play a structural role in
modulating pit structure and permeability (Plavcová and Jansen
2015, Abedi et al. 2020). Vessel-associated cells (VACs) and
parenchyma cells are in symplasmic continuity in species such
as Robinia pseudoacacia L. and walnut (Fromard et al. 1995,
Alves et al. 2001, Améglio et al. 2004, Alves et al. 2007). More
recently, it has been suggested that while the xylem transports
C, this transport would be driven by the Münch flow in the
phloem when the transpiration is limited (Tixier et al. 2017). In some species such as walnut, the C is converted into starch During winter, stored C is used to synthetize compatible
solutes such as sucrose, polyols and RFOs to preserve tissues
from cold/frost damage and to maintain respiration, membrane
stabilization and xylem refilling, among other life preserving
functions (Cox and Stushnoff 2001, Tarkowski and Van den
Ende 2015, Hacke and Laur 2016, Tixier et al. 2019). The
nature of the compatible solutes that are accumulated during
winter depends on the species. Rosaceae produce polyols
(Sakai 1966). In aspen, raffinose and stachyose levels increase
in early winter, while GolSII has been associated to seasonal
mobilization of carbohydrates (Unda et al. 2012, Unda et
al. 2017). Juglans sp. (walnut) produces sucrose, glucose
and fructose (Améglio et al. 2004). Pinus strobus L., willow
and Japanese white birch (Betula platyphylla var. japonica)
accumulate sucrose and raffinose during winter (Hinesley et
al. 1992, Ögren 1999, Kasuga et al. 2007). The accumulation
of sugars and polyols in the xylem during winter likely occurs
through facilitated or active transport (Sauter 1988, Améglio
et al. 2004, Decourteix et al. 2006, Ito et al. 2012) and may
be derived from the redistribution of the C between vessels
and cells around them instead of being a consequence of long-
distance transport (Noiraud et al. 2001, Améglio et al. 2004). Other mechanisms also contribute to the accumulation of C
such as the activation of a phosphatase in chestnut (Castanea
sativa Mill.) xylem (CSDSP4) related to starch catabolism
during autumn (Berrocal-Lobo et al. 2011). The accumulated
compatible solutes are consumed when the temperature
increases as part of the dehardening process (Charrier et al. 2018). The metabolic changes during dormancy may also be
combined with anatomical changes affecting C transport in the
stem. Carbon transport during growth-dormancy
transitions in trees Trees of temperate and cold regions have an annual growth
cycle controlled by environmental conditions such as light and
temperature that prevents growth under unfavorable conditions
(Campoy et al. 2011, Maurya and Bhalerao 2017, Tixier et al. 2019). In winter, trees undergo a process of dormancy, charac-
terized by changes in the metabolic activity, the arrest of growth
and cold acclimatation, while in spring, growth is resumed. A
particularly important metabolic pool in these growth-dormancy
transitions is the stored C. The wood C storage sites can be
the axial parenchyma cells and/or the ray cells (Spicer 2014). In the model trees Populus sp., axial parenchyma cells are rare
so most of the reserves are found in the ray cells (Sauter
and Kloth 1987, Sauter and van Cleve 1994, Larisch et al. Tree Physiology Volume 42, 2022 Mobile forms of carbon in trees 475 2012). Another important C storage tissue in trees is roots
(Loescher et al. 1990). In trees, the main storage form of C
is starch, which is synthetized from ADP-glucose in plastids
(reviewed by Noronha et al. 2018). Starch can be transformed
into sucrose, and other C forms such as glucose, fructose or
polyols (mainly mannitol and sorbitol) to be used by sink tissues
(Loescher et al. 1990, Witt and Sauter 1994, Moing 2000). Many trees also store lipids, and in aspen, a possible relationship
between lipid storage and annual cycle was observed (Grimberg
et al. 2018, Watanabe et al. 2018). Genera such as Pinus
and Picea accumulate high levels of lipids in wood, but no
variations across seasons were detected in these trees (Hoch et
al. 2003). Furthermore, RFOs such as raffinose are also believed
to contribute to the C storage pool in some species (Moing
2000). growth-promoting
signals
from
accessing
the
meristem
(Tylewicz et al. 2018). During the spring, the metabolic activity is characterized by
the nutrient remobilization from the storage compartments in
the main trunk, branches and roots, and the transport to the
sink tissues to sustain growth (Sauter and Ambrosius 1986,
Witt and Sauter 1994, Maurya and Bhalerao 2017, Noronha
et al. 2018). Especially important sink tissues and organs
during this period are the cambial zones, the leaf buds, which
will flush and develop into leaves (also called the bud break
process or budburst) (Young et al. 2018), and flower buds,
which will form flowers (Goeckeritz and Hollender 2021). Carbon transport during growth-dormancy
transitions in trees During growth resumption, the circulation of C from the storage
compartments in the stem to the cambium is characterized by
the degradation of starch or other forms of C storage followed
by the transport to the cambial cells (Figure 4C) (Chaffey
and Barlow 2001). In addition, already prior to bud flush
photosynthetically active tissues such as green branches can
contribute C to cambium zone via phloem as described above. In other species, growth resumption occurs after bud flush,
so C is mainly obtained from photosynthetically active leaves
(Klein et al. 2016). If the C storage is in the axial parenchyma
cells, their breakdown products are transported to the ray cells
symplasmically (Figure 4C) (Chaffey and Barlow 2001). Carbon
is also transported symplasmically through the xylem rays to the
cambium rays together with the breakdown products of the C
storage located in the ray cells themselves. The other important
route is that of the C delivery to the growing buds (Figure 4C). It
involves the loading of the C into the xylem vessels, the transport
inside the vessels and the unloading of the C in the buds (Sauter
1982, Maurel et al. 2004, Decourteix et al. 2006, Alves et al. 2007). The amount of C in the xylem sap before the leaves
develop depends on the balance between the efflux of stored
sugars from xylem parenchyma into the vessels and the influx of
C from the vessels to the parenchyma cells, possibly mediated
by the VACs (Sauter 1980, Améglio et al. 2004, Alves et al. 2007, Bonhomme et al. 2010, Morris et al. 2018). Xylem
vessels and VACs are connected through pits and the solute
exchange is likely mediated by transporters and pit anatomy. These pits contain an amorphous layer, possibly to enlarge the
exchange surface; this layer is enriched in arabinogalactan-rich
glycoproteins and extensins, which may play a structural role in
modulating pit structure and permeability (Plavcová and Jansen
2015, Abedi et al. 2020). Vessel-associated cells (VACs) and
parenchyma cells are in symplasmic continuity in species such
as Robinia pseudoacacia L. and walnut (Fromard et al. 1995,
Alves et al. 2001, Améglio et al. 2004, Alves et al. 2007). More
recently, it has been suggested that while the xylem transports
C, this transport would be driven by the Münch flow in the
phloem when the transpiration is limited (Tixier et al. 2017). Carbon transport during growth-dormancy
transitions in trees I
i
h
l
t th
C i
t d i t
t
h During the spring, the metabolic activity is characterized by
the nutrient remobilization from the storage compartments in
the main trunk, branches and roots, and the transport to the
sink tissues to sustain growth (Sauter and Ambrosius 1986,
Witt and Sauter 1994, Maurya and Bhalerao 2017, Noronha
et al. 2018). Especially important sink tissues and organs
during this period are the cambial zones, the leaf buds, which
will flush and develop into leaves (also called the bud break
process or budburst) (Young et al. 2018), and flower buds,
which will form flowers (Goeckeritz and Hollender 2021). During growth resumption, the circulation of C from the storage
compartments in the stem to the cambium is characterized by
the degradation of starch or other forms of C storage followed
by the transport to the cambial cells (Figure 4C) (Chaffey
and Barlow 2001). In addition, already prior to bud flush
photosynthetically active tissues such as green branches can
contribute C to cambium zone via phloem as described above. In other species, growth resumption occurs after bud flush,
so C is mainly obtained from photosynthetically active leaves
(Klein et al. 2016). If the C storage is in the axial parenchyma
cells, their breakdown products are transported to the ray cells
symplasmically (Figure 4C) (Chaffey and Barlow 2001). Carbon
is also transported symplasmically through the xylem rays to the
cambium rays together with the breakdown products of the C
storage located in the ray cells themselves. The other important
route is that of the C delivery to the growing buds (Figure 4C). It
involves the loading of the C into the xylem vessels, the transport
inside the vessels and the unloading of the C in the buds (Sauter
1982, Maurel et al. 2004, Decourteix et al. 2006, Alves et al. 2007). The amount of C in the xylem sap before the leaves
develop depends on the balance between the efflux of stored
sugars from xylem parenchyma into the vessels and the influx of
C from the vessels to the parenchyma cells, possibly mediated
by the VACs (Sauter 1980, Améglio et al. 2004, Alves et al. 2007, Bonhomme et al. 2010, Morris et al. 2018). Xylem
vessels and VACs are connected through pits and the solute
exchange is likely mediated by transporters and pit anatomy. Carbon transport during growth-dormancy
transitions in trees In Populus sp., there is a pronounced reduction of the
number of plasmodesmata in the cambium during dormancy,
while their number increases when growth is reassumed (Fuchs
et al. 2010, Spicer 2014). Sealing of plasmodesmata with
callose may also occur during dormancy in hybrid aspen buds
in an abscisic acid (ABA)-dependent manner, which prevents Tree Physiology Online at http://www.treephys.oxfordjournals.org 476 Dominguez and Niittylä again once it arrives to the buds, and it is later degraded for use
in the shoot apical meristems (Young et al. 2018). Another factor affecting C storage is wood anatomy. For
example, Quercus, which bears ring-porous wood, is thought to
require a greater amount of C reserves due to the increased
hydraulic risk associated with this anatomy compared with
diffuse-porous wood anatomy (Klein et al. 2016). Besides, tree
height correlates positively with non-structural carbohydrates
(NSC) and inversely with leaf water and osmotic potentials,
suggesting that taller trees require more NSC probably to cope
with stress (Woodruff and Meinzer 2011, Sala et al. 2012). Moreover, trees can undergo growth arrest even in conditions
of high C availability (Körner 2015), which indicates that there
are several growth-limiting constraints (stress, nitrogen deficit,
etc.) in addition to photosynthesis. This emphasizes that studies
should focus on the whole plant and not just on individual
tissues/organs such as buds to fully understand the mobilization
of C during the different phenological phases of trees. The preferred source of C (either stored C or recently fixed C)
during the onset of growth in spring depends on the species. Huang et al. (2014) modeled the growth of two conifers, P. mariana and Abies balsamea (L.) Mill., and observed that in
these evergreen species, the activation of the cambium and
the formation of the new xylem tissue is initiated prior to
the budburst, and therefore, C produced by older needles and
reserves (starch, lipids) are used as a source of C and energy. This is in agreement with what was observed in other conifers
(Rossi et al. 2009, Zhai et al. 2012). Following budburst in
deciduous trees, the C transported from the newly formed leaves
is used as energy source. In poplar, where the activation of
the cambium and the budburst occur at the same time while
flowering precedes them, the main source for the development
of the xylem at the onset of growth also comes from reserves
(Linkosalo 1999, Begum et al. Carbon transport during growth-dormancy
transitions in trees 2007, Deslauriers et al. 2009). The dependence of sink tissues on C from xylem is evidenced by
the increased starch degrading enzyme activities and decreased
starch content in the xylem during the bud break in several trees
including peach and walnut (Alves et al. 2007, Bonhomme et
al. 2010, Tixier et al. 2017) and the increased C levels in the
xylem sap (Sauter 1980, Alves et al. 2007, Bonhomme et al. 2010, Ito et al. 2012). On the other hand, in species such as
Fagus sylvatica L. and Quercus petraea (Matt.) Liebl., where the
budburst occurs before the activation of the cambium, the onset
of the xylem formation coincides with the C assimilation by the
newly formed leaves (Klein et al. 2016). The onset of growth
is also affected by the differential responses of leaves and
cambium to the environment, which can lead to unsynchronized
behavior (Delpierre et al. 2016), and the existence of additional
sources of C such as stem photosynthesis (Aschan and Pfanz
2003, Vandegehuchte et al. 2015). The preference in the use
of either stem starch, which requires long-distance transport, or
branchlet starch, which is C stored closer to buds, varies also
between species (Klein et al. 2016, Tixier et al. 2019). Origin of mobile forms of carbon and distribution The capacity of sucrose synthesis in plants has a procaryotic
origin associated with the symbiotic bacteria that evolved into
organelles. Sucrose can be synthesized by both cyanobacteria,
which gave rise to chloroplasts, and proteobacteria, which gave
rise to mitochondria in eucaryotes. However, only plant cells
can synthesize sucrose (MacRae and Lunn 2012). Therefore,
the evolutionary origin of sucrose production by plants is
thought to date back to the cyanobacterial ancestors of chloro-
plasts (Salerno and Curatti 2003, MacRae and Lunn 2012). In cyanobacteria, sucrose plays a role as a compatible solute to
fight stress (MacRae and Lunn 2012). During evolution, the site
of sucrose synthesis was transferred from chloroplasts to the
cytosol, possibly to maintain an osmotic balance between the
chloroplasts and the cytosol (MacRae and Lunn 2012). Hence,
the sucrose synthesis capacity existed in charophyte algae prior
to the evolution of the plant vascular system, and therefore, it
is believed that sucrose influenced the evolution of the vascular
tissue system (Cho et al. 2017). The presence of sucrose as a
transport molecule in all higher plant species further supports its
early evolutionary origin. This also means that the evolution of
the entire enzymatic and regulatory system of sucrose occurred
simultaneously with the development of the vascular tissues. For
example, the characteristics that distinguish the SPSs of plants
from those of cyanobacteria (such as the phosphorylation at
Ser229 and Ser424 sites) arose during the divergence of the
algae of the stratophyte lineage, which is associated to the
use of sucrose in long-distance C transport (MacRae and Lunn
2012). Studies on the evolution of plant invertases revealed that
CINs are ancient, stable and highly conserved, probably related
to their functions in sugar homeostasis, while CWINVs (and
their inhibitors) have coevolved with vascular plants, probably
related to their functions in phloem unloading (Wan et al. 2018). Carbon export to the rhizosphere The
co-evolution of these fungi and plants have generated forms of
reciprocal regulation of the exchange of nutrients, which allows
them to have a stable symbiotic relationship (Wang et al. 2017). In a model of the establishment of the AM–plant symbiotic
relationship, the plant increases the transport of C to the fungus
only if it detects an increase in the flow of phosphorus which
would establish a positive feedback in the transport of C to
the fungus (Wang et al. 2017). A similar control mechanism
for EM could occur (Bogar et al. 2019), but it would not
be universal (Näsholm et al. 2013, Stuart and Plett 2020). On the other hand, mycorrhizae can affect the characteristics
of the roots (Sheng et al. 2009, Chen et al. 2016), and in
turn this can affect the functionality of the leaves (Freschet et
al. 2015, Jing et al. 2018), which would imply the existence
of a leaf–root–symbiont connection. Furthermore, it is believed
that it is the same nutrients transported between trees and
symbionts that act as signals controlling exchange (Garcia et al. 2015), although more studies are needed to provide evidence
in this regard. Besides, trees with associations with EM are
connected to each other through the mycelium of the EM Knowledge of the different mobile forms of C and the
transport mechanisms among plant species and families remains
limited (Tables 1–4). The number of large-scale studies eluci-
dating the distribution of mobile forms of C and their transport
mechanisms is scarce. The aforementioned works by Mer-
chant et al. (2007), who studied the distribution of polyols
in various species of eucalyptus, and by Rennie and Tur-
geon (2009) and Fu et al. (2011), who studied the phloem
loading mechanism in various species using biochemical and
physiological techniques, stand out as the sole large-scale
studies in the field. Macroevolutionary studies of the differ-
ent forms of C transport and associated mechanisms are
lacking. The role of polyols and RFOs in stress responses
and the metabolic flexibility that comes with the ability to
transport several C forms could be the reasons of the emer-
gence of these compounds as mobile C forms together with
sucrose. Carbon export to the rhizosphere Trees export 6–20% of the assimilated C from roots to the sur-
rounding rhizosphere to the benefit of symbiotic rhizobacteria
and mycorrhizal fungi (Bago et al. 2000, Wang et al. 2017,
Schiestl-Aalto et al. 2019). This makes these bacteria and fungi
into a strong C sink and, thus, into a major phloem unloading
driver. Mycorrhizal fungi are ubiquitous, being present in ∼80%
of angiosperms and in all gymnosperms (Wilcox 1991). In
exchange for C, these fungi provide other nutrients to the plants
(phosphorus, nitrogen, ionic metals), which are inaccessible to
them due to their chemical forms or to their physical distance
(Phillips et al. 2013, Wang et al. 2017). It is generally believed
that the plant provides C to mycorrhizal fungi in the form of
hexoses, organic acids and/or lipids (Garcia et al. 2016, Wang
et al. 2017). Other forms of C, such as sucrose, are converted
by plants into the forms used by fungi. Unlike polyols, RFOs are only present in higher plants (Sen-
gupta et al. 2015), and therefore, their acquisition as a transport
form of C did not precede sucrose. The phylogenetic analyses of
the RFO synthesis-related enzymes, GolS, RafS and StaS, have
revealed that dicots clearly separate from monocots in relation
to the protein sequence of GolS and RafS, unlike StaS, which
does not show a clear separation between those plant types
(Sengupta et al. 2015). This suggests that the initial synthesis
of RFO until raffinose evolved separately from the latter steps of
RFO synthesis. Most tree species form symbioses with either arbuscular myc-
orrhizal (AM) fungi, which is the most common type of endomy-
corrhiza, or ectomycorrhizal (EM) fungi, nearly all of which asso-
ciate only with trees (Phillips et al. 2013). Broadly speaking,
EM predominate in boreal forests and AM in tropical zones,
while temperate zones have forests with both predominant EM
species and predominant AM species (Phillips et al. 2013, Chen
et al. 2016, Hasselquist et al. 2016, Rog et al. 2020). The way
AM and EM interact with plants is different and there may be
differences even among taxa within each group (Phillips et al. 2013). The AM–plant relationship is believed to have arisen
over 400 million years ago, while the EM–plant relationship
originated over 100 million years ago (Brundrett 2002). hys/article/42/3/458/6372535 by Sveriges Lantbruksuniversitet user on 06 October 2022 The origin of the C used during the phenological phases of
trees is not always well understood (Sala et al. 2012, Körner
2015). The difficulty lies in the variety of factors that affect the
coordination between starch metabolism, photosynthesis, the
availability of C and the activity of the cambium. Trees exhibit
high constitutive C storage, which is not always proportional to
the balance of C supply and demand. This suggests that stored C
has additional functions to its established role of compensation
for periods of photoassimilate shortage (Sala et al. 2012). This
is evidenced by the formation of starch even at the cost of
reducing growth (Wiley and Helliker 2012) and by the existence
of starch pools that are not available for the day-to-day needs
(Carbone et al. 2013). The latter type of stored C, which
has a slow metabolism, is probably reserved for contingencies
(Carbone et al. 2013). Tree Physiology Volume 42, 2022 Mobile forms of carbon in trees 477 The reason why sucrose is the main C transport molecule and
not polyols, which have similar physicochemical properties and
also exist in bacteria, is unclear (MacRae and Lunn 2012). The
species that transport polyols as main C source (Rosaceae such
as peach, apple and pear trees) have a greater number of genes
associated with the metabolism of polyols, mostly generated
by gene duplications (Velasco et al. 2010, Verde et al. 2013,
Wu et al. 2013, Qiao et al. 2018). In contrast, strawberries
(which belong to the Rosaceae family, Rosoideae subfamily, and
do not accumulate polyols) do not differ in the number of genes
associated with sorbitol metabolism in comparison with other
species that do not transport sorbitol as a major C source (Verde
et al. 2013). This suggests that the expansion of the gene family
involved in the metabolism of polyols in Rosaceae is associated
with their capacity to accumulate these compounds and that the
drivers for this gene expansion arose after sucrose had already
become the major sugar transported by plants. influenced long-distance C transport, perhaps most notably in
the rhizosphere. Conclusions and prospects Studies on the signaling role of molecules
transported in the xylem sap are incipient in plants. Due to
its potential impact on plant nutrition and biomass generation,
sink–source communication is a promising field that will help
us to understand the regulation of C transport and allocation
at the whole-tree level. The evolutionary origin of polyols and
RFOs as transport molecules is also still obscure. In addition
to evolution studies, large-scale studies aimed to study the
relationship between the transported C forms, the species and
the environment could help to understand the evolutionary
origin of the different mobile C forms and the associated long-
distance transport mechanisms. In this regard, we propose that
rhizosphere could have played an active role in the evolution of
long-distance transport of C in trees. The fact that mycorrhizal fungi form a strong sink tissue,
the existence of a leaf–root–mycorrhiza communication, the
reciprocal regulation of nutrient transport between trees and
mycorrhizae, the distribution of nutrients and signals from the
EM mycelia among the trees of a forest, added to the long
history of co-evolution of trees and mycorrhizae, allow us to
hypothesize that these symbiotic relationships have also had an
effect on the composition, flow and transport mechanisms of
C from source tissues to sink tissues in trees. Furthermore, the
fact that the symbiosis between an individual mycelium and a
tree increases the chances that the mycelium associates with
other seedlings of the same genotype (Pickles et al. 2017),
could hypothetically be associated with the specialization in C
transport to reduce the risk of losing C to competing species. In the plant rhizosphere (the area around the roots rich in
components exuded by them including sugars, amino acids,
organic acids, vitamins and other compounds) free-living bac-
teria and fungi also proliferate that benefit from the root exu-
dated compounds (Singh et al. 2019). These micro-organisms
growing in close relationship with the roots have a central
role in ecosystem processes and nutrient cycling. An important
group of rhizosphere bacteria are the plant growth promot-
ing rhizobacteria (PGPR), defined as those bacteria that have
at least one characteristic that increases plant growth, e.g.,
being involved in nitrogen fixation, or phytohormone production
(Diaz-Garza et al. 2020). Thus, the presence of these micro-
organisms is beneficial for plants, and throughout evolution,
they have developed various ways of plant–microorganism com-
munication, including hormonal and volatile compound control
(Singh et al. 2019). Conclusions and prospects Due to
its potential impact on plant nutrition and biomass generation,
sink–source communication is a promising field that will help
us to understand the regulation of C transport and allocation
at the whole-tree level. The evolutionary origin of polyols and
RFOs as transport molecules is also still obscure. In addition
to evolution studies, large-scale studies aimed to study the
relationship between the transported C forms, the species and
the environment could help to understand the evolutionary
origin of the different mobile C forms and the associated long-
distance transport mechanisms. In this regard, we propose that
rhizosphere could have played an active role in the evolution of
long-distance transport of C in trees. How trees metabolize and transport C is a central part of
biomass formation in trees. The main form of transported C
in plants is sucrose. The focus on sucrose has led to signif-
icant advances in our understanding of sucrose metabolism
and transport. In many tree species, sucrose is transported
together with other metabolites, especially polyols and RFOs. Our knowledge on the biochemistry and physiology related
to these metabolites has significant gaps, especially in the
stems. Studies on the synthesis and degradation of polyols
and ROFs are essentially based on measurements of enzymatic
activities of tissue extracts. Despite the availability of gene
sequences in data repositories, their use to study different
aspects of polyols and ROFs has been limited. Therefore, there is
a large study field that deserves to be explored further, including
phylogenetic analyses, advanced enzymatic biochemical studies
and functional studies utilizing transgenic plants. Moreover,
existing studies employing transgenics focus on the role of
polyols and RFOs in stress responses and do not explore
their role in C transport, with the exception of studies on
tree fruits that transport polyols. Some of the topics that need
further attention include the quercitol synthesis pathway; the
catabolism pathways of the majority of polyols in the trunk;
the major synthesis and degradation pathways of ROFs in
trees; the identity of polyol and RFO transporters in the trunk;
the role of SWEET transporters whose function has not been
described in trees, although they have been studied in several
herbaceous species; and the co-regulation of the transport of
different forms of C in species that simultaneously transport
sucrose, polyols and/or RFOs under different conditions, and
their possible impact on the generation of wood biomass and C
allocation to wood. Carbon export to the rhizosphere Comparison of the available studies (Tables 1–4) does
not reveal obvious association between types of transported
molecules, transport mechanisms, species and their growth
environments, and in general, there are no notable differences
in the C transport between angiosperms and gymnosperms. Possibly, this is due to the multiplicity of environmental factors
that have been shaping the transport of C in the different
species throughout evolution. It is also possible that there are
unrecognized or unevaluated evolutionary forces that have Tree Physiology Online at http://www.treephys.oxfordjournals.org 478 Dominguez and Niittylä hys/article/42/3/458/6372535 by Sveriges Lantbruksuniversitet user on 06 October 2022 The relationships between trees and underground bacteria
and fungi have co-evolved over millions of years with the aim
of sharing nutrients. Understanding these relationships and their
possible effect on the development of long-distance C transport
could shed further light on the evolution of long-distance C
transport and the factors that affect it. Conclusions and prospects fungi, in a structure aptly named the ‘wood wide web’, through
which they share nutrients and signals (Simard et al. 1997). Through this fungal network, trees direct more resources to
their offspring than they do to unrelated trees (Pickles et al. 2017). fungi, in a structure aptly named the ‘wood wide web’, through
which they share nutrients and signals (Simard et al. 1997). Through this fungal network, trees direct more resources to
their offspring than they do to unrelated trees (Pickles et al. 2017). How trees metabolize and transport C is a central part of
biomass formation in trees. The main form of transported C
in plants is sucrose. The focus on sucrose has led to signif-
icant advances in our understanding of sucrose metabolism
and transport. In many tree species, sucrose is transported
together with other metabolites, especially polyols and RFOs. Our knowledge on the biochemistry and physiology related
to these metabolites has significant gaps, especially in the
stems. Studies on the synthesis and degradation of polyols
and ROFs are essentially based on measurements of enzymatic
activities of tissue extracts. Despite the availability of gene
sequences in data repositories, their use to study different
aspects of polyols and ROFs has been limited. Therefore, there is
a large study field that deserves to be explored further, including
phylogenetic analyses, advanced enzymatic biochemical studies
and functional studies utilizing transgenic plants. Moreover,
existing studies employing transgenics focus on the role of
polyols and RFOs in stress responses and do not explore
their role in C transport, with the exception of studies on
tree fruits that transport polyols. Some of the topics that need
further attention include the quercitol synthesis pathway; the
catabolism pathways of the majority of polyols in the trunk;
the major synthesis and degradation pathways of ROFs in
trees; the identity of polyol and RFO transporters in the trunk;
the role of SWEET transporters whose function has not been
described in trees, although they have been studied in several
herbaceous species; and the co-regulation of the transport of
different forms of C in species that simultaneously transport
sucrose, polyols and/or RFOs under different conditions, and
their possible impact on the generation of wood biomass and C
allocation to wood. Studies on the signaling role of molecules
transported in the xylem sap are incipient in plants. Supplementary data for this article are available at Tree
Physiology Online. Conclusions and prospects It has been documented that some herba-
ceous species can alter the surrounding rhizosphere microbiota
according to their stage of development upon the control of the
compounds exudated to the soil (Zhalnina et al. 2018). In trees,
PGPR have been characterized in orange trees (Diaz-Garza et
al. 2020), apple trees (Guo et al. 2014), birch (Zappelini et
al. 2018) and Lauraceae species (Báez-Vallejo et al. 2020). Therefore, as with mycorrhizae, it could be proposed that these
microorganisms that have co-evolved with plants (Lambers et al. 2009) have also influenced the development of long-distance
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Diaquabis(2-chlorobenzoato-κ<i>O</i>)bis(<i>N</i>,<i>N</i>-diethylnicotinamide-κ<i>N</i><sup>1</sup>)nickel(II)
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Acta crystallographica. Section E
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metal-organic compounds
Acta Crystallographica Section E
Experimental
Structure Reports
Online
Crystal data
ISSN 1600-5368
Diaquabis(2-chlorobenzoato-jO)bis(N,N-diethylnicotinamide-jN1)nickel(II)
Tuncer Hökelek,a* Hakan Dal,b Barış Tercan,c F. Elif
Özbekd and Hacali Necefoğlud
a
Department of Physics, Hacettepe University, 06800 Beytepe, Ankara, Turkey,
Department of Chemistry, Faculty of Science, Anadolu University, 26470
Yenibağlar, Eskişehir, Turkey, cDepartment of Physics, Karabük University, 78050
Karabük, Turkey, and dDepartment of Chemistry, Kafkas University, 63100 Kars,
Turkey
Correspondence e-mail: merzifon@hacettepe.edu.tr
b
Received 7 April 2009; accepted 16 April 2009
Key indicators: single-crystal X-ray study; T = 100 K; mean (C–C) = 0.002 Å;
R factor = 0.031; wR factor = 0.084; data-to-parameter ratio = 19.4.
In the monomeric and centrosymmetric title NiII complex,
[Ni(C7H4ClO2)2(C10H14N2O)2(H2O)2], the NiII ion is located
on an inversion center. The asymmetric unit contains one
2-chlorobenzoate ligand, one diethylnicotinamide (DENA)
ligand and one coordinating water molecule, the ligands being
monodentate. The four O atoms in the equatorial plane
around the NiII ion form a slightly distorted square-planar
arrangement, while the slightly distorted octahedral coordination is completed by two N atoms of the DENA ligands in
axial positions. The dihedral angle between the benzene ring
and the attached carboxylate group is 87.36 (10) , while the
pyridine and benzene rings are oriented at an angle of
41.90 (5) . In the crystal structure, intermolecular O—H O
hydrogen bonds link the molecules into a two-dimensional
network parallel to (101).
Related literature
For general backgroud, see: Antolini et al. (1982); Bigoli et al.
(1972); Nadzhafov et al. (1981); Shnulin et al. (1981). For
related structures, see: Hökelek et al. (1995, 1997, 2007, 2008);
Hökelek & Necefoğlu (1996, 1997, 2007).
[Ni(C7H4ClO2)2(C10H14N2O)2(H2O)2]
Mr = 762.31
Monoclinic, P21 =n
a = 12.7505 (2) Å
b = 10.3565 (2) Å
c = 14.9673 (3) Å
= 114.046 (1)
V = 1804.92 (6) Å3
Z=2
Mo K radiation
= 0.74 mm1
T = 100 K
0.27 0.18 0.11 mm
Data collection
Bruker Kappa APEXII CCD areadetector diffractometer
Absorption correction: multi-scan
(SADABS; Bruker, 2005)
Tmin = 0.828, Tmax = 0.923
16593 measured reflections
4519 independent reflections
3781 reflections with I > 2(I)
Rint = 0.034
Refinement
R[F 2 > 2(F 2)] = 0.031
wR(F 2) = 0.084
S = 1.06
4519 reflections
233 parameters
2 restraints
H atoms treated by a mixture of
independent and constrained
refinement
max = 1.08 e Å3
min = 0.39 e Å3
Table 1
Selected bond lengths (Å).
Ni1—O1
Ni1—O4
2.0336 (10)
2.0867 (10)
Ni1—N1
2.1181 (12)
Table 2
Hydrogen-bond geometry (Å, ).
D—H A
i
O4—H41 O2
O4—H42 O3ii
D—H
H A
D A
D—H A
0.82 (2)
0.85 (2)
1.86 (2)
1.93 (2)
2.6267 (17)
2.7826 (15)
155 (2)
172 (2)
Symmetry codes: (i) x; y þ 1; z; (ii) x þ 12; y 12; z þ 12.
Data collection: APEX2 (Bruker, 2007); cell refinement: SAINT
(Bruker, 2007); data reduction: SAINT; program(s) used to solve
structure: SHELXS97 (Sheldrick, 2008); program(s) used to refine
structure: SHELXL97 (Sheldrick, 2008); molecular graphics:
ORTEP-3 for Windows (Farrugia, 1997); software used to prepare
material for publication: WinGX (Farrugia, 1999).
The authors are indebted to Anadolu University and the
Medicinal Plants and Medicine Research Centre of Anadolu
University, Eskişehir, Turkey, for the use of the X-ray
diffractometer.
Supplementary data and figures for this paper are available from the
IUCr electronic archives (Reference: CI2776).
References
Antolini, L., Battaglia, L. P., Corradi, A. B., Marcotrigiano, G., Menabue, L.,
Pellacani, G. C. & Saladini, M. (1982). Inorg. Chem. 21, 1391–1395.
Bigoli, F., Braibanti, A., Pellinghelli, M. A. & Tiripicchio, A. (1972). Acta
Cryst. B28, 962–966.
Bruker (2005). SADABS. Bruker AXS Inc., Madison, Wisconsin, USA.
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Bruker (2007). APEX2 and SAINT. Bruker AXS Inc., Madison, Wisconsin,
USA.
Farrugia, L. J. (1997). J. Appl. Cryst. 30, 565.
Farrugia, L. J. (1999). J. Appl. Cryst. 32, 837–838.
Hökelek, T., Budak, K. & Necefoğlu, H. (1997). Acta Cryst. C53, 1049–1051.
Hökelek, T., Çaylak, N. & Necefoğlu, H. (2007). Acta Cryst. E63, m2561–
m2562.
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Hökelek, T. & Necefoğlu, H. (1996). Acta Cryst. C52, 1128–1131.
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Hökelek, T. & Necefoğlu, H. (1997). Acta Cryst. C53, 187–189.
Hökelek, T. & Necefoğlu, H. (2007). Acta Cryst. E63, m821–m823.
Hökelek, T., Necefoğlu, H. & Balcı, M. (1995). Acta Cryst. C51, 2020–2023.
Nadzhafov, G. N., Shnulin, A. N. & Mamedov, Kh. S. (1981). Zh. Strukt. Khim.
22, 124–128.
Sheldrick, G. M. (2008). Acta Cryst. A64, 112–122.
Shnulin, A. N., Nadzhafov, G. N., Amiraslanov, I. R., Usubaliev, B. T. &
Mamedov, Kh. S. (1981). Koord. Khim. 7, 1409–1416.
Acta Cryst. (2009). E65, m545–m546
supporting information
supporting information
Acta Cryst. (2009). E65, m545–m546
[doi:10.1107/S1600536809014226]
Diaquabis(2-chlorobenzoato-κO)bis(N,N-diethylnicotinamide-κN1)nickel(II)
Tuncer Hökelek, Hakan Dal, Barış Tercan, F. Elif Özbek and Hacali Necefoğlu
S1. Comment
Transition metal complexes with biochemically active ligands frequently show interesting physical and/or chemical
properties, as a result they may find applications in biological systems (Antolini et al., 1982). The structural functions and
coordination relationships of the arylcarboxylate ion in transition metal complexes of benzoic acid derivatives change
depending on the nature and position of the substituent groups on the benzene ring, the nature of the additional ligand
molecule or solvent, and the medium of the synthesis (Nadzhafov et al., 1981; Shnulin et al., 1981). The nicotinic acid
derivative N,N-diethylnicotinamide (DENA) is an important respiratory stimulant (Bigoli et al., 1972).
The structure determination of the title compound, (I), a nickel complex with two 2-chlorobenzoate (CB), two diethylnicotinamide (DENA) ligands and two water molecules, was undertaken in order to determine the properties of the
ligands and also to compare the results obtained with those reported previously.
Compound (I) is a monomeric complex, with the NiII ion on a centre of symmetry. It contains two CB, two DENA
ligands and two water molecules (Fig. 1). All ligands are monodentate. The four O atoms (O1, O4, and the symmetryrelated atoms O1′, O4′) in the equatorial plane around the NiII ion form a slightly distorted square-planar arrangement,
while the slightly distorted octahedral coordination is completed by the two N atoms of the DENA ligands (N1, N1′) in
the axial positions (Table 1 and Fig. 1).
The near equality of the C1—O1 [1.2616 (17) Å] and C1—O2 [1.2435 (18) Å] bonds in the carboxylate group indicates
a delocalized bonding arrangement, rather than localized single and double bonds, and may be compared with the
corresponding distances: 1.256 (6) and 1.245 (6) Å in [Mn(DENA)2(C7H4ClO2)2(H2O)2], (II) (Hökelek et al., 2008),
1.265 (6) and 1.275 (6) Å in [Mn(C9H10NO2)2(H2O)4].2(H2O), (III) (Hökelek & Necefoğlu, 2007), 1.260 (4) and 1.252 (4)
Å in [Zn(DENA)2(C7H4FO2)2(H2O)2],(IV) (Hökelek et al., 2007), 1.259 (9) and 1.273 (9) Å in Cu2(DENA)2(C6H5COO)4,
(V) (Hökelek et al., 1995), 1.279 (4) and 1.246 (4) Å in [Zn2(DENA)2(C7H5O3)4].2H2O, (VI) (Hökelek & Necefoğlu,
1996), 1.251 (6) and 1.254 (7) Å in [Co(DENA)2(C7H5O3)2(H2O)2], (VII) (Hökelek & Necefoğlu, 1997) and 1.278 (3) and
1.246 (3) Å in [Cu(DENA)2(C7H4NO4)2(H2O)2], (VIII) (Hökelek et al., 1997). In (I), the average Ni—O bond length is
2.0602 (10) Å and the Ni1 atom is displaced out of the least-squares plane of the carboxylate group (O1/C1/O2) by
-0.276 (1) Å. The dihedral angle between the planar carboxylate group and the benzene ring A (C2—C7) is 87.36 (10)°,
while that between rings A and B (N1/C8—C12) is 41.90 (5)°.
In the crystal structure, intermolecular O—H···O hydrogen bonds (Table 2) link the molecules into a two-dimesional
network parallel to the (1 0 1).
S2. Experimental
The title compound was prepared by the reaction of Ni(SO4).6(H2O) (1.31 g, 5 mmol) in H2O (20 ml) and DENA (1.78 g,
10 mmol) in H2O (20 ml) with sodium 2-chlorobenzoate (1.785 g, 10 mmol) in H2O (50 ml). The mixture was filtered and
set aside to crystallize at ambient temperature for 3 d, giving blue single crystals.
Acta Cryst. (2009). E65, m545–m546
sup-1
supporting information
S3. Refinement
H atoms of water molecule were located in a difference Fourier map and refined isotropically, with a O-H restraint. The
remaining H atoms were positioned geometrically with C-H = 0.93, 0.97 and 0.96 Å, for aromatic, methylene and methyl
H atoms and constrained to ride on their parent atoms, with Uiso(H) = xUeq(C), where x = 1.5 for methyl H and x = 1.2 for
all other H atoms.
Figure 1
The molecular structure of the title compound with the atom-numbering scheme. Displacement ellipsoids are drawn at
the 50% probability level. Primed atoms are generated by the symmetry operator (- x, 1 -y, -z).
Diaquabis(2-chlorobenzoato-κO)bis(N,N- diethylnicotinamide-κN1)nickel(II)
Crystal data
[Ni(C7H4ClO2)2(C10H14N2O)2(H2O)2]
Mr = 762.31
Monoclinic, P21/n
Hall symbol: -P 2yn
a = 12.7505 (2) Å
b = 10.3565 (2) Å
c = 14.9673 (3) Å
β = 114.046 (1)°
V = 1804.92 (6) Å3
Z=2
Acta Cryst. (2009). E65, m545–m546
F(000) = 796
Dx = 1.403 Mg m−3
Mo Kα radiation, λ = 0.71073 Å
Cell parameters from 7783 reflections
θ = 2.5–28.4°
µ = 0.74 mm−1
T = 100 K
Block, blue
0.27 × 0.18 × 0.11 mm
sup-2
supporting information
Data collection
Bruker Kappa APEXII CCD area-detector
diffractometer
Radiation source: fine-focus sealed tube
Graphite monochromator
φ and ω scans
Absorption correction: multi-scan
(SADABS; Bruker, 2005)
Tmin = 0.828, Tmax = 0.923
16593 measured reflections
4519 independent reflections
3781 reflections with I > 2σ(I)
Rint = 0.034
θmax = 28.4°, θmin = 1.8°
h = −17→17
k = −13→12
l = −20→18
Refinement
Refinement on F2
Least-squares matrix: full
R[F2 > 2σ(F2)] = 0.031
wR(F2) = 0.084
S = 1.06
4519 reflections
233 parameters
2 restraints
Primary atom site location: structure-invariant
direct methods
Secondary atom site location: difference Fourier
map
Hydrogen site location: inferred from
neighbouring sites
H atoms treated by a mixture of independent
and constrained refinement
w = 1/[σ2(Fo2) + (0.0456P)2 + 0.2566P]
where P = (Fo2 + 2Fc2)/3
(Δ/σ)max = 0.001
Δρmax = 1.08 e Å−3
Δρmin = −0.39 e Å−3
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.
Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2)
Ni1
Cl1
O1
O2
O3
O4
H41
H42
N1
N2
C1
C2
C3
H3
C4
x
y
z
Uiso*/Ueq
0.0000
−0.31050 (4)
−0.03038 (8)
−0.13227 (11)
0.43014 (9)
0.11786 (9)
0.1299 (19)
0.1100 (18)
0.13740 (10)
0.48379 (11)
−0.09075 (13)
−0.11211 (13)
−0.03393 (14)
0.0299
−0.05013 (15)
0.5000
0.35043 (4)
0.44605 (10)
0.61462 (11)
0.61598 (10)
0.34845 (10)
0.338 (2)
0.2746 (17)
0.61957 (11)
0.49545 (12)
0.50440 (14)
0.42820 (14)
0.42920 (16)
0.4830
0.35049 (17)
0.0000
0.08471 (3)
0.11794 (7)
0.13322 (9)
0.39621 (7)
0.03504 (8)
−0.0143 (13)
0.0570 (15)
0.08958 (9)
0.29580 (9)
0.15415 (11)
0.23115 (11)
0.32862 (12)
0.3480
0.39746 (12)
0.00853 (8)
0.02678 (11)
0.0128 (2)
0.0268 (3)
0.0151 (2)
0.0123 (2)
0.042 (6)*
0.043 (6)*
0.0114 (2)
0.0152 (3)
0.0135 (3)
0.0148 (3)
0.0202 (3)
0.024*
0.0259 (4)
Acta Cryst. (2009). E65, m545–m546
sup-3
supporting information
H4
C5
H5
C6
H6
C7
C8
H8
C9
H9
C10
H10
C11
C12
H12
C13
C14
H14A
H14B
C15
H15A
H15B
H15C
C16
H16A
H16B
C17
H17A
H17B
H17C
0.0027
−0.14508 (16)
−0.1551
−0.22513 (15)
−0.2895
−0.20830 (14)
0.14003 (12)
0.0804
0.22780 (12)
0.2255
0.31847 (12)
0.3776
0.31889 (12)
0.22679 (12)
0.2267
0.41594 (12)
0.47757 (15)
0.5535
0.4278
0.43258 (16)
0.4372
0.3541
0.4781
0.58189 (13)
0.5967
0.5628
0.68933 (14)
0.7512
0.6750
0.7098
0.3517
0.27062 (17)
0.2169
0.27003 (16)
0.2170
0.34949 (15)
0.74683 (14)
0.7833
0.82654 (14)
0.9148
0.77378 (14)
0.8255
0.64090 (14)
0.56819 (14)
0.4799
0.58190 (13)
0.45998 (18)
0.4670
0.5205
0.32361 (19)
0.3023
0.3187
0.2638
0.44280 (15)
0.3551
0.4401
0.52372 (17)
0.4876
0.6105
0.5242
0.4624
0.36887 (13)
0.4145
0.27308 (13)
0.2541
0.20560 (11)
0.07426 (11)
0.0209
0.13450 (11)
0.1223
0.21268 (11)
0.2547
0.22689 (10)
0.16507 (10)
0.1762
0.31243 (11)
0.19868 (12)
0.1989
0.1507
0.16849 (14)
0.1078
0.1605
0.2181
0.38015 (11)
0.3657
0.4366
0.40425 (13)
0.4600
0.4188
0.3492
0.031*
0.0270 (4)
0.032*
0.0235 (4)
0.028*
0.0183 (3)
0.0136 (3)
0.016*
0.0151 (3)
0.018*
0.0139 (3)
0.017*
0.0119 (3)
0.0124 (3)
0.015*
0.0120 (3)
0.0254 (4)
0.031*
0.031*
0.0368 (5)
0.055*
0.055*
0.055*
0.0166 (3)
0.020*
0.020*
0.0233 (4)
0.035*
0.035*
0.035*
Atomic displacement parameters (Å2)
Ni1
Cl1
O1
O2
O3
O4
N1
N2
C1
C2
C3
C4
C5
C6
U11
U22
U33
U12
U13
U23
0.00850 (13)
0.0248 (2)
0.0143 (5)
0.0457 (8)
0.0180 (5)
0.0136 (5)
0.0114 (6)
0.0143 (6)
0.0158 (7)
0.0181 (7)
0.0185 (8)
0.0288 (9)
0.0380 (10)
0.0293 (9)
0.00775 (13)
0.0307 (2)
0.0135 (5)
0.0154 (6)
0.0122 (5)
0.0093 (5)
0.0109 (6)
0.0175 (7)
0.0125 (7)
0.0132 (7)
0.0232 (9)
0.0322 (10)
0.0253 (9)
0.0199 (9)
0.00768 (13)
0.0230 (2)
0.0110 (5)
0.0323 (7)
0.0103 (5)
0.0121 (5)
0.0102 (6)
0.0098 (6)
0.0126 (7)
0.0166 (7)
0.0194 (8)
0.0175 (8)
0.0269 (9)
0.0286 (9)
0.00027 (9)
−0.00606 (17)
0.0022 (4)
0.0119 (5)
0.0014 (4)
0.0011 (4)
−0.0004 (5)
0.0038 (5)
−0.0021 (6)
0.0037 (6)
0.0028 (6)
0.0105 (8)
0.0072 (8)
−0.0026 (7)
0.00159 (10)
0.00789 (18)
0.0055 (4)
0.0293 (6)
0.0008 (4)
0.0034 (4)
0.0027 (5)
0.0009 (5)
0.0061 (6)
0.0106 (6)
0.0081 (7)
0.0104 (7)
0.0228 (9)
0.0193 (8)
0.00033 (9)
−0.00259 (17)
0.0018 (4)
0.0083 (5)
−0.0013 (4)
0.0006 (4)
0.0001 (5)
0.0004 (5)
−0.0011 (6)
0.0016 (6)
0.0026 (7)
0.0082 (7)
0.0114 (7)
0.0018 (7)
Acta Cryst. (2009). E65, m545–m546
sup-4
supporting information
C7
C8
C9
C10
C11
C12
C13
C14
C15
C16
C17
0.0212 (8)
0.0125 (7)
0.0169 (7)
0.0137 (7)
0.0117 (7)
0.0144 (7)
0.0114 (7)
0.0223 (9)
0.0259 (10)
0.0159 (7)
0.0169 (8)
0.0181 (8)
0.0120 (7)
0.0093 (7)
0.0125 (7)
0.0120 (7)
0.0097 (7)
0.0089 (7)
0.0383 (10)
0.0442 (12)
0.0144 (8)
0.0271 (9)
0.0175 (8)
0.0131 (7)
0.0164 (7)
0.0133 (7)
0.0097 (7)
0.0113 (7)
0.0118 (7)
0.0129 (8)
0.0291 (10)
0.0147 (7)
0.0214 (9)
0.0008 (6)
0.0019 (6)
−0.0002 (6)
−0.0022 (6)
0.0005 (5)
0.0006 (5)
−0.0023 (5)
0.0119 (7)
0.0087 (8)
0.0054 (6)
0.0006 (7)
0.0098 (7)
0.0018 (6)
0.0039 (6)
0.0033 (6)
0.0022 (6)
0.0032 (6)
0.0009 (6)
0.0042 (7)
−0.0003 (8)
0.0012 (6)
0.0033 (7)
0.0002 (6)
0.0028 (6)
0.0010 (6)
−0.0019 (6)
−0.0006 (5)
0.0012 (5)
−0.0005 (5)
−0.0016 (7)
−0.0213 (9)
0.0021 (6)
−0.0046 (7)
Geometric parameters (Å, º)
Ni1—O1i
Ni1—O1
Ni1—O4
Ni1—O4i
Ni1—N1i
Ni1—N1
Cl1—C7
O1—C1
O2—C1
O3—C13
O4—H41
O4—H42
N1—C8
N1—C12
N2—C14
N2—C16
C2—C3
C2—C1
C3—C4
C3—H3
C4—H4
C5—C4
C5—H5
C6—C5
C6—H6
2.0336 (10)
2.0336 (10)
2.0867 (10)
2.0867 (10)
2.1181 (12)
2.1181 (12)
1.7466 (17)
1.2616 (17)
1.2435 (18)
1.2429 (17)
0.820 (15)
0.854 (15)
1.3405 (19)
1.3452 (18)
1.470 (2)
1.4728 (19)
1.392 (2)
1.510 (2)
1.394 (2)
0.93
0.93
1.382 (3)
0.93
1.380 (2)
0.93
C7—C2
C7—C6
C8—C9
C8—H8
C9—H9
C10—C9
C10—H10
C11—C10
C11—C12
C12—H12
C13—N2
C13—C11
C14—H14A
C14—H14B
C15—C14
C15—H15A
C15—H15B
C15—H15C
C16—H16A
C16—H16B
C17—C16
C17—H17A
C17—H17B
C17—H17C
1.390 (2)
1.386 (2)
1.387 (2)
0.93
0.93
1.379 (2)
0.93
1.392 (2)
1.384 (2)
0.93
1.3369 (18)
1.501 (2)
0.97
0.97
1.522 (3)
0.96
0.96
0.96
0.97
0.97
1.519 (2)
0.96
0.96
0.96
O1i—Ni1—O1
O1i—Ni1—O4
O1—Ni1—O4
O1i—Ni1—O4i
O1—Ni1—O4i
O1i—Ni1—N1i
O1—Ni1—N1i
O1i—Ni1—N1
180.0
93.00 (4)
87.00 (4)
87.00 (4)
93.00 (4)
90.70 (4)
89.30 (4)
89.30 (4)
C6—C7—C2
C6—C7—Cl1
N1—C8—C9
N1—C8—H8
C9—C8—H8
C8—C9—H9
C10—C9—C8
C10—C9—H9
121.94 (15)
119.13 (13)
122.93 (14)
118.5
118.5
120.3
119.41 (14)
120.3
Acta Cryst. (2009). E65, m545–m546
sup-5
supporting information
O1—Ni1—N1
O4—Ni1—O4i
O4—Ni1—N1i
O4i—Ni1—N1i
O4—Ni1—N1
O4i—Ni1—N1
N1i—Ni1—N1
C1—O1—Ni1
Ni1—O4—H41
Ni1—O4—H42
H41—O4—H42
C8—N1—Ni1
C8—N1—C12
C12—N1—Ni1
C13—N2—C14
C13—N2—C16
C14—N2—C16
O1—C1—C2
O2—C1—O1
O2—C1—C2
C3—C2—C1
C7—C2—C1
C7—C2—C3
C2—C3—C4
C2—C3—H3
C4—C3—H3
C3—C4—H4
C5—C4—C3
C5—C4—H4
C6—C5—C4
C6—C5—H5
C4—C5—H5
C5—C6—C7
C5—C6—H6
C7—C6—H6
C2—C7—Cl1
90.70 (4)
180.0
92.55 (4)
87.45 (4)
87.45 (4)
92.55 (4)
180.00 (7)
127.35 (9)
104.4 (15)
125.8 (15)
109 (2)
122.58 (10)
117.45 (12)
119.97 (9)
124.98 (13)
118.44 (12)
116.10 (12)
114.14 (12)
127.09 (14)
118.77 (13)
121.41 (14)
120.65 (14)
117.87 (14)
120.80 (16)
119.6
119.6
120.1
119.74 (16)
120.1
120.52 (15)
119.7
119.7
119.08 (16)
120.5
120.5
118.94 (12)
C9—C10—C11
C9—C10—H10
C11—C10—H10
C10—C11—C13
C12—C11—C10
C12—C11—C13
N1—C12—C11
N1—C12—H12
C11—C12—H12
O3—C13—N2
O3—C13—C11
N2—C13—C11
N2—C14—C15
N2—C14—H14A
N2—C14—H14B
C15—C14—H14A
C15—C14—H14B
H14A—C14—H14B
C14—C15—H15A
C14—C15—H15B
C14—C15—H15C
H15A—C15—H15B
H15A—C15—H15C
H15B—C15—H15C
N2—C16—C17
N2—C16—H16A
N2—C16—H16B
C17—C16—H16A
C17—C16—H16B
H16A—C16—H16B
C16—C17—H17A
C16—C17—H17B
C16—C17—H17C
H17A—C17—H17B
H17A—C17—H17C
H17B—C17—H17C
118.10 (13)
121.0
121.0
118.90 (13)
119.09 (13)
121.91 (13)
122.94 (13)
118.5
118.5
122.60 (13)
118.39 (13)
119.01 (13)
112.80 (15)
109.0
109.0
109.0
109.0
107.8
109.5
109.5
109.5
109.5
109.5
109.5
111.60 (13)
109.3
109.3
109.3
109.3
108.0
109.5
109.5
109.5
109.5
109.5
109.5
O4—Ni1—O1—C1
O4i—Ni1—O1—C1
N1i—Ni1—O1—C1
N1—Ni1—O1—C1
O1i—Ni1—N1—C8
O1—Ni1—N1—C8
O1i—Ni1—N1—C12
O1—Ni1—N1—C12
O4—Ni1—N1—C8
O4i—Ni1—N1—C8
O4—Ni1—N1—C12
172.55 (12)
−7.45 (12)
−94.85 (12)
85.15 (12)
58.61 (11)
−121.39 (11)
−121.15 (11)
58.85 (11)
151.65 (11)
−28.35 (11)
−28.11 (11)
C1—C2—C3—C4
C7—C2—C3—C4
C2—C3—C4—C5
C6—C5—C4—C3
C7—C6—C5—C4
Cl1—C7—C2—C1
Cl1—C7—C2—C3
C6—C7—C2—C1
C6—C7—C2—C3
Cl1—C7—C6—C5
C2—C7—C6—C5
−174.81 (14)
2.0 (2)
−0.2 (2)
−1.3 (3)
0.8 (3)
−5.36 (19)
177.78 (12)
174.35 (14)
−2.5 (2)
−179.16 (12)
1.1 (2)
Acta Cryst. (2009). E65, m545–m546
sup-6
supporting information
O4i—Ni1—N1—C12
Ni1—O1—C1—O2
Ni1—O1—C1—C2
Ni1—N1—C8—C9
C12—N1—C8—C9
Ni1—N1—C12—C11
C8—N1—C12—C11
C13—N2—C14—C15
C16—N2—C14—C15
C13—N2—C16—C17
C14—N2—C16—C17
C3—C2—C1—O1
C3—C2—C1—O2
C7—C2—C1—O1
C7—C2—C1—O2
151.89 (11)
−9.8 (2)
170.38 (9)
177.78 (11)
−2.5 (2)
−179.49 (11)
0.7 (2)
−110.16 (17)
77.88 (18)
−90.03 (17)
82.48 (17)
85.51 (18)
−94.30 (19)
−91.23 (17)
88.96 (19)
N1—C8—C9—C10
C11—C10—C9—C8
C12—C11—C10—C9
C13—C11—C10—C9
C10—C11—C12—N1
C13—C11—C12—N1
O3—C13—N2—C14
O3—C13—N2—C16
C11—C13—N2—C14
C11—C13—N2—C16
O3—C13—C11—C10
O3—C13—C11—C12
N2—C13—C11—C10
N2—C13—C11—C12
1.6 (2)
1.1 (2)
−2.7 (2)
−179.11 (13)
1.8 (2)
178.16 (13)
−174.33 (15)
−2.5 (2)
5.4 (2)
177.22 (12)
61.59 (19)
−114.75 (16)
−118.19 (15)
65.48 (19)
Symmetry code: (i) −x, −y+1, −z.
Hydrogen-bond geometry (Å, º)
D—H···A
i
O4—H41···O2
O4—H42···O3ii
D—H
H···A
D···A
D—H···A
0.82 (2)
0.85 (2)
1.86 (2)
1.93 (2)
2.6267 (17)
2.7826 (15)
155 (2)
172 (2)
Symmetry codes: (i) −x, −y+1, −z; (ii) −x+1/2, y−1/2, −z+1/2.
Acta Cryst. (2009). E65, m545–m546
sup-7
|
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https://openalex.org/W3016003958
|
https://ora.ox.ac.uk/objects/uuid:242d66f6-adec-4e0a-9f7c-f1d4edd10ff1/download_file?safe_filename=Abeler_et_al_2020.pdf&file_format=pdf&type_of_work=Journal+article
|
English
| null |
COVID-19 Contact Tracing and Data Protection Can Go Together
|
JMIR mhealth and uhealth
| 2,020
|
cc-by
| 3,558
|
KEYWORDS COVID-19; app; contact tracing; proximity tracing; privacy; data protection; Bluetooth COVID-19; app; contact tracing; proximity tracing; privacy; data protection; Bluetooth person was in close proximity with. The risk of infection is
highest if one has been within 1.5-2 m of an infected person for
at least 10-15 minutes. If it could be determined who had been
in such close proximity, then one could ask freshly infected,
presymptomatic people to self-isolate and thus stop them from
infecting more people. Mathematical models of the pandemic [6]
show that fast contact tracing combined with a large-scale
virus-testing program might be able to not just delay the
epidemic but to stop it entirely. This would also mean that the
lockdown measures currently in place around the world could
be slowly loosened up again. However, such fast contact tracing
is not possible manually. Only a digital, largely automatic
solution would help. Why Is Contact Tracing Useful? The coronavirus disease (COVID-19) pandemic is the greatest
public health threat that the world has seen in the last 100
years. In response, countries have introduced various levels of
“lockdown” to reduce the number of new infections. Lockdowns, however, come at a great cost to workers, firms,
and families. Recent epidemiological models also predict that
the epidemic will start anew, once the lockdown is lifted [1]. Scientists have thus discussed a second approach to keeping
the epidemic in check: app-based contact tracing. Several apps
are currently in development (eg, in the United Kingdom [2],
by a pan-European initiative [3], and in a joint Google and Apple
venture [4]), or have already been launched (eg, in Singapore
[5]). Related Article: Related Article:
Comment on: https://mhealth.jmir.org/2020/4/e18936/ (JMIR Mhealth Uhealth 2020;8(4):e19359) doi: 10.2196/19359 (JMIR Mhealth Uhealth 2020;8(4):e19359) doi: 10.2196/19359 JMIR Mhealth Uhealth 2020 | vol. 8 | iss. 4 | e19359 | p. 1
(page number not for citation purposes) Guest Editorial Guest Editorial http://mhealth.jmir.org/2020/4/e19359/ Johannes Abeler1*, PhD; Matthias Bäcker2*, PhD, LLM; Ulf Buermeyer3*, PhD, LLM; Hannah Zillessen1*, MPhil Johannes Abeler1*, PhD; Matthias Bäcker2*, PhD, LLM; Ulf Buermeyer3*, PhD, LLM; Hannah Zillessen1*, MPhil 1Department of Economics, University of Oxford, Oxford, United Kingdom
2Rechts- und Wirtschaftswissenschaften, Johannes Gutenberg-Universität Mainz, Mainz, Germany
3Gesellschaft für Freiheitsrechte (German Society for Civil Rights), Berlin, Germany
*all authors contributed equally Corresponding Author:
Johannes Abeler, PhD
Department of Economics
University of Oxford
Manor Road
Oxford
United Kingdom
Phone: 44 1865281440
Email: johannes.abeler@economics.ox.ac.uk Abstract We discuss the implementation of app-based contact tracing to control the coronavirus disease (COVID-19) pandemic and discuss
its data protection and user acceptability aspects. We discuss the implementation of app-based contact tracing to control the coronavirus disease (COVID-19) pandemic and discuss
its data protection and user acceptability aspects. JMIR MHEALTH AND UHEALTH JMIR MHEALTH AND UHEALTH Abeler et al Epidemiology Meets Data Protection However, even in the face of an existential threat, we should
interfere with fundamental rights as little as possible. Among
the effective approaches, we should choose the one that least
compromises fundamental rights. In particular, we believe that
swift and efficient contact tracing is possible without collecting
extensive amounts of data in a central database. Another example for this kind of “privacy by design” COVID-19
tracing approach is the TraceTogether app [5] from the
Singaporean government. Unlike the contact point system, it
only requires users to enable Bluetooth on their phone. Pan-European Privacy-Preserving Proximity Tracing (PEPP-PT)
by the European consortium [3], as well as Google and Apple’s
recently announced joint initiative [4], are following a very
similar concept. We present a slightly modified version below. A contact tracing system can be set up in a way that would allow
for most data processing to happen locally on users’ mobile
phones rather than on a central server. Only the notification of
users who have been in contact with an infected person would
need to be coordinated centrally. Even in this case, the necessary
data could be processed in a way that would effectively preclude
the central server from identifying users. The system would
also not require collecting any location data. In order to detect whether two people have come into close
enough physical proximity to risk an infection, one can use
Bluetooth low energy technology. The general drawback of
Bluetooth—that it can only reach across a few meters—becomes
an advantage here. The tracking itself would work as follows:
as many people as possible voluntarily install the app on their
phone. The app cryptographically generates a new temporary
ID every half hour. As soon as another phone with the same
app is in close proximity, both phones receive the temporary
ID of the respective other app and record it. This list of logged
IDs is encrypted and stored locally on the users’ phones (Figure
1) The fundamental idea is simple: it does not matter where people
get in contact with an infected person. Be it on the bus or at
work—what matters is proximity to a contagious person. This
means that particularly sensitive location data, such as GPS or
radio cell data, is actually neither necessary nor useful. Instead,
the only data that matters is whether two people have come into
close enough contact to risk an infection. Epidemiology Meets Data Protection Some might argue that the demands of the COVID-19 crisis
justify even extreme countermeasures. After all, this is about
saving the lives and preserving the health of as many people as
possible. Weakening data protection might be preferable to the
far-reaching restrictions of personal freedom and to the
economic costs of the current lockdown. In keeping with this,
many countries have started tracking their citizens’ phones and
using location data to monitor the spread of the virus as well as Why would such an app be useful at all? We still don’t know
many things about COVID-19. The data so far suggest, however,
that about half of all infections occur before the dreaded
symptoms of fever or a persistent cough appear. It is therefore
not enough to quarantine people only after they show symptoms. To reduce infections, one would need to act quickly when a
person is diagnosed with COVID-19 to find all people this XSL•FO
RenderX JMIR MHEALTH AND UHEALTH Abeler et al interaction, be it among friends and family or in public spaces
like a restaurant, then the app could use both this information
and voluntary notifications from users should they be diagnosed
with the virus to compute transmission graphs. These graphs in
turn could let every user know if there were any possible
transmission paths leading up to the checkpoints they visited
and thus their risk of being infected. The app would not need
any location data and in fact wouldn’t even require users to
register. It would, however, require the active participation of
users who would need to either create or join a checkpoint
whenever they get close to someone outside their household. Thus, this approach relies on high levels of vigilance and
willingness to participate among at least a majority of the
population—not only initially but also as the pandemic
continues. to enforce both lockdown and early isolation restrictions. The
most prominent example of this is China [7], where entry to
many public places is restricted to people who can show a green
health code on their smartphones and thus demonstrate they
have not been in contact with a confirmed case of COVID-19. However, countries like Israel [8] or South Korea [9] use
location data as well—in the former case, to enforce quarantine
rules and notify the contacts of an infected person, and in the
latter to warn people before they enter “high risk” zones. JMIR Mhealth Uhealth 2020 | vol. 8 | iss. 4 | e19359 | p. 2
(page number not for citation purposes) http://mhealth.jmir.org/2020/4/e19359/ Epidemiology Meets Data Protection One example of this
would be contact tracing based on “contact points” as suggested
by Yasaka et al [10]. The smartphone app they propose would
allow users to create “checkpoints” by generating a QR code
that can be scanned by all other app users when joining their
checkpoint. If checkpoints were created for any social http://mhealth.jmir.org/2020/4/e19359/ XSL•FO
RenderX Abeler et al JMIR MHEALTH AND UHEALTH Figure 1. A COVID-19 tracing approach via Bluetooth. Every mobile phone stores a list of mobile phones that were within 2 m for at least 15 minutes. IDs are temporary but can be decrypted by the server. Figure 1. A COVID-19 tracing approach via Bluetooth. Every mobile phone stores a list of mobile phones that were within 2 m for at least 15 minutes. IDs are temporary but can be decrypted by the server. s are temporary but can be decrypted by the server. As soon as an app user is diagnosed with COVID-19, the doctor
making the diagnosis asks the user to share their locally stored
data with the central server (Figure 2). If the user complies, the
central server receives information on all the temporary IDs the
“infected” phone has been in contact with. The server is not
able to decrypt this information in a way that allows for the
identification of individuals. However, it is able to notify all
affected phones. This is because the server does not need any
personal data to send a message to someone’s phone. The server
only needs a so-called PushToken, a kind of digital address of an app installation on a particular phone. This PushToken is
generated when the app is installed on the user’s phone. At the
same time, the app will send a copy of the PushToken, as well
as the temporary IDs it sends out over time, to a central server. The server could be hosted, for example, by the Robert Koch
Institute for Germany or by the National Health Service for the
United Kingdom. This way, it would be possible to contact
phones solely based on temporary IDs and PushTokens whilst
completely preserving the privacy of the person using the phone. an app installation on a particular phone. This PushToken is
generated when the app is installed on the user’s phone. JMIR Mhealth Uhealth 2020 | vol. 8 | iss. 4 | e19359 | p. 3
(page number not for citation purposes) Epidemiology Meets Data Protection At the
same time, the app will send a copy of the PushToken, as well
as the temporary IDs it sends out over time, to a central server. The server could be hosted, for example, by the Robert Koch
Institute for Germany or by the National Health Service for the
United Kingdom. This way, it would be possible to contact
phones solely based on temporary IDs and PushTokens whilst
completely preserving the privacy of the person using the phone. Figure 2. A user can share their data with the server after receiving a COVID-19 diagnosis. The server then alerts all phones that have been in close
proximity to the infected phone. The alerted people would still need to contact their local health authorities, as their identity is not linked to the app. If a phone has been in close proximity to an “infected” phone,
the user of that phone receives a notification together with the
request to immediately go into quarantine at home. The user
will then need to contact the local health authorities to get tested
for the virus as soon as possible so that, depending on the
outcome, the user is either able to stop quarantining or all their
contacts can be informed (Figure 2). JMIR Mhealth Uhealth 2020 | vol. 8 | iss. 4 | e19359 | p. 3
http://mhealth.jmir.org/2020/4/e19359/
(page number not for citation purposes
FO If a phone has been in close proximity to an “infected” phone,
the user of that phone receives a notification together with the
request to immediately go into quarantine at home. The user
will then need to contact the local health authorities to get tested for the virus as soon as possible so that, depending on the
outcome, the user is either able to stop quarantining or all their
contacts can be informed (Figure 2). JMIR Mhealth Uhealth 2020 | vol. 8 | iss. 4 | e19359 | p. 3
(page number not for citation purposes) JMIR Mhealth Uhealth 2020 | vol. 8 | iss. 4 | e19359 | p. 3
(page number not for citation purposes) XSL•FO
RenderX Abeler et al JMIR MHEALTH AND UHEALTH cooperate (by installing the app and carrying their phones with
them) for any chance of success. Consequently, the effectiveness
of any contact-tracing system depends on public support. Epidemiology Meets Data Protection There
is reason to believe that the level of support can be increased
by opting for a data-minimizing solution. A representative
survey [11] across the United States, United Kingdom, Germany,
Italy, and France shows that about 70% of respondents would
install an app like the one described above on their phones
(disclosure: co-author JA was also the lead author of the survey
study). The reason most frequently brought up against an
installation is the worry that the government could use the app
as an excuse for greater surveillance after the end of the
epidemic. If the government wants as many people as possible
to install the app, it should take these concerns seriously and
refrain from using location data. Contact tracing works without
it. During the entire process, no one learns the identity of the app
user (eg, other users who got in close contact with them, the
local health authorities, the central server) since the app is not
linked to an identity. Location data is neither recorded nor stored
at any point of the process. As mentioned above, we did not come up with this concept. Singapore introduced a very similar app, and several European
countries [3] are working on comparable apps as well. We do
not agree with all aspects of the Singaporean app and their
practice of contact tracing. For example, every app installation
in Singapore is linked with the user’s telephone number, making
the user identifiable—something that is not strictly necessary
and thus, for data protection reasons, should be rejected. Nevertheless, we like the general concept. The recently
published PEPP-PT [3] looks promising and might prove to be
a legitimate implementation of the privacy-friendly tracing
approach outlined above. Conclusion: Proportionality Instead of
“Whatever It Takes” Such an app could implement contact tracing much more
effectively than a system that relies on radio cell or location
data, since neither of these two data sources permit determining
a person’s position with the necessary precision of 2 m
maximum. At the same time, such a concept would comply with
existing data protection regulations. Finally, it would work even
without users paying constant attention to potentially risky
interactions as would be necessary in a contact-point system. Thus, this concept is potentially more robust to fatigue or
inattentiveness. In the current crisis, we will have to endure more and deeper
encroachments on fundamental rights than we are used to. Still,
there is no reason to tolerate such encroachments to a greater
extent than strictly necessary. Even under the current time
pressure, it is important to find solutions that minimize data
processing as far as possible. We have shown above that this is
possible for the case of contact tracing. As the pandemic
progresses, many other challenges will emerge. For each of
them, one will have to check which data processing is necessary
to address them and which ones can be avoided. Data Minimization Begets Acceptance Trying to find a data-minimizing solution does not just protect
fundamental rights. Such solutions will often increase the
effectiveness and efficiency of the respective data-processing
system. Only if people trust a system—because it does not spy
on them—will the system find broad support in the population. In the case of contact tracing, the approach that requires the
least amount of data also seems to be the most effective
epidemiologically. This is because an app like the one described
above would be better suited to determine who actually was in
close proximity than any of the other proposed solutions. Moreover, even digital contact-tracing systems need users to http://mhealth.jmir.org/2020/4/e19359/ References 1. Ferguson N, Laydon D, Nedjati-Gilani G, Imai N, Ainslie K, Baguelin M, Imperial College COVID-19 Response Team. Impact of non-pharmaceutical interventions (NPIs) to reduce COVID19 mortality and healthcare demand. Imperial College
London 2020:1-20 [FREE Full text] [doi: 10.25561/77482]
2. Rapson J. HSJ. 2020 Mar 18. NHS developing coronavirus contact tracking app URL: https://www.hsj.co.uk/
free-for-non-subscribers/nhs-developing-coronavirus-contact-tracking-app/7027163.article
3. PEPP-PT. Pan-European Privacy-Preserving Proximity Tracing. 2020. URL: https://www.pepp-pt.org/
4. Google Inc. Apple and Google partner on COVID-19 contact tracing technology. 2020 Apr 10. URL: https://www.blog.google/
inside-google/company-announcements/apple-and-google-partner-covid-19-contact-tracing-technology/
5. Government of Singapore. TraceTogether. URL: https://www.tracetogether.gov.sg/
6. Ferretti L, Wymant C, Kendall M, Zhao L, Nurtay A, Abeler-Dörner L, et al. Quantifying SARS-CoV-2 transmission
suggests epidemic control with digital contact tracing. Science 2020 Mar 31 [FREE Full text] [doi: 10.1126/science.abb6936]
[Medline: 32234805]
7. Associated Press. Time. 2020 Apr 02. Inside China's Smartphone 'Health Code' System Ruling Post-Coronavirus Life URL:
https://time.com/5814724/china-health-code-smartphones-coronavirus/
8. Tidy J. BBC News. 2020 Mar 17. Coronavirus: Israel enables emergency spy powers URL: https://www.bbc.com/news/
technology-51930681
JMIR Mhealth Uhealth 2020 | vol. 8 | iss. 4 | e19359 | p. 4
http://mhealth.jmir.org/2020/4/e19359/
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FO 1. Ferguson N, Laydon D, Nedjati-Gilani G, Imai N, Ainslie K, Baguelin M, Imperial College COVID-19 Response Team. Impact of non-pharmaceutical interventions (NPIs) to reduce COVID19 mortality and healthcare demand. Imperial College
London 2020:1-20 [FREE Full text] [doi: 10.25561/77482]
2. Rapson J. HSJ. 2020 Mar 18. NHS developing coronavirus contact tracking app URL: https://www.hsj.co.uk/
free-for-non-subscribers/nhs-developing-coronavirus-contact-tracking-app/7027163.article
3. PEPP-PT. Pan-European Privacy-Preserving Proximity Tracing. 2020. URL: https://www.pepp-pt.org/
4. Google Inc. Apple and Google partner on COVID-19 contact tracing technology. 2020 Apr 10. URL: https://www.blog.google/
inside-google/company-announcements/apple-and-google-partner-covid-19-contact-tracing-technology/
5. Government of Singapore. TraceTogether. URL: https://www.tracetogether.gov.sg/
6. Ferretti L, Wymant C, Kendall M, Zhao L, Nurtay A, Abeler-Dörner L, et al. Quantifying SARS-CoV-2 transmission
suggests epidemic control with digital contact tracing. Science 2020 Mar 31 [FREE Full text] [doi: 10.1126/science.abb6936]
[Medline: 32234805]
7. Associated Press. Time. 2020 Apr 02. Inside China's Smartphone 'Health Code' System Ruling Post-Coronavirus Life URL:
https://time.com/5814724/china-health-code-smartphones-coronavirus/
8. Tidy J. BBC News. 2020 Mar 17. Coronavirus: Israel enables emergency spy powers URL: https://www.bbc.com/news/
technology-51930681
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London 2020:1-20 [FREE Full text] [doi: 10.25561/77482] 1. Ferguson N, Laydon D, Nedjati-Gilani G, Imai N, Ainslie K, Baguelin M, Imperial College COVID-19 Response Team. Impact of non-pharmaceutical interventions (NPIs) to reduce COVID19 mortality and healthcare demand. Imperial College
London 2020:1-20 [FREE Full text] [doi: 10.25561/77482] [
] [
]
2. Rapson J. HSJ. 2020 Mar 18. NHS developing coronavirus contact tracking app URL: https://www.hsj.co.uk/
free-for-non-subscribers/nhs-developing-coronavirus-contact-tracking-app/7027163.article
3. PEPP-PT. Pan-European Privacy-Preserving Proximity Tracing. 2020. URL: https://www.pepp-pt.org/
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3. PEPP-PT. Pan-European Privacy-Preserving Proximity Tracing. 2020. URL: https://www.pepp-pt.org/
4. Google Inc. Apple and Google partner on COVID-19 contact tracing technology. 2020 Apr 10. URL: https://www.blog.google/
inside-google/company-announcements/apple-and-google-partner-covid-19-contact-tracing-technology/
5. Government of Singapore. TraceTogether. URL: https://www.tracetogether.gov.sg/ 6. Ferretti L, Wymant C, Kendall M, Zhao L, Nurtay A, Abeler-Dörner L, et al. Quantifying SARS-CoV-2 transmission
suggests epidemic control with digital contact tracing. Science 2020 Mar 31 [FREE Full text] [doi: 10.1126/science.abb6936]
[Medline: 32234805] 7. Associated Press. Time. 2020 Apr 02. Inside China's Smartphone 'Health Code' System Ruling Post-Coronavirus Life URL:
https://time.com/5814724/china-health-code-smartphones-coronavirus/ p
p
8. Tidy J. BBC News. 2020 Mar 17. Coronavirus: Israel enables emergency spy powers URL: https://www.bbc.com/news/
technology-51930681 JMIR Mhealth Uhealth 2020 | vol. 8 | iss. 4 | e19359 | p. 4
(page number not for citation purposes) http://mhealth.jmir.org/2020/4/e19359/ JMIR Mhealth Uhealth 2020 | vol. 8 | iss. 4 | e19359 | p. 5
(page number not for citation purposes) JMIR MHEALTH AND UHEALTH Abeler et al 9. Cuthbertson A. Independent. Coronavirus Apps Let People Avoid High-Risk Locations in South Korea URL: https://www. independent.co.uk/life-style/gadgets-and-tech/news/coronavirus-news-app-south-korea-latest-cases-deaths-location-a9371651. html 10. Yasaka TM, Lehrich BM, Sahyouni R. Peer-to-Peer Contact Tracing: Development of a Privacy-Preserving Smartphone
App. JMIR Mhealth Uhealth 2020 Apr 07;8(4):e18936 [FREE Full text] [doi: 10.2196/18936] [Medline: 32240973]
11. OSF. 2020 Mar 26. covid19_app_survey URL: https://osf.io/7vgq9/ Abbreviations Edited by G Eysenbach; this is a non–peer-reviewed article. Submitted 14.04.20; accepted 15.04.20; published 20.04.20. Please cite as:
Abeler J, Bäcker M, Buermeyer U, Zillessen H
COVID-19 Contact Tracing and Data Protection Can Go Together
JMIR Mhealth Uhealth 2020;8(4):e19359
URL: http://mhealth.jmir.org/2020/4/e19359/
doi: 10.2196/19359
PMID: 32294052 Edited by G Eysenbach; this is a non–peer-reviewed article. Submitted 14.04.20; accepted 15.04.20; published 20.04.20. Please cite as:
Abeler J, Bäcker M, Buermeyer U, Zillessen H
COVID-19 Contact Tracing and Data Protection Can Go Together
JMIR Mhealth Uhealth 2020;8(4):e19359
URL: http://mhealth.jmir.org/2020/4/e19359/
doi: 10.2196/19359
PMID: 32294052 ©Johannes Abeler, Matthias Bäcker, Ulf Buermeyer, Hannah Zillessen. Originally published in JMIR mHealth and uHealth
(http://mhealth.jmir.org), 20.04.2020. This is an open-access article distributed under the terms of the Creative Commons Attribution
License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any
medium, provided the original work, first published in JMIR mHealth and uHealth, is properly cited. The complete bibliographic
information, a link to the original publication on http://mhealth.jmir.org/, as well as this copyright and license information must
be included. http://mhealth.jmir.org/2020/4/e19359/ http://mhealth.jmir.org/2020/4/e19359/ XSL•FO
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Determination of the complex frequencies for the normal modes below 1mHz after the 2010 Maule and 2011 Tohoku earthquakes
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Annals of geophysics
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cc-by
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ANNALS OF GEOPHYSICS, 56, 5, 2013, S0563; doi:10.4401/ag-6400 ANNALS OF GEOPHYSICS, 56, 5, 2013, S0563; doi:10.4401/ag-6400 ABSTRACT ture [Chao and Gilbert 1980, He and Tromp 1996]. In
addition, determining the quality factors Qs of those
modes may also help to improve the knowledge of the
3D attenuation model of the Earth [Roult et al. 2006]. Based upon SG (superconducting gravimeter) records, the autoregressive
method proposed by Chao and Gilbert [1980] is used to determine the fre-
quencies of the singlets of seven spheroidal modes (0S2, 2S1, 0S3, 0S4, 1S2,
0S0, and 3S1) and the degenerate frequencies of three toroidal modes (0T2,
0T3, and 0T4) below 1 mHz after two recent huge earthquakes, the 2010
Mw8.8 Maule earthquake and the 2011 Mw9.1 Tohoku earthquake. The
corresponding quality factors Qs are also determined for those modes, of
which the Qs of the five singlets of 1S2 and the five singlets (m=0,
m=±2, and m=±3) of 0S4 are estimated for the first time using the SG
observations. The singlet m=0 of 3S1 is clearly observed from the power
spectra of the SG time series without using other special spectral analy-
sis methods or special time series of polar station records. In addition,
the splitting width ratio R of 3S1 is 0.99, and consequently we conclude
that 3S1 is normally split. The frequencies and Qs of the modes below
1mHz can contribute to refining the 3D density and attenuation models
of the Earth. Numerous studies have dedicated to obtaining the
frequencies of all of the singlets of the modes below
1mHz. All of the singlets of 0S2 and 0S3 were completely
observed by Buland et al. [1979] using seismic datasets
from the International Deployment of Accerometers
(IDA) network, and the triplet of 2S1 was first observed
by Rosat et al. [2003] based on five SG records. The
triplet of 3S1 was first observed by Chao and Gilbert
[1980] using seismic datasets from IDA network. The
above mentioned four modes and the modes 1S2, 0S4,
0S5 have been extensively investigated by other scholars
[e.g., Ritzwoller et al. 1986, Giardini et al. 1988, Wid-
mer-Schnidrig et al. 1992a, Resovsky and Ritzwoller
1998, Hu et al. 2006, Widmer-Schnidrig and Laske 2007,
Roult et al. 2010, Deuss et al. 2011, Shen and Wu 2012,
Ding and Shen 2013]. Article history Article history
Received August 18, 2013; accepted November 21, 2013. Subject classification:
Normal modes, Splitting, Frequency, Quality factor Q. Determination of the complex frequencies for the normal modes
below 1mHz after the 2010 Maule and 2011 Tohoku earthquakes Hao Ding1, WenBin Shen1,2,* 1 Wuhan University, Department of Geophysics, School of Geodesy and Geomatics, Key Laboratory of Geospace
Environment and Geodesy of the Ministry of Education, Wuhan, China
2 State Key Laboratory of Information Engineering in Surveying, Mapping and Remote Sensing, China 1 Wuhan University, Department of Geophysics, School of Geodesy and Geomatics, Key Laboratory of Geospace
Environment and Geodesy of the Ministry of Education, Wuhan, China
2 State Key Laboratory of Information Engineering in Surveying, Mapping and Remote Sensing, China 1 Wuhan University, Department of Geophysics, School of Geodesy and Geomatics, Key Laboratory of G
Environment and Geodesy of the Ministry of Education, Wuhan, China
2 State Key Laboratory of Information Engineering in Surveying, Mapping and Remote Sensing, China 1. Introduction As for
the toroidal modes below 1mHz, the degenerate fre-
quencies of 0T2, 0T3 and 0T4, and Qs of 0T3 and 0T4
have been determined [e.g., Widmer-Schnidrig et al. 1992b, Tromp and Zanzerkia 1995, Hu et al. 2006, Abd
El-Gelil et al. 2010], and Roult et al. [2010] even have
determined the five split frequencies of 0T2. Due to the
fact that the frequencies of 0T5, 2S2 and 1S3 are very
close to each other, it is hard to distinguish them from
the spectra of the chosen time series. Since the singlets
m=0 of 2S1 and 3S1 are difficult to clearly observe to
date, the main purpose of this paper is to re-determine
or newly determine the frequencies and Qs of the
whole set of the singlets of the modes 0S0, 0S2, 1S2, 2S1,
0S3, 3S1, 0S4, 0T2, 0T3, and 0T4. needs not following that criterion [e.g., Rosat et al. 2005, Roult et al. 2010]. Because the noise levels of
those SG stations are different from each other [Rosat
and Hinderer 2011], we choose different groups of
records for different modes; and because different
events may excite different amplitudes even for the
same mode, we choose different groups of records for
a given mode (see details in Section 3).We note that, in
this study, the sampling interval of all records is 1min,
and for each of the target modes except for 3S1, all
records start 5h after the earthquakes; as for 3S1, data
sets with a 50h later starting point after the earthquake
are used to weaken the interference of 1S3 [Masters and
Gilbert 1983]. For each of the chosen records, after re-
moving the tidal and local atmospheric pressure effects,
we obtain the corresponding residual gravity time se-
ries for further use. Since SGs deployed in the frame of the Global Ge-
odynamics Project (GGP) can outmatch the best seis-
mometers in the frequency band below 1mHz [e.g.,
Widmer-Schnidrig 2003], and since the 2010 Mw8.8
Maule earthquake and the 2011 Mw9.1 Tohoku earth-
quake have strongly excited the normal modes (see Fig-
ure 1), SG records after the two events are used to
determine the relevant frequencies and Qs. 1. Introduction The normal modes below 1mHz are very sensitive
to density heterogeneities of the Earth, and precise de-
termination of the splitting frequencies of these modes
can provide further information to constrain the 3D
models, especially the core and mantle density distri-
bution and structures. For instance, the observations of
the splitting of 0S2, 0S3, and 0S4 may provide further in-
formation to constrain the mantle structure [Okal 1978,
He and Tromp 1996, Roult et al. 2010]; the observations
of the splitting of 2S1 may help to constrain the outer
core and mantle models [Rosat et al. 2003]; the obser-
vations of 1S2 may provide additional constraints on the
3D models, especially the mantle structure [Roult et al. 2010, Deuss et al. 2011]; and the observations of 3S1
may constrain the Earth’s density and the core struc- Except for the mode 0S0, only few studies con-
cerned about the quality factors Qs of the whole set of
the singlets of those modes. The Q of 0S0 was deter-
mined by various authors [e.g., Sailor and Dziewonski
1978, Buland et al. 1979, Chao and Gilbert 1980,
Riedesel et al. 1980, Dziewonski and Anderson 1981,
Roult et al. 2006, Rosat et al. 2007, Xu et al. 2008, Abd
El-Gelil et al. 2010, Zábranová et al. 2012]. The Qs of all
of the five singlets of 0S2 were first estimated by Tani-
moto [1990], and then re-estimated by Roult et al. [2006], Rosat et al. [2008] and Abd El-Gelil et al. [2010]. The Qs of all of the singlets of 0S3 and 2S1 were first es-
timated by Roult et al. [2006]. Rosat et al. [2008] and
Abd El-Gelil et al. [2010] further provided the refined S0563 DING AND SHEN
Figure 1. The amplitude spectra of the residual gravity sequences from CB station after the 2010 Maule event (gray area) and 2011 Tohoku
event (black area), both of the sequences starting 5h after the earthquake with a length of 200h. 0S2
2S1
0S3
0T5/2S2/1S3/3S1 DING AND SHEN Figure 1. The amplitude spectra of the residual gravity sequences from CB station after the 2010 Maule event (gray area) and 2011 Tohoku
event (black area), both of the sequences starting 5h after the earthquake with a length of 200h. measurements of Qs of the seven singlets of 0S3. 1. Introduction In this study,
after the 2010 event, we use 13 SG records from differ-
ent stations, listing as Apache Point (AP), Bad Homburg
(BH), Schiltach (BF, the Black Forest Observatory),
Cantley (CA), Canberra (CB), Membach (MB), Medic-
ina (MC), Moxa (MO), Ny-Alesund (NY), Pecny (PE),
Sutherland (SU), Strasbourg (ST), and Wettzell (WE). After the 2011 event, we use the records from 13 SG sta-
tions, listing as AP, BF, BH, CB, Conrad (CO), MB, MC,
MO, NY, PE, ST, Concepcion (TC), and WE. Accord-
ing to Dahlen [1982], in general cases, the optimum
record length should be about 1.1Q-cycle of the modes. However, in order to obtain a sufficient frequency res-
olution, for each mode, the selection of the data length COMPLEX FREQUENCIES OF THE NORMAL MODES However, considering that Xi(~k) has a real part and an
imaginary part, if K=1, Equation (6) can be rewritten as
the matrix form For a discrete time series (with N data points), set
vj = ~j + iaj and Aj = Ajeizj, then Equation (1) can be
rewritten as, (3)
,
, ,...,
exp
exp
a n
A
in
A
in
n
N
1 2
j
j
j
j
j
M
1
v
v
=
+
=
)
)
=
^
^
^
h
h
h
6
@
/ (3) (8)
Re
Im
Re
Im
Re
Im
X
X
X
X
X
X
S
S
2
1
2
1
1
1
1
1
1
1
0
0
1
2
$
~
~
~
~
~
~
=
_
_
_
_
_
_
i
i
i
i
i
i
7
7
7
7
>
<
7
7
>
A
A
A
AH
F
A
AH (8) where ‘ * ’ denotes complex conjugate, M denotes the
number of the complex exponential functions. Then
a(n) can be expressed as the following linear equation, Hence, taking K=M is enough to solve out {Si}
from Equation (7). And if K > M, a simple way to solve
Equation (7) is the linear least-squares estimate. After
{Si} are found, {vj} can be determined by using some
numerical methods. The estimation of the complex
amplitudes {Aj} is another problem of linear least-
squares estimate, which is referred to Chao and Gilbert
[1980] for details. (4)
,
,...,
a n
S a n
n
M
N
1
2
1
i
i
M
1
2
=
-
=
+
=
^
^
h
h
/ (4) where Si is a real constant coefficient (i = 1,2,...,2M). According to the Prony method [e.g., Chao and Gilbert
1980, Chao 1990a], the relationship between {vj} and
{Si} can be constructed through the following polyno-
mial equation The above process is known as the AR method. And from Equation (7), one can see that this method has
the capability to process multiple records simultane-
ously to enhance the signal to noise ratio (SNR), namely,
one can stack the matrix from different records obtained
from different stations. Furthermore, the records even
can be obtained after different earthquakes. COMPLEX FREQUENCIES OF THE NORMAL MODES complex frequency estimation methods. Smith [1972]
reviewed the early observations of aj, most of which
were obtained by the time lapse method. Chao and
Gilbert [1980] proposed a method, referred to as the au-
toregressive (AR) method, to estimate the complex fre-
quencies. Masters and Gilbert [1983] proposed a
nonlinear least-squares fitting method, named as least-
squares (LS) algorithm. Roult and Clévédé [2000] pro-
posed an improved method, which is based on the ‘time
lapse’ method, to estimate the attenuation aj. Rosat et
al. [2008] proposed a non-linear damped harmonic
analysis (NLDHA) method, which can be also used to
estimate the complex frequencies of a normal mode. In addition, the complex frequencies can be also esti-
mated by fitting a Lorentzian function [e.g., Smylie
1992, Abd El-Gelil et al. 2010]. Here we should note
that we just want to give a short review about those
presented methods, rather than to explain their strengths
and weaknesses. In this study, due to the fact that the
validity of the AR method has been verified by previous
studies [Chao 1983, Masters and Gilbert 1983], we use
this method to estimate the complex frequencies of
those modes. Because the details of the estimation
process have been introduced in Chao and Gilbert
[1980], here we only provide a brief introduction of the
basic idea. not solve Equation (4) for {Si} in the time domain. Fortu-
nately, different modes could be separated into individual
peaks in the frequency domain (for most of the normal
modes). The technique is stated as follows. The Fourier
transform (FT) of Equation (4) can be expressed as (6)
,
, ,...,
X
X
S
k
K
1 2
k
k
i
M
i
0
1
1
2
~
~
=
=
=
^
^
h
h
/ (6) where {Xi(~k), i = 1,2,...,2M} corresponds to the Fourier
transform of {a(n − i), n = 2M + 1,...,N}. 2. The estimate method for frequencies and Qs For a vertical acceleration record, it can be simply
expressed as a sum of decaying cosinusoids [e.g., Aki
and Richards 1980, Chao and Gilbert 1980, Master and
Gilbert 1983, Master and Widmer-Schnidrig 1995]: (1)
,
cos
a t
A
t
e
j
j
M
j
j
tj
~
z
=
+
a
-
^
^
h
h
/ (1) where Aj is the amplitude, aj the attenuation, ~j the fre-
quency and zj the phase of the jth mode. And the qual-
ity factor Q of the jth mode can be estimated through
~j and aj, by the following equation (2)
2
Qj
j
j
a
~
= (2) According to Equation (2), one needs only to know
the complex frequency of a mode, and then the corre-
sponding Q can be estimated. There are numerous 2 COMPLEX FREQUENCIES OF THE NORMAL MODES It can be also
written as the matrix form (7)
X
X
X
X
X
X
X
X
X
S
S
S
X
X
X
K
K
M
M
M
K
M
K
1
1
1
2
1
2
1
2
2
2
2
1
2
2
2
1
2
2
0
1
0
2
0
h
h
j
h
h
h
~
~
~
~
~
~
~
~
~
~
~
~
=
_
_
_
_
_
_
_
_
_
_
_
_
i
i
i
i
i
i
i
i
i
i
i
i
R
T
S
S
S
S
S
R
T
S
S
S
SS
R
T
S
S
S
S
S
V
X
W
W
W
W
W
V
X
W
W
W
WW
V
X
W
W
W
W
W (7) It should be noted that a Hanning taper should be
multiplied by each new time sequence prior to FT to
weaken the spectral leakage. In addition, M becomes
the number of the cosinusoids residing in the target fre-
quency band. It is 1 for a single peak, and it is a small
number for a group of interfering peaks with close fre-
quencies and for some strongly coupled normal modes
or splitting of a multiplet [Chao and Gilbert 1980, Chao
1990a, 1990b]. Here we only take M=1 as an example,
and then, there are only two AR coefficients S1 and S2. COMPLEX FREQUENCIES OF THE NORMAL MODES It is noted
that the AR method is very powerful in estimating the
parameters (complex frequency and amplitude) of the
singlets of a multiplet simultaneously [Chao 1990a]. (5)
... exp
exp
Z
S Z
S Z
S
Z
i
Z
i
Z
p Z
q
M
M
M
M
j
j
M
j
j
j
M
j
2
1
2
1
2
2
2
2
1
2
1
v
v
-
-
-
-
=
=
-
-
=
=
+
+
)
-
-
=
=
^
^
h
h
6
6
6
@
@
@
%
% ... Z
S Z
S Z
S
M
M
M
M
2
1
2
1
2
2
2
2
-
-
-
-
=
-
- (5) where aj = −0.5lnqi and
Therefore, if {Si} are obtained, {vj} can be determined
by Equation (3). However, for a time series after an
earthquake, M is usually unknown; that means one can-
. cos
p
q
2
j
j
i
1
~ =
-
-
^
`
hj 3 DING AND SHEN Figure 2. The power spectra of 0S0. (a) The results of 13 residual gravity time series after the 2010 Maule event; (b) the results of 12 resid-
ual gravity time series after the 2011 Tohoku; (c) the estimated frequencies from different series, the red symbols (e.g., circle point, squares,
etc.) indicate the results obtained from the 2010 event, and the green ones indicate the results obtained from the 2011 event (hereafter the
same). The corresponding theoretical frequency based on PREM is denoted by vertical dashed lines. Figure 2. The power spectra of 0S0. (a) The results of 13 residual gravity time series after the 2010 Maule event; (b) the results of 12 resid-
ual gravity time series after the 2011 Tohoku; (c) the estimated frequencies from different series, the red symbols (e.g., circle point, squares,
etc.) indicate the results obtained from the 2010 event, and the green ones indicate the results obtained from the 2011 event (hereafter the
same). The corresponding theoretical frequency based on PREM is denoted by vertical dashed lines. 0S0
Frequency (mHz)
Q
PREM
0.81431
5327
Sailor and Dziewonski [1978]
0.814564
4229
Buland et al. [1979]
0.8146346±2.4e-5
4100±1066
Riedesel et al. [1980]
0.814664±3.3e-6
5700±285
Chao and Gilbert [1980]
0.814695±2.0e-5
5280±25
Roult et al. [2006]
0.814661±5.0e-6
5489.1±19.0
Rosat et al.[2007]
0.8146566±1.6e-6
5506±19
Xu et al. COMPLEX FREQUENCIES OF THE NORMAL MODES 1
1
i
i
N
2
v =
/ (10)
,
f
P f
e f
P e f
f
i
i
n
i
f
i
i
i
n
1
2
1
$
$
=
=
=
=
^
^
^
h
hh
/
/ (10) where ff is the finally estimated frequency value for a
given singlet, e( ff ) its error, Pi the weight given by
Equation (9), fi and e( fi ) are the estimates of the corre-
sponding frequency value and its corresponding error
bar for each chosen SG series. COMPLEX FREQUENCIES OF THE NORMAL MODES out to get a final estimation. out to get a final estimation. 1981], but our result is very close to those given by pre-
vious studies. The Q value is about 5500, which is in
consistent with PREM prediction and previous results. out to get a final estimation. For a given singlet after the two earthquakes, 1/v2
is used as a weight to obtain the finally estimated fre-
quency. The weight for the i-th observed singlet fre-
quency from a chosen SG series is given by Concerning the mode 0S2, we use nine residual
gravity time series after the 2010 Maule event and 11
residual gravity time series after the 2011 Tohoku event
(see Figure 3), with a length of 300h. The other records
are not used because the SNRs of them are not high
enough (hereafter the same reason for using different
records after the two events). The corresponding power
spectra are plotted in Figure 3. Comparing with the re-
sults excited by the 2004 Sumatra earthquake [e.g.,
Rosat et al. 2005, Roult et al. 2010], the singlets m=0 ex-
cited by the 2010 Maule event and 2011 Tohoku event
are both quite weak, and there is no detectable peak for
the singlet m=0 in the SG time series after the 2010
event, whereas it can be only weakly observed in three
SG time series (from BF, MB, and ST stations) after the
2011 event. In addition, excited by the 2004 Sumatra
event, the amplitudes of m=0 and m=±2 are larger than
the amplitudes of m=±1; whereas excited by the 2010
and 2011 event, the amplitudes of m=±1 are much
larger than the amplitudes of m=0 and m=±2. This
phenomenon is related to the focal mechanism and ge-
ographical locations of different earthquakes. (9)
P
1
1
i
i
i
i
N
2
2
v
v
= ^
h / (9) where N is the number of all of the chosen series after
the two events, vi is the estimated error of the estimated
frequency, and the denominator is applied to
make For each singlet, we use the follow-
ing formula [Taylor 1997] to obtain a final estimate of
the corresponding frequency based on the records after
both earthquakes:
1
i
i
N
2
v
/
. 3.1. The frequencies and Qs of the modes 0S0, 0S2, 0S3 3.1. The frequencies and Qs of the modes 0S0, 0S2, 0S3
Concerning the mode 0S0, we use the SG residual
time series from the first group of 13 stations (see Sec-
tion 1) after the 2010 Maule event and the second group
of 12 stations (except for the MC station) after the 2011
Tohoku event, with a length of 750h. The power spec-
tra are shown in Figure 2, and for the same event,
owing to the fact that the amplitudes of different
records are not the same, the normalized power spec-
tra are formulated for comparison (hereafter the same). The final estimation provided by this study is the
weighted average of the 23 results estimated separately
from each of all of the time series (hereafter, the same
rule holds throughout this paper except for a special
statement), which is provided in Table 1. For a com-
parative purpose, the results of previous studies and the
theoretical predictions are also listed in Table 1. From
Figure 2a,b, we find that the observed peaks of 0S0 from
different records are almost overlapped, and from Fig-
ure 2c and Table 1, there is a large deviation between
our frequency estimate and the corresponding theoret-
ical prediction of PREM [Dziewonski and Anderson We use eight residual gravity time series after the
2010 event and 12 residual gravity time series after the
2011 event to detect the mode 0S3 (see Figure 4). The
length of each sequence is 350 h. The power spectra
and the estimated frequencies are shown in Figure 4. Comparing with Rosat et al. [2005]’s result (see Figure
2 therein) observed at the ST station after the 2004
Sumatra event, the mode 0S3 observed at the ST station
after three different earthquakes are quite different. The
amplitudes of m=±2 excited by the 2010 and 2011 event
are relatively larger. The observed average values of the
frequencies and Qs of 0S3 are listed in Table 3. COMPLEX FREQUENCIES OF THE NORMAL MODES [2008]
0.8146565±1.2e-6
5400.94±22.4
Abd El-Gelil et al. [2010]
0.814666±0.7e-6
5551.0 ± 9.1
Zábranová et al. [2012]
--
5500±140
This paper (Average value)
0.8146568±6.5e-7
5566.6±18.8
Table 1. Observed weighted average values of the frequencies and Qs of 0S0, and the model predictions and observations of previous studies. Table 1. Observed weighted average values of the frequencies and Qs of 0S0, and the model predictions and observations of previous studies. the estimates as the final estimation. Due to the fact that the SNRs of different records
from the same event and those of the records from the
same station after the two events are different, and
some singlets of a mode may appear in a record but not
appear in some other records, we use AR method to es-
timate the singlets of one target mode from each sepa-
rated record, and take the weighted average of all of As for the estimated error, we prefer to use the
bootstrap method [see e.g., Efron and Tibshirani 1986]
as suggested by Häfner and Widmer-Schnidrig [2013],
where the details were presented. After the estimated
errors vj of a singlet frequency from different chosen
SG records are obtained, the following step is carried 4 4 3. The observations of different modes based on SG
records The observed weighted average values of the fre-
quencies and Qs of 0S2, and the corresponding theoret-
ical predictions of PREM [Millot-Langet et al. 2003,
Rosat et al. 2008] and some previous estimates are listed
in Table 2. The estimated results of the frequencies and
Qs are very close to the results of the previous studies
as well as the PREM predictions. In this study, as mentioned in Section 1, concern-
ing observing different modes, we use the records from
different stations, due to the fact that different stations
are differently sentsitive to different modes. 3.2. The frequencies and Qs of the modes 2S1 and 3S1 We use five residual gravity time series after the
2010 event and another five residual gravity time series
after the 2011 event to detect the mode 2S1 (see Figure 5),
with a length of 168h. From Figure 5, we find that the
singlet m=0 is clearly observed based on the SG records
with high SNRs after the two earthquakes, whereas
there are no records by which this singlet is observed 5 DING AND SHEN Figure 3. The power spectra of 0S2 based on the residual gravity time series after the 2010 and 2011 events. (a) The results of 9 residual grav-
ity time series after the 2010 Maule event; (b) the results of 11 residual gravity time series after the 2011 Tohoku; (c) the estimated frequen-
cies from different series. The PREM predictions (from Rosat et al. [2008]) are denoted by vertical dashed lines. Figure 3. The power spectra of 0S2 based on the residual gravity time series after the 2010 and 2011 events. (a) The results of 9 residual grav-
ity time series after the 2010 Maule event; (b) the results of 11 residual gravity time series after the 2011 Tohoku; (c) the estimated frequen-
cies from different series. The PREM predictions (from Rosat et al. [2008]) are denoted by vertical dashed lines. 0S2
m=−2
m=−1
m=0
m=+1
m=+2
PREM*
f
0.30000117
0.30449303
0.30906353
0.31371556
0.31845238
Q
494.6
501.8
509.3
517.0
525.0
Buland et al. [1979]
f
0.30001
0.30480
0.30949
0.31400
0.31850
Rosat et al. [2005]
f
0.29997±6.3e-6
0.30458±4.7e-6
0.30924±6.0e-6
0.31381±1.1e-5
0.31843±4.6e-6
Roult et al. [2006]
f
0.299779±7.4e-5
0.304624±2.9e-5
0.309397±1.57e-4
0.313892±6.9e-5
0.318465±8.9e-5
Q
490.6±14.0
562.9±4.0
395.6±11.3
495.3±4.0
480.2±14.9
Rosat et al. [2008] §
f
0.299951±1.5e-6
0.304599±1.6e-6
0.3092607±2.5e-7
0.3138446±2.6e-7
0.3184385±2.8e-7
Q
449.3±0.1
481.5±0.1
506.7±0.4
457.7±0.3
518.7±0.4
Abd El-Gelil et al. [2010]
f
0.300001±1.2e-6
0.304533±1.1e-6
0.309296±1.1e-6
0.313882±0.5e-6
0.318402±1.0e-6
Q
509.9±3.9
677.9±11.5
512.3±3.9
592.7±8.1
520.3±3.1
Roult et al. [2010]
f
0.29998±3.313e-4
0.30447±4.985e-4
0.30922±3.560e-4
0.31374±4.480e-4
0.31835±3.548e-4
Deuss et al. [2011]
f
0.29993
0.30463
0.30928
0.31386
0.31840
Rosat et al. [2012] #
f
0.29996±2.2e-5
0.30458±5.1e-5
0.30925±3.3e-5
0.31383±4.6e-5
0.31844±2.1e-5
Häfner and Widmer-
Schnidrig [2013]
f
0.299948±9.0e-6
0.304612±6.0e-6
0.309269±1.6e-5
0.313840±5.0e-6
0.318429±9.0e-6
This paper
(average value)
f
0.299958±8.1e-6
0.304588±4.6e-6
0.309263±1.1e-5
0.313835±1.4e-6
0.318422±7.4e-6
Q
509.4±12.1
484.7±9.3
394.4±14.3
520.2±8.1
532.7±10.1
Table 2. The observed weighted average values of the frequencies and Qs of 0S2, compared with the previous estimates and the PREM pre-
dictions. (*): from Millot-Langet et al. [2003], Rosat et al. [2008]. Table 2. The observed weighted average values of the frequencies and Qs of 0S2, compared with the previous estimates and the PREM pre-
dictions. (*): from Millot-Langet et al. [2003], Rosat et al. [2008]. (§): the results using the NLDHA method based on one SG record (ST) after
the 2004 Sumatra earthquake. (#): correction to Rosat et al. [2005]. 3.2. The frequencies and Qs of the modes 2S1 and 3S1 (§): the results using the NLDHA method based on one SG record (ST) after
the 2004 Sumatra earthquake. (#): correction to Rosat et al. [2005]. 6 COMPLEX FREQUENCIES OF THE NORMAL MODES Table 3. The observed and predicted values of the frequencies (mHz) and Qs of 0S3. (*): from Millot-Langet et al. [2003], Rosat et al. [2008].
(§): the results using the NLDHA method based on one SG record (ST) after the 2004 Sumatra earthquake. (#): correction to Rosat et al. [2005]. (§): the results using the NLDHA method based on one SG record (ST) after the 2004 Sumatra earthquake. (#): correction to Rosat et al. [2005]. ved and predicted values of the frequencies (mHz) and Qs of 0S3. (*): from Millot-Langet et al. [2003], Rosat et al. [2008]. Table 3. The observed and predicted values of the frequencies (mHz) and Qs of 0S3. (*): from Millot-Langet et al. [2003], Rosat et al. [2008].
(§): the results using the NLDHA method based on one SG record (ST) after the 2004 Sumatra earthquake. (#): correction to Rosat et al. [2005]. COMPLEX FREQUENCIES OF THE NORMAL MODES Figure 4. The power spectra of 0S3 based on the residual gravity time series after the 2010 and 2011 events. (a) The results of 8 residual grav-
ity time series after the 2010 Maule event; (b) the results of 12 residual gravity time series after the 2011 Tohoku; (c) the estimated frequen-
cies from different series. The PREM predictions (from Rosat et al. [2008]) are denoted by vertical dashed lines. Figure 4. The power spectra of 0S3 based on the residual gravity time series after the 2010 and 2011 events. (a) The results of 8 residual grav-
ity time series after the 2010 Maule event; (b) the results of 12 residual gravity time series after the 2011 Tohoku; (c) the estimated frequen-
cies from different series. The PREM predictions (from Rosat et al. [2008]) are denoted by vertical dashed lines. 0S3
m=−3
m=−2
m=−1
m=0
m=+1
m=+2
m=+3
PREM*
f
0.4618
0.4641
0.4664
0.4686
0.4707
0.4728
0.4748
Q
411.7
413.6
415.5
417.4
419.3
421.3
423.3
Buland et al. [1979]
f
0.46167
0.46408
0.46617
0.46883
0.47091
0.47317
0.47450
Rosat et al. [2005]
f
0.46167
±3.0e-5
0.46424
±2.0e-5
0.46639
±1.8e-5
-
0.47084
±1.1e-5
0.47266
±4.8e-5
0.47474
±2.1e-5
Roult et al. [2006]
f
0.461646
±8.7e-5
0.464329
±7.1e-5
0.466236
±8.3e-5
0.468560
±4.2e-5
0.470877
±5.2e-5
0.472659
±1.33e-4
0.474269
±4.68e-4
Q
450.5±63.3
442.5±46.3
456.6±93.0
434.2±19.3
339.9±68.9
493.9±56.3
382.6±39.2
Rosat et al. [2008] §
f
0.4615728
±4.9e-6
0.4642270
±4.9e-6
0.4664168
±4.6e-7
-
0.4707937
±3.8 e-6
0.4727150
±6.9e-6
0.4747854
±1.1 e-6
Q
380.3±0.04
412.7±0.1
447.5±0.5
-
446.1±0.1
477.2±0.2
328.2±0.3
Abd El-Gelil et al. [2010]
f
0.461662
±0.4e-6
0.464377
±0.5e-6
0.466121
±0.2e-6
0.468456
±0.2e-6
0.470920
±0.3e-6
0.472505
±0.7e-6
0.474535
±2.1e-6
Q
393.2±8.2
474.5±24.7
466.6±25.6
468.5±18.4
501.5±32.4
573.4±41.5
605.3±49.4
Roult et al. [2010]
f
0.46169
±3.515e-4
0.46417
±3.480e-4
0.46640
±4.036e-4
0.46860
±4.220e-4
0.47076
±1.760e-4
0.47275
±4.059e-
0.47470
±1.786e-4
Rosat et al. [2012] #
f
0.46167
±5.4e-5
0.46424
±7.8e-5
0.46639
±3.6e-5
-
0.47084
±3.0e-5
0.47266
±7.8e-5
0.47474
±6.8e-5
This paper
(average value)
f
0.461623
±4.9e-6
0.464219
±1.8e-6
0.466535
±2.6e-6
0.468549
±5.2e-6
0.470657
±2.4e-6
0.472843
±1.7e-6
0.474831
±3.5e-6
Q
351.5±19.1
418.5±9.2
348.4±15.8
424.4±22.5
356.9±14.5
397.7±10.0
417.2±16.3
Table 3. The observed and predicted values of the frequencies (mHz) and Qs of 0S3. (*): from Millot-Langet et al. [2003], Rosat et al. [2008]. §
# 7 7 DING AND SHEN Figure 5. The power spectra of 2S1 based on the residual gravity time series after the 2010 and 2011 events. COMPLEX FREQUENCIES OF THE NORMAL MODES (a) The results of 5 residual grav-
ity time series after the 2010 Maule event; (b) the results of 5 residual gravity time series after the 2011 Tohoku; (c) the estimated frequen-
cies from different series. The PREM predictions are denoted by vertical dashed lines (for 2S1, the three values are from Roult et al. [2006]
denoted by black vertical dashed lines; for 0T2, the value is from Dziewonski and Anderson [1981], denoted by red vertical dashed line). Figure 5. The power spectra of 2S1 based on the residual gravity time series after the 2010 and 2011 events. (a) The results of 5 residual grav-
ity time series after the 2010 Maule event; (b) the results of 5 residual gravity time series after the 2011 Tohoku; (c) the estimated frequen-
cies from different series. The PREM predictions are denoted by vertical dashed lines (for 2S1, the three values are from Roult et al. [2006]
denoted by black vertical dashed lines; for 0T2, the value is from Dziewonski and Anderson [1981], denoted by red vertical dashed line). 2S1
PREM*
Rosat et al. [2003]
Rosat et al. [2005]
Roult et al. [2006]
Roult et al. [2010]
Deuss et al. [2011]
This paper
(average value)
f (mHz)
Q
f (mHz)
f (mHz)
f (mHz)
Q
f (mHz)
f (mHz)
f (mHz)
Q
m=−1
0.39803582
391.1
0.3986
±1.9e-4
0.39821
±6.0e-5
0.39510
368.8
0.39779
±2.543e-3
0.39792
0.398662
±8.5e-6
365.9±20.3
m=0
0.40368712
396.6
0.4049
±2.1e-4
-
0.40645
428.2
0.40394
±1.352e-3
0.40518
0.405014
±2.7e-6
448.3±15.7
m=+1
0.41022196
403.1
0.4111
±1.8e-4
0.41080
±4.2e-5
0.41184
322.1
0.41063
±1.012e-3
0.41045
0.410768
±1.2e-6
385.6±11.8
Table 4. The observed and predicted values of frequencies and Qs of 2S1. (*): from Millot-Langet et al. [2003], Roult et al. [2006]. after the 2004 Sumatra event [see Rosat et al. 2005]. In
addition, the toroidal mode 0T2, which is coupled with
the mode 2S1, is also observed in the spectra, and there
is a very clear peak around 0.3826 mHz, which may cor-
respond to the singlet m=2 of 0T2 (see Roult et al. [2010]). From Figure 5c and Table 4, there is a large de-
viation between our frequency estimates with the re-
sults of Roult et al. [2006], but our results are very close
to the results of Rosat et al. [2003, 2005] and Deuss et
al. [2011]. Moreover, comparing with the PREM pre-
dictions [Millot-Langet et al. 2003, Roult et al. COMPLEX FREQUENCIES OF THE NORMAL MODES COMPLEX FREQUENCIES OF THE NORMAL MODES Figure 6. The power spectra of 3S1 based on the SG sequences after the 2010 and 2011 events. (a) The results of 3 residual gravity time se-
ries after the 2010 Maule event; (b) the results of 7 residual gravity time series after the 2011 Tohoku; (c) the estimated frequencies from dif-
ferent series. The PREM predictions (from Roult et al. [2010]) are denoted by vertical dashed lines. 2S2
1S3 Figure 6. The power spectra of 3S1 based on the SG sequences after the 2010 and 2011 events. (a) The results of 3 residual gravity time se-
ries after the 2010 Maule event; (b) the results of 7 residual gravity time series after the 2011 Tohoku; (c) the estimated frequencies from dif-
ferent series. The PREM predictions (from Roult et al. [2010]) are denoted by vertical dashed lines. 3S1
PREM*
Chao and Gilbert
[1980]
Roult et al. [2010]
Shen and Wu
[2012]
This paper
(average value)
f (mHz)
f (mHz)
Q
f (mHz)
f (mHz)
f (mHz)
Q
m=−1
0.942267
0.94270±5.5e-5
884±0.11
0.94256±1.241e-4
0.942598 ±4.20e-4
0.942426±2.5e-6
943.8±12.5
m=0
0.944217
0.94535±9.0e-5
1450±0.2
0.94419±3.444e-4
0.944113 ±2.65e-4
0.944713±1.7e-6
773.6±10.1
m=+1
0.945472
0.94563±4.0e-5
890±0.08
0.94579±1.493e-4
0.945864 ±2.13e-4
0.945612±4.6e-6
629.5±18.4
Table 5. The predictions and observations of frequencies and Qs of 3S1. (*): from Roult et al. [2010]. observed in the direct power spectra of the series. observed in the direct power spectra of the series. larger than that of m=±1 (Figure 6). In addition, our re-
sults show a systematic shift from the PREM predic-
tions toward a higher frequency (see Figure 6c and
Table 5). The observed weighted average values of Qs
of 3S1 are also listed in Table 5. If we accept the state-
ment that a multiplet is anomalously split if the split-
ting width ratio R (ratio of observed splitting width to
the predicted splitting width) is larger than 1.5 [Wid-
mer-Schnidrig et al. 1992a], then according to Table 5,
our results (R=0.99) suggest that 3S1 is a normally split
mode (consistent with the result of Roult et al. [2010]). Some studies [e.g., Giardini et al. 1988, Widmer-
Schnidrig et al. 1992a] considered that the 3S1 was
anomalously split. For example, Widmer-Schnidrig et
al. 3.3. The frequencies and Qs of the modes 0S4 and 1S2
In order to identify all of the singlets of 0S4, a very
long record should be used. However, the SNRs of
the modes will gradually decrease with the increase COMPLEX FREQUENCIES OF THE NORMAL MODES 2006],
our results perform a total shift toward a higher fre- quency. Concerning the observed Q-values of 2S1, dif-
ferent authors give different results (see Table 4), and
our results show that the Q-values are between 360 and
450. Here we note that the estimated frequency and Q
value of 0T2 are listed in Table 7. As for the mode 3S1, in order to weaken the inter-
ference of 1S3, we use three SG records starting 50h after
the 2010 event and seven SG records starting 50h after
the 2011 event (see Figure 6), the data length of each
record is taken as 680h. From Figure 6, there are no ob-
servable peaks for 2S2 and 1S3 (blue dotted curves), but
the triplet (especially the singlet m=0) of 3S1 is clearly 8 COMPLEX FREQUENCIES OF THE NORMAL MODES [1992a] declared that 3S1 was anomalously split based
upon the observations of the m=0 singlet using only
the record from the Amundsen-Scott South Pole sta-
tion, but the SNR of their observations is not high, and
the length of the data is only 100h long (far less than
the 1.1Q-cycle), which may not provide a sufficient fre-
quency resolution. Moreover, according to the results
of Roult et al. [2010], there are only the singlets m=±1
which are excited to the detectable level by the 2004
Sumatra event. Our results show that all of the three
singlets are excited to the detectable level by the 2010
and 2011 events, and the amplitude of m=0 is even In order to identify all of the singlets of 0S4, a very
long record should be used. However, the SNRs of
the modes will gradually decrease with the increase 9 DING AND SHEN Figure 7. The spectra of 0S4. (a) The results after the 2010 Maule event; (b) the results after the 2011 Tohoku; (c) the product spectra of the
residual gravity time series after each of the two events; (d) the distributions of the estimated frequencies and the corresponding PREM pre-
dictions. The dashed vertical lines indicate the observed frequencies of the singlets m=±4 observed by Roult et al. [2010]. Figure 7. The spectra of 0S4. (a) The results after the 2010 Maule event; (b) the results after the 2011 Tohoku; (c) the product spectra of the
residual gravity time series after each of the two events; (d) the distributions of the estimated frequencies and the corresponding PREM pre-
dictions. The dashed vertical lines indicate the observed frequencies of the singlets m=±4 observed by Roult et al. [2010]. f (mHz)
Q
f (mHz)
Q
0S4
m=−3
0.631015±2.2e-6
292.7±14.1
1S2
m=−2
0.673533±3.2e-6
325.7±9.5
m=−2
0.644624±2.4e-6
354.3±12.5
m=−1
0.677671±1.2e-6
312.3±5.8
m=0
0.64715±5.5e-6
325.1±15.7
m=0
0.680915±7.4e-6
269.9±16.7
m=+2
0.649545±4.5e-7
349.6±6.8
m=+1
0.683541±1.7e-6
270.4±6.4
m=+3
0.650534±5.9e-7
462.8±7.2
m=+2
0.685372±3.4e-6
279.3±10.3
Table 6. The observed weighted average values of frequencies and Qs of 0S4 and 1S2 in this study. Table 6. The observed weighted average values of frequencies and Qs of 0S4 and 1S2 in this study. cies and Qs are listed in Table 6. Here, the Qs of five
singlets of 0S4 were determined for the first time by
observation. of the length of data. COMPLEX FREQUENCIES OF THE NORMAL MODES Hence, there must be a trade-
off between the chosen data length and the SNR. In
this paper, 13 residual gravity time series with a
length of 550h are chosen to observe the 0S4 modes
(see Figure 7). The power spectra of the 0S4 modes
are shown in Figure 7a,b, and the product spectra of
the series after each of the two events are shown in
Figure 7c. The two dashed vertical lines (Figure 7a,b)
indicate the observed frequencies of the singlets
m=±4 given by Roult et al. [2010], and the PREM pre-
dictions [Roult et al. 2010] are shown in Figure 7d. There are only five singlets that can be found from
the spectra, and according to Figure 7d, they are cor-
responding to the singlets m=0, m=±2, and m=±3. The estimated values of the corresponding frequen- Concerning the observation of the mode 1S2, we
use two SG records after the 2010 event and six SG
records after the 2011 event (see Figure 8). Each of the
time series has a length of 330h. The corresponding
spectra are shown in Figure 8, and we find that there is
no observable peak for the singlet m=0 after the 2010
event (Figure 8a), whereas all of the five singlets are ob-
served after the 2011 Tohoku earthquake (Figure 8b). The PREM predictions are plotted in Figure 8 (dashed
vertical lines), where the effects of rotation and ellip-
ticity have been taken into account. The estimated
weighted average values of the frequencies and Qs of 10 3.4. The frequencies and Qs of the toroidal modes 0T2,
0T3 and 0T4
The power spectra of 0T2 have been shown in Fig-
ure 5, and the estimated weighted average values of the
frequencies and Qs are listed in Table 7. COMPLEX FREQUENCIES OF THE NORMAL MODES COMPLEX FREQUENCIES OF THE NORMAL MODES COMPLEX FREQUENCIES OF THE NORMAL MODES Figure 8. The power spectra of 1S2 based on the residual gravity time series after the 2010 and 2011 events. (a) The results of two residual
gravity time series after the 2010 Maule event; (b) the results of 6 residual gravity time series after the 2011 Tohoku; (c) the estimated fre-
quencies from different series. The PREM predictions are denoted by vertical dashed lines. Figure 8. The power spectra of 1S2 based on the residual gravity time series after the 2010 and 2011 events. (a) The results of two residual
gravity time series after the 2010 Maule event; (b) the results of 6 residual gravity time series after the 2011 Tohoku; (c) the estimated fre-
quencies from different series. The PREM predictions are denoted by vertical dashed lines. 0T2
0T3
0T4
PREM
f (mHz)
0.37917
0.58616
0.76566
Q
250.4
240.0
228.2
Widmer-Schnidrig et al. [1992b]
f (mHz)
0.3766
0.58726
0.76673
Q
--
222.2
265.3
Tromp and Zanzerkia [1995]
f (mHz)
--
--
0.76583
Q
--
--
195
Hu et al. [2006]
f (mHz)
0.37823
0.58634
0.76586
Abd El-Gelil et al. [2010]
f (mHz)
0.37760
0.58690
0.76460
This paper (average value)
f (mHz)
0.379130±2.4e-6
0.586918±4.9e-7
0.765803±5.6e-7
Q
271.6±14.2
232.9±8.4
242.4±9.5
Table 7. The observed weighted average values of frequencies and Qs of 0T2, 0T3, and 0T4, compared to the PREM predictions and the re-
sults of some previous studies. Table 7. The observed weighted average values of frequencies and Qs of 0T2, 0T3, and 0T4, compared to the PREM predictions and the re-
sults of some previous studies. 1S2 are listed in Table 6. The PREM predictions of 0S4
and 1S2 are not tabulated in Table 6, which are referred
to Roult et al. [2010]. It is noted that the Qs of the
whole set of the singlets of 1S2 are determined by ob-
servation for the first time in this study. 3.4. The frequencies and Qs of the toroidal modes 0T2,
0T3 and 0T4 The power spectra of 0T2 have been shown in Fig-
ure 5, and the estimated weighted average values of the
frequencies and Qs are listed in Table 7. 11 DING AND SHEN as parts of the estimates of the frequencies and Qs of
the mode. Hence, the final results (see Table 7) of the
frequencies and Qs are given by the weighted average
of the 2010 and 2011 results. For the purpose of observing the 0T3 mode, we
use five residual gravity time series after the 2010 event
and four residual gravity time series after the 2011
event, each of them with a length of 70h. The power
spectra are shown in Figure 9, where one can find that
the results are larger than the theoretical predictions of
PREM [Dziewonski and Anderson 1981]. The observed weighted average values of the fre-
quencies and Qs of 0T2, 0T3, and 0T4 are listed in Table
7. Comparing with the results of Widmer-Schnidrig et
al. [1992b], Tromp and Zanzerkia [1995], Hu et al. [2006] and Abd El-Gelil et al. [2010], we provide addi-
tional estimates of the Q-values of the three modes,
which are comparable with the corresponding PREM
predictions. Concerning the mode 0T4, we use five residual
gravity time series after the 2010 event (Figure 10a),
each of the records with a length of 120h, and seven
residual gravity time series after the 2011 event (Fig-
ure 10b), each of the series starting 5h after the event
with a length of 80h. Here we choose the records
with different lengths, because the SNRs of the
records after the two events are significantly different. Due to different lengths (120h and 80h) of the
records, the discrepancy of the frequency resolutions
of the spectra of the two events is about 1.16µHz. From Figure 10c, we find that the discrepancy be-
tween the weighted average values obtained respec-
tively from the records after the 2010 event and the
2011 event is only 0.48µHz, which is far less than
1.16µHz. This implies that both the results (based on
respectively the records after the 2010 and 2011
events) are physically meaningful and should be used COMPLEX FREQUENCIES OF THE NORMAL MODES COMPLEX FREQUENCIES OF THE NORMAL MODES Figure 10. The power spectra of 0T4. (a) The results of 5 residual gravity time series after the 2010 Maule event; (b) the results of 7 residual grav-
ity time series after the 2011 Tohoku; (c) the estimated frequencies from different series. The residual gravity time series after the 2010 event
have a length of 120h, and the series after the 2011 event have a length of 80h. The PREM prediction is denoted by the vertical dashed line. Figure 10. The power spectra of 0T4. (a) The results of 5 residual gravity time series after the 2010 Maule event; (b) the results of 7 residual grav-
ity time series after the 2011 Tohoku; (c) the estimated frequencies from different series. The residual gravity time series after the 2010 event
have a length of 120h, and the series after the 2011 event have a length of 80h. The PREM prediction is denoted by the vertical dashed line. vide further valuable information for constraining and
refining the 3D density structure and attenuation mod-
els of the Earth. In addition, the singlets m=0 of the modes 2S1 and 3S1
were clearly observed in the direct spectra of the resid-
ual gravity time series without using other special spec-
tral analysis methods or special series of polar station
records, especially for 3S1. Due to the fact that, some
studies considered that 3S1 was anomalously split [e.g.,
Giardini et al. 1988, Widmer-Schnidrig et al. 1992a],
while others [e.g., Ritzwoller et al. 1986, He and Tromp
1996, Roult et al. 2010] considered normal, a clear ob-
servation of the singlet m=0 may provide further evi-
dence to judge whether 3S1 is normally split. Taking
into account the fact that the splitting width ratio R of
3S1 obtained in this study is 0.99, we conclude that 3S1
is normally split. In addition, the singlets m=0 of the modes 2S1 and 3S1
were clearly observed in the direct spectra of the resid-
ual gravity time series without using other special spec-
tral analysis methods or special series of polar station
records, especially for 3S1. Due to the fact that, some
studies considered that 3S1 was anomalously split [e.g.,
Giardini et al. 1988, Widmer-Schnidrig et al. 1992a],
while others [e.g., Ritzwoller et al. 1986, He and Tromp
1996, Roult et al. COMPLEX FREQUENCIES OF THE NORMAL MODES 2010] considered normal, a clear ob-
servation of the singlet m=0 may provide further evi-
dence to judge whether 3S1 is normally split. Taking
into account the fact that the splitting width ratio R of
3S1 obtained in this study is 0.99, we conclude that 3S1
is normally split. Acknowledgements. The authors are grateful to the GGP
station managers for the high-quality records; thank Professor B. F. Chao for his valuable comments during the preparation of the man-
uscript, and thank Editor Dr. Francesca Bianco and an anonymous
Reviewer for their valuable comments and suggestions, which
greatly improved the manuscript. This study is supported by NSFC
(grant No. 41174011), National 973 Project China (grant No. 2013CB733305), NSFC (grant Nos. 41210006, 41128003, 41021061,
40974015), the Fundamental Research Funds for the Central Uni-
versities (grant No. 2012214020203), the Open Research Fund Pro-
gram of the Key Laboratory of Geospace Environment and
Geodesy, Ministry of Education, China (No. 12-02-04, 12-02-02), and
the Open Fund of the State Key Laboratory of Geodesy and Earth’s
Dynamics, Institute of Geodesy and Geophysics, CAS (No. SKLGED2013-2-3-E). Comparing with previous studies, our results again
clearly show that the amplitudes of the singlets of a
given mode excited by different earthquakes (which
have similar magnitude) are different (of course, it is a
well-known statement). For instance, the singlet m=0
of 2S1 cannot be observed using the SG records after
the 2004 Mw9.0 Sumatra earthquake whereas it can be
clearly observed from the SG residual sequences after
the 2010 Mw8.8 Maule earthquake and the 2011 Mw9.1
Tohoku earthquake. 4. Conclusions After the 2010 Mw8.8 Maule earthquake and the
2011 Mw9.1 Tohoku earthquake, we used the SG
records to determine the frequencies and Qs of the sin-
glets of the modes below 1mHz based on the AR
method proposed by Chao and Gilbert [1980]. The fre-
quencies and Qs of all the singlets of the modes 0S2, 2S1,
0S3, 0S0, and 3S1, and the frequencies of 0S4, 1S2, 0T2,
0T3, and 0T4 were re-estimated. Moreover, the Qs of all
of the five singlets of 1S2, the Qs of the five singlets
(m=0, m=±2, and m=±3) of 0S4 and the Qs of 0T2, 0T3,
and 0T4 were first time or re-estimated by observation. Figure 9. The power spectra of 0T3 based on the residual gravity time series after the 2010 and 2011 events. (a) The results of 5 residual grav-
ity time series after the 2010 Maule event; (b) the results of 4 residual gravity time series after the 2011 Tohoku; (c) the estimated frequen-
cies from different series. The PREM prediction is denoted by the vertical dashed line. The red symbols indicate the results obtained from
the 2010 event, and the green symbols indicate the results obtained from the 2011 event. Figure 9. The power spectra of 0T3 based on the residual gravity time series after the 2010 and 2011 events. (a) The results of 5 residual grav-
ity time series after the 2010 Maule event; (b) the results of 4 residual gravity time series after the 2011 Tohoku; (c) the estimated frequen-
cies from different series. The PREM prediction is denoted by the vertical dashed line. The red symbols indicate the results obtained from
the 2010 event, and the green symbols indicate the results obtained from the 2011 event. 12 References Abd El-Gelil, M., S. Pagiatakis and A. El-Rabbany
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rights reserved. *Corresponding author: WenBin Shen,
Wuhan University, Department of Geophysics, School of Geodesy
and Geomatics, China; email: wbshen@sgg.whu.edu.cn. *Corresponding author: WenBin Shen,
Wuhan University, Department of Geophysics, School of Geodesy
and Geomatics, China; email: wbshen@sgg.whu.edu.cn.
© 2013 by the Istituto Nazionale di Geofisica e Vulcanologia. All
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Tripartite Motif-Containing 3 (TRIM3) Enhances ER Signaling and Confers Tamoxifen Resistance in Breast Cancer
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Tripartite Motif-Containing 3 (TRIM3) Enhances ER
Signaling and Confers Tamoxifen Resistance in
Breast Cancer Signaling and Confers Ta
Breast Cancer
Run-yi Ye
Sun Yat-sen University First Affiliated Hospital
Xia-ying Kuang
Sun Yat-sen University First Affiliated Hospital
Hui-juan Zeng
Sun Yat-sen University First Affiliated Hospital
Nan Shao
Sun Yat-sen University First Affiliated Hospital
ying lin
Sun Yat-sen University First Affiliated Hospital
pian liu
Wuhan Union Hospital
shenming wang
(
wshenm@163.com
)
Sun Yat-sen University First Affiliated Hospital
Research
Keywords: breast cancer, tamoxifen resistance, ES
Posted Date: November 18th, 2020
DOI: https://doi.org/10.21203/rs.3.rs-105983/v1
License:
This work is licensed under a Creat
Read Full License Run-yi Ye
Sun Yat-sen University First Affiliated Hospital
Xia-ying Kuang
Sun Yat-sen University First Affiliated Hospital
Hui-juan Zeng
Sun Yat-sen University First Affiliated Hospital
Nan Shao
Sun Yat-sen University First Affiliated Hospital
ying lin
Sun Yat-sen University First Affiliated Hospital
pian liu
Wuhan Union Hospital
shenming wang
(
wshenm@163.com
)
Sun Yat-sen University First Affiliated Hospital Run-yi Ye
Sun Yat-sen University First Affiliated Hospital
Xia-ying Kuang
Sun Yat-sen University First Affiliated Hospital
Hui-juan Zeng
Sun Yat-sen University First Affiliated Hospital
Nan Shao
Sun Yat-sen University First Affiliated Hospital
ying lin
Sun Yat-sen University First Affiliated Hospital
pian liu
Wuhan Union Hospital
shenming wang
(
wshenm@163.com
)
Sun Yat-sen University First Affiliated Hospital Run-yi Ye
Sun Yat-sen University First Affiliated Hospital
Xia-ying Kuang
Sun Yat-sen University First Affiliated Hospital
Hui-juan Zeng
Sun Yat-sen University First Affiliated Hospital
Nan Shao
Sun Yat-sen University First Affiliated Hospital
ying lin
Sun Yat-sen University First Affiliated Hospital
pian liu
Wuhan Union Hospital
shenming wang
(
wshenm@163.com
)
Sun Yat-sen University First Affiliated Hospital Abstract Background: Tamoxifen resistance remains a tricky clinical problem in estrogen receptor (ER)-positive
breast cancer. SUMOylation of ERα enhances ERα-induced transcription activity. TRIM proteins are a new
class of SUMO E3 ligases, regulating SUMOylation of proteins. However, the precise molecular
mechanism and function of TRIM3 in SUMOylation and response to tamoxifen remains unclear. Methods: The expression of TRIM3 was assessed in 48 cases with breast cancer which received
tamoxifen therapy. The protein and RNA levels of TRIM3 in 12 ER+ breast cancer cell lines were assessed
by Western blotting and qRT-PCR assays. Effects of TRIM3 in regulating tamoxifen resistance were
evaluated both in vitro and in vivo via Cell viability, Colony formation, Anchorage-independent growth
ability assays and Tumor xenografts. Molecular mechanism of TRIM3 in regulating SUMOylation of ERα
was analyzed by Western blotting, Immunoprecipitation, Far Western blotting and Luciferase activity
assays. Results: We reported that TRIM3 was dramatically overexpressed in breast cancer, which correlated with
tamoxifen resistance., Furthermore,TRIM3 overexpression significantly correlated with poor survival of
ER+ breast cancer treated with tamoxifen. TRIM3 overexpression conferred cell survival and
tumorigenesis, whereas knocking downTRIM3 reduced these capabilities. Moreover, TRIM3, as a UBC9
binding protein, promoted SUMO modification of ESR1 and activated ER pathway. Silencing UBC9
abolished the function of TRIM3 in regulating tamoxifen resistance. Conclusions: These results suggest a novel biomarker for breast cancer therapy, indicating that inhibiting
TRIM3 combined with tamoxifen may provide a potential treatment for breast cancer. Research 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 Oncogenesis on September 1st, 2021. See
the published version at https://doi.org/10.1038/s41389-021-00350-x. Page 1/30 Background With almost 25% cancer cases among women, Breast cancer remains a global challenge among females
in worldwide1. Estrogen receptor alpha (ERα) is the main molecular target for endocrine therapies, which
antagonize ER and suppress estrogen synthesis to inhibit tumor growth 2. Tamoxifen as one of adjuvant
endocrine therapy, is widely used as ER antagonists in breast cancer, which blocked the binding of
estrogen and ER and suppressed ER-α target genes 3. Unfortunately, approximately 30%-40% patients of
ER+ breast cancer fail to response (de novo resistance) or become resistance (acquired resistance), and
with a deadly outcome, which presents a huge clinical challenge for breast cancer4. Therefore, to unveil
the underlying molecular mechanism of endocrine resistance and potential therapies for breast cancer
are of great importance. Emerging evidences have proved that several mechanisms contribute to tamoxifen resistance, such as
activation of RTKs signal transduction pathways, activation of oncogenic signaling pathways
(PI3K/Akt/mTOR, NF-κB) and modulation of ER signaling5–8. ER is widely expressed in breast cancer
(almost 70%), which is a strong predictor for tamoxifen therapy 9. Notably, loss of ESR1 expression Page 2/30 confers tamoxifen resistance, which is reported in ~ 15%-20% of breast cancer9, 10. However, the
expression of ER in most of tamoxifen-resistance cases, remains to be expressed and active9, suggesting
that there might other mechanism regulate ESR1 to confer tamoxifen resistance. Emerging evidence
shows that ESR1 expression is regulated by diverse aspect, including histone modification11, DNA
methylation12, somatic mutation9, ESR1 fusion genes13 and post-translational modifications (PTMs)14. SUMOylation, as important PTM, effects subcellular localization, protein-protein interaction, protein
stability and transcriptional activity, which regulated by three important enzymes, including activating
enzyme (E1), conjugating enzyme (E2) and ligases (E3). Stephanie Sentis and his colleagues proved that
small ubiquitin-like modifier (SUMO)-1 modifies ERα SUMOylation, which enhances ERα-induced
transcription activity via improving ERα’ DNA binding property15. However, the mechanism and biology
function of ESR1 SUMOylation in breast cancer remains unclear. Tripartite motif-containing (TRIM) proteins belong to RING type E3 ubiquitin ligase, which involved
cellular signaling, cell progression and tumorigenesis16. Furthermore, it has reported that TRIM proteins
are a new class of SUMO E3 ligases, transferring small ubiquitin-related modifier (SUMO) to substrates16,
17. TRIM27, TRIM32 and TRIM36 are well-known SUMO E3 ligases, which modify SUMOylation via
binding to ubiquitin-conjugating enzyme 9 (UBC9)16, 17. TRIM3, as one of TRIM proteins, is located at
chromosome 11p15, which is thought to harbor tumor suppressor genes18–20. Background However, the precise
molecular mechanism and function of TRIM3 in SUMOylation and response to tamoxifen remains
unclear. Herein, we found that TRIM3 was significantly upregulated in tamoxifen-resistant breast cancer, and
associated with poor survival in breast cancer during tamoxifen therapy. Overexpressed of TRIM3
conferred estrogen-independent growth and contributed tamoxifen resistance. Experiments revealed that
TRIM3 upregulated ER SUMO modification and activated ER signaling pathway via binding to UBC9,
which could be abolished by deSUMOylation enzyme SENP1. Taken together, our results unveil the crucial
role of TRIM3 in SUMOylation of ESR1 and modulation of tamoxifen response, which identify a potential
target to improve clinical outcomes of breast cancer. Cell culture The human breast cancer cell lines BT-474, BT-483, CAMA-1, HCC1428, HCC1500, MCF7, MDA-MB-134-VI,
MDA-MB-175-VII, MDA-MB-361, T-47D, ZR-75-1 and ZR-75-30 were cultured according to the
manufacturer’s instruction. Short tandem repeat (STR) profiling were uesd and authenticated in all cell
lines. Mycoplasma eradication was evaluated by PCR. Chemical reagents Tissue specimens and immunohistochemistry A cohort of paraffin-embedded breast cancer (48 cases) which receved tamoxifen therapy was used to
detecte the expression of TRIM3 via Anti-TRIM3 (ab111840). The specimens were obtained from the First
Affiliated Hospital of Sun Yat-sen University between 2010 and 2015. 10 freshly breast cancer tissues
with tamoxifen sensitive and tamoxifen resistant were collected. Prior patient consent and approval were
obtained. Immunoprecipitation analysis Immunopresipitation assay was performed according to described previously21. Lysates were
incubutated with Flag or His affinity beads (Sigma-Aldrich). The agarose beads were washed with wash
buffer. Then the eluations were detected using appropriate antibodies. 17β-estradiol (E2) and 4-hydroxytamoxifen (TAM) were purchased from Sigma-Aldrich (St. Louis, MO,
USA). 17β-estradiol (E2) and 4-hydroxytamoxifen (TAM) were purchased from Sigma-Aldrich (St. Louis, MO,
USA). 17β-estradiol (E2) and 4-hydroxytamoxifen (TAM) were purchased from Sigma-Aldrich (St. Louis, MO,
USA) 17β-estradiol (E2) and 4-hydroxytamoxifen (TAM) were purchased from Sigma-Aldrich (St. Louis, MO,
USA). Constructs and transfection Genomic DNA of TRIM3 was PCR-amplified and cloned into a pBABE-puro retroviral vector. The pSUPER-
puro shRNA of TRIM3 and UBC9 were purchased from Transheep Bio. All clone primer and siRNA
obigonucleotides are listed as Supplemental Table 1. Estrogen Response Element (ERE) was cloned into
a pGL3 basic vector (Progema). The Renilla luciferase TK was used as transfection control. All cells
overexpressing TRIM3 or silencing TRIM3 were selected with 0.5 µg/ml puromycine. Far Western blotting Far immunoblotting were performed by using the proteins immunoprecipitated by anti-His antibody. The
proteins were detected using western blotting. Empty vector or Flag-ESR1 cell lysate was added to the
PVDF membrane and incubated overnight. Then the membrane was subjected to immunoblotting
analysis by indicated antibody. Western blotting analysis Western blotting was performed using antibodies against TRIM3 (ab111840), ESR1 (ab108398), UBC9
(ab75854), SENP1 (ab108981) and SAE1 (ab185552).The menbranes were re-probed with an anti-GAPDH
(BOSTER, BM3876) as the loading control. Chemical reagents Page 3/30 Page 3/30 Quantitative real-time reverse transcription PCR (qPCR) Quantitative real-time reverse transcription PCR (qPCR) Total RNA was isolated with TRIzol reagent (Invitrogen) according to manufacturer’s instructions. RNA
was reverse-transcribed into cDNA and carried out via Real-time PCR with SYBR Green Master (Roche). The data were assessed base on the threshold cycle (Ct), and calculated as 2−[(C t of gene) − (C t of GAPDH)],
which was normalized to GAPDH expression. All primers are listed as Supplemental Table 1. Cell viability assay Anchorage-independent growth ability assay Indicated cells were suspended with mediun plus 0.33% agar, and then plated on top of 0.66% agar
medium mix. After 10 days, colonies > 0.1 mm in diameter were counted. The expriments was perfrmed in
triplicates. Tumor xenografts Under the guideline of National Institutes of Health Guide for Care and Use of Laboretory Animals,
xenografts were performed using athymic nude female mice (4–5 weeks of age, 18–20 g). 5 × 106 of
indicated cells were injected into the left and right dorcal flank of mice implantd with E2 pellets
(0.72 mg/peller; 60-day release). After one week, a subcutaneous injuection with or without tamoxifen
pellet (5 mg/pellet; 60-day release). Tumor were examined twice a week, and tumor volum was calculated
as (L × W2)/2. Cell viability assay Page 4/30 Page 4/30 Indicated cells were treated with Estrogen and/or tamoxifen for 48 h. The cells were added MTT dye
(Sigma-Aldrich), and then the cells were resuspended with dimethl sulfoxide (Sigma-Aldrich) and
measured with automatic microplate reader.. Colony formation assay Indicated cells were incubated at a level of 5% CO2 at a temperature of 37 ℃ for 2 weeks. Then the cells
were fixed, stained with crystal violet stain, and counted. Indicated cells were incubated at a level of 5% CO2 at a temperature of 37 ℃ for 2 weeks. Then the cells
were fixed, stained with crystal violet stain, and counted. Data processing and viaualization The datasets is available in The Cancer Genome Atlas (TCGA) (https://tcga-data.nci.nih.gov/tcga/). Gene
set enrichment analysis (GSEA) was performed on GSEA 2.0. 9 (http://www.broadinstitute.org/gsea/.)
The relationship between the expression of TRIM3 and ESR1 was determined by correlation coefficient. For the relationship between TRIM3 and the OS, RFS and DMFS of breast cancers, Kaplan-Meier Plotter
((http://kmplot.com/analysis) was used. Luciferase activity assay Luciferase reporter plasmid and pRL-TK Renilla plasmid were transfected into indicated cells. After 48 h,
the cells were lysis and measured using a Dual Luciferase Reporter Assay (Promega) according to the
f
t
’ i
t
ti Luciferase reporter plasmid and pRL-TK Renilla plasmid were transfected into indicated cells. After 48 h,
the cells were lysis and measured using a Dual Luciferase Reporter Assay (Promega) according to the
macufacturer’s instructions. Statisticas Unpaired Student’s test was used to evaluate the statistical significance of the differences between two
groups. Spearman’s correlation analysis and Chi-square were used to evaluate the correlations. All P
values were two-siled, and a value of P < 0.05 was considered statistically significant. All data analyze
were performed with SPSS19.0 software and presented with GraphPad Prism 8.0. TRIM3 contributes tamoxifen-resistant in breast cancer To determine the molecular mechanism which contributes to breast cancer tamoxifen resistance, we
analysis the genes expression in breast cancer patients from TCGA database. As shown in Fig. 1a, the
expression of TRIM3 is significantly higher in the patients without response to tamoxifen therapy [stable
disease (SD), progressive disease (PD)] than those response to tamoxifen treatment [complete remission
(CR), partial remission (PR)]. The mRNA of TRIM3 was higher expressed in ER+ breast cancer than ER−
breast cancer (Supplemental Fig. 1a, p = 1.365E-68). Furthermore, 48 cases clinical breast cancer
specimens with ER+ after tamoxifen treatment were used to examine the expression of
TRIM3(Supplemental table 2). As expect, TRIM3 expression was significantly higher in tamoxifen-
resistant ER + breast cancer than that in tamoxifen-sensitive ER+ breast cancer (Fig. 1b). Statistical
analysis revealed that the expression of TRIM3 was closely correlated with recurrence of patients with
tamoxifen treatment (Fig. 1c). By analyzing published expression profiles obtained from breast cancer
(TCGA) data, we found that levels of TRIM3 mRNA is positively correlated with levels of TRIM3 protein,
and TRIM3 mRNA expression was upregulated in ER+ breast cancer tissues compared to ER− cancer
tissues and normal breast tissue (Supplemental Fig. 1b and 2a). Consistently, Kaplan-Meier plotter
showed that the higher expression of TRIM3 was positively correlated with poorer overall survival (OS),
relapse-free survival (RFS) and distant metastasis-free survival (DMSF) in cases of ER+ breast cancer
with tamoxifen treatment and/or chemotherapy, but not in ER− breast cancer (Fig. 1c and Supplemental
Fig. 1c).Herein, these results reveal that TRIM3 might correlate with the tamoxifen resistance of ER+
breast cancer. Results Results Page 5/30 TRIM3 promoted tamoxifen resistance in ER + breast cancer in vitro To further determine the role of TRIM3 in regulating tamoxifen resistance, we detected the expression
levels of TRIM3 in 12 ER+ breast cancer cell lines. As shown in Fig. 2a, TRIM3 was high expressed in
MDA-MB-134-VI, MDA-MB-175-VII and MDA-MB-361, and low expressed in BT-483, CAMA-1and MCF7. Consistent with the clinical evidence, higher expression of TRIM3 in MDA-MB-134-VI, MDA-MB-175-VII
and MDA-MB-361 resistance to tamoxifen than lower expression of TRIM3 in BT-483, CAMA-1and MCF7
(Fig. 2b-2c), suggesting that TRIM3 might contribute to tamoxifen resistance in breast cancer. Furthermore, we established BT-483 and MCF7 stably overexpressed TRIM3, MDA-MB-134-VI and MDA-
MB-361 stably silencing TRIM3 (Fig. 3a). As expected, overexpressed TRIM3 induced tamoxifen
resistance and formed more colony formation in BT-483 and MCF7, whereas silencing TRIM3 expression
promoted tamoxifen sensitivity and formed less colony formation in MDA-MB-134-VI and MDA-MB-361
(Fig. 3b-3c). Therefore, these results revealed that TRIM3 induced tamoxifen resistance in ER+ breast
cancer in vitro. TRIM3 conferred tamoxifen resistance in ER + breast cancer in vivo TRIM3 conferred tamoxifen resistance in ER + breast cancer in vivo Page 6/30 To further investigate the function of TRIM3 in regulating tamoxifen resistance in vivo, we first examined
the anchor-independent growth ability in manipulation of TRIM3 expression cells compared with control
cells. As shown in Fig. 4a, TRIM3 overexpression promoted cell growth and formed more and larger
colonies compared with vector cells, whereas silencing TRIM3 expression inhibited cell growth and
formed less and smaller colonies compared with vector cells. Furthermore, we subcutaneously inoculated
TRIM3-deregulated cells into NOD/SCID mice. Consistently, TRIM3 overexpressed cells formed larger
tumors compared with control vector cells (Fig. 4a). Taken together, these results revealed that TRIM3
response to tamoxifen resistance and promotes tumorigenicity of breast cancer in vivo. TRIM3 corrlateds with ESR1 SUMOylation and tamoxifen
resistance in berast cancer To further investigate the correlation of TRIM3 and ESR1 in regulating tamoxifen resistance, 10 freshly
collected clinical breast cancer samples were examined. As shown in Figure in 8a, TRIM3 expression was
upregulated in tamoxifen resistant breast cancer than tamoxifen sensitive breast cancer, and strongly
associated with ESR1 SUMOylation. Consistently, TRIM3 promoted the transcription levels of ER-targeted
genes in tamoxifen resistant ER+ breast cancer (Fig. 8b). Collectively, these results support that TRIM3
upregulation promotes ESR1 SUMO modification and ER signaling pathway, leading to tamoxifen
resistance in ER+ breast cancer. Ubc9 Is Required For Tamoxifen Resistance Effect Of Trim3 To further investigate whether SUMOylation of ESR1 is required for promoting tamoxifen resistance
effect of TRIM3, the effect of silencing of UBC9 and overexpressed SENP1 in TRIM3-induced tamoxifen
resistance were examine. As shown in Fig. 7a-7b, silencing UBC9 and overexpressed SENP1 in TRIM3-
transduced cells formed less colony formation, which promoted tamoxifen sensitivity. Furthermore,
silencing UBC9 in TRIM3-transduced cells reversed the ability of TRIM3-induced promoted tamoxifen
sensitivity and decreased tumortigenicity in vivo (Fig. 7c). Taken together, these results suggest that
UBC9 is required for the effext of TRIM3 regulating ESR1 SUMOylationg and promoting tamoxifen
resistance in breast cancer. Trim3 Promoted Sumoylation Of Esr1 Via Binding To Ubc9 To further investigate the mechanism by which TRIM3 response to tamoxifen resistance, gene set
enrichment analysis was analyzed in published ER+ breast cancer expression profiles from TCGA data. We found that TRIM3 levels were positively correlated with estrogen receptor 1 (ESR1) expression,
suggesting that TRIM3 might be involved in regulating ESR signaling pathway (Fig. 5a). As expect, we
found that overexpressed of TRIM3 increased estrogen response element (ERE) luciferase reporter
activity and promoted ER-regulated genes upon tamoxifen treatment, whereas silencing of TRIM3
expression decreased (Fig. 5b-5c). However, analysis of breast cancer in TCGA showed that levels of
TRIM3 mRNA and protein were not correlated with levels of ESR1 mRNA and protein (Supplemental
Fig. 2a and 3a). Furthermore, we found that E2 treatment did not induce ESR1 and TRIM3 expression in
breast cancer (Supplemental Fig. 2b). Overexpressed of ESR1 did not affect the expression of TRIM3
(Supplemental Fig. 2c). Collectively, TRIM3 induced ESR1 transcription activity, but not induced ESR1
expression. The E3 ubiquitin-protein ligase TRIM proteins are identified as a new class of SUMO ligases (E3s), which
regulate SUMOylation specificity17. It has been reported that transcription activity of ERα is upregulated
by SUMOylation via binding to UBC9, the unique SUMO E2-conjugating enzyme, and repressed by
tamoxifen treatment 15, 22–24. Here, we found that TRIM3 overexpression increased SUMO modification of
ESR1, whereas silencing TRIM3 decreased (Fig. 6a). Overexpressed SENP1, a deSUMOylase enzymes,
decreases SUMOylation of ESR1 and ERE luciferase reporter activity in TRIM3-transduced cells compared
with control cells upon tamoxifen treatment (Fig. 6b-6c). Furthermore, immunoprecipitation and western
blot assay showed that TRIM3 formed complex with ESR1 and UBC9, suggesting that TRIM3 might
regulate SUMOylation of ESR1 via binding to ESR1, UBC9 and SAE1 (Fig. 6d). Silencing UBC9 in TRIM3-
transduced cells reduced the SUMOylation of ESR1 and ERE luciferase reporter activity (Supplemental
Fig. 3b-3c). To further investigate the binding of TRIM3 and ESR1, three truncated ESR1 fragments were
constructed (Fig. 6e). Next, immunoprecipitation and far-western blot assay showed that TRIM3
interacted with DNA binding fragment of ESR1 (ER2 fragment) and directly interacted with Flag-tagged
ESR1 cell lysate (Fig. 6f-6 g). Taken together, these data reveal that TRIM3 promoted SUMOylation and
activated transcription activity of ESR1 via interacting with ESR1 and UBC9. Page 7/30 Discussion Though SUMO
modification is widely known to repress transcription via binding with co-repressors, including HDACs and
DAXX36, 37, a growing number of evidence has been showed that SUMOylation involved in promoting
transcription activity. For example, the transcription activity of ERα is upregulated in breast cancer via
binding with SUMOylation proteins, such as CLOCK22 and ZFP28238. Furthermore, ERα is proved to be
SUMOylated via binding with UBC9, and blocking its SUMOylation impaired the transcription activity of
ERα through decreased DNA binding without influencing ERα cellular localization15. Therefore, the precise
molecular mechanism by which SUMO modification enhances transcription activity of ERα in breast
cancer needs further investigation. In current study, we identified that TRIM3 played an important role in regulating ER signaling pathway
and conferred tamoxifen resistance in breast cancer. Overexpression of TRIM3 enhanced ERE luciferase
reporter activity and upregulated ER-regulated genes upon tamoxifen treatment, whereas silencing of
TRIM3 expression decreased (Fig. 5b-5c). Furthermore, TRIM proteins are proved to be a new class of
SUMO E3 ligases, regulating SUMO modification via transferring SUMO1 from UBC9 to substrate16, 17. Consistently, the SUMOylation and transcription activity of ESR1 was upregulated in TRIM3-transduced
cells upon tamoxifen treatment, which abolished by overexpression SENP1 (Fig. 6a-6c). Furthermore,
immunoprecipitation analysis demonstrated that TRIM3 interacted with ESR1 UBC9 and SAE1 In current study, we identified that TRIM3 played an important role in regulating ER signaling pathway
and conferred tamoxifen resistance in breast cancer. Overexpression of TRIM3 enhanced ERE luciferase
reporter activity and upregulated ER-regulated genes upon tamoxifen treatment, whereas silencing of
TRIM3 expression decreased (Fig. 5b-5c). Furthermore, TRIM proteins are proved to be a new class of
SUMO E3 ligases, regulating SUMO modification via transferring SUMO1 from UBC9 to substrate16, 17. Consistently, the SUMOylation and transcription activity of ESR1 was upregulated in TRIM3-transduced
cells upon tamoxifen treatment, which abolished by overexpression SENP1 (Fig. 6a-6c). Furthermore,
immunoprecipitation analysis demonstrated that TRIM3 interacted with ESR1, UBC9 and SAE1,
suggesting that TRIM3 acted as SUMO E3 ligase via transferring SUMO from SAE1 (E1) and UBC9 (E2) to
ESR1 (substrate). Importantly, TRIM3 interacted with ESR1 directly binding to its DNA-binding domain
(Fig. 6e-6 g), which might induce ESR1 transcription. Overexpression of UBC9 promotes tumorigenesis of
breast cancer, while a dominant negative UBC9 decreases39. Similarly, blocking UBC9 expression also
decreased cell survival and tumorigenesis in TRIM3-transduced cells upon E2 and tamoxifen treatment in
vitro and in vivo (Fig. 7). Discussion Taken together, our results confirm that TRIM3 acted as a SUMO E3 ligase in
regulating ESR1 SUMO modification and transcription activity, and conferred tamoxifen resistance via the
TRIM3/UBC9/ESR1 axis. Discussion Tamoxifen is a widely used treatment for ER-positive breast cancer patients, but de novo resistance or
acquired resistance results in failure for tamoxifen therapy, which is a clinical challenge 4. In the present
study, we found that TRIM3 was upregulated in tamoxifen resistant breast cancer tissues compared to
tamoxifen sensitive breast cancer tissues, and was closely correlated with poorer survival in ER+ breast
cancer. Overexpression of TRIM3 contributed to cell survival upon tamoxifen treatment both in vivo and
in vitro. Moreover, TRIM3 promoted ESR1 SUMO modification and activated transcription of ER target
genes via binding with UBC9, which could be abolished with overexpression of SENP1. Taken together,
our results demonstrated that TRIM3 modify SUMOylation and confer tamoxifen resistance, which
provided a novel therapeutic target for breast cancer therapy. TRIM3, a member of RING-type E3 ubiquitin ligases, plays diverse roles in regulating neoplastic
processes20, 25–27. TRIM3 is identified as a novel RING finger protein expressed in the rat brain, which
involved in regulating neuronal outgrowth18, 28. TRIM3 is a candidate brain tumor suppressor gene, which
suppresses brain tumorigenesis via attenuating Notch signaling and suppressing c-MYC expression29, 30. Furthermore, TRIM3 is downregulated in various types of cancer, including liver cancer19, esophageal
squamous cell carcinoma27 and colon cancer31, and inhibited cell proliferations, invasion and Page 8/30 Page 8/30 Page 8/30 Page 8/30 metastasis. However, the correlation of TRIM3 and tamoxifen resistance in breast cancer remains
unclear. In current study, we found that TRIM3 was significantly higher expression in recurrence ER +
breast cancer than that in non-recurrence ER+ breast cancer, which consistent to the analysis of TRIM3
expression response to tamoxifen treatment in ER-positive breast cancer data obtained from TCGA
(Fig. 1a-1b, Fig. 8a). Furthermore, the expression of TRIM3 correlated with poorer OS, RFS and DMSF in
ER+ breast cancer upon tamoxifen treatment, but not in ER− breast cancer (Fig. 1c and Supplemental
Fig. 1b), suggesting that TRIM3 might be a potential predictive marker for tamoxifen response in ER+
breast cancer. ERα has been recognized as a favorable prognostic biomarker and a determinative role for breast cancer
therapy32. ERα belongs to nuclear receptor superfamily, and activates the transcription of targeted genes
to conquer tamoxifen treatment33, 34. SUMOylation as a post-translational modification (PTMs), is
hyperactivated in breast cancer, and correlated with ERα signaling pathways35. Conclusions Page 9/30 In conclusion, we demonstrated that TRIM3, as a novel SUMO E3 ligase, promoted SUMO modification
and transcription activity of ESR1 via binding to UBC9, which contributed to tamoxifen resistance in ER+
breast cancer. Silencing UBC9 in TRIM3-transduced cells conferred tamoxifen sensitive in breast cancer. Importantly, overexpression of TRIM3 was correlated with poor survival of ER+ breast cancer upon
tamoxifen resistance, which present a potential biomarker for treatment of ER+ breast cancer. Abbreviations ER+
Estrogen receptor -positive
ERα
Estrogen receptor alpha
SUMO
small ubiquitin-like modifier
TRIM3
Tripartite motif-containing 3
UBC9
ubiquitin-conjugating enzyme 9
SD
stable disease
PD
progressive disease
CR
complete remission
PR
partial remission
OS
overall survival
RFS
relapse-free survival
DMSF
distant metastasis-free survival ER+
Estrogen receptor -positive
ERα
Estrogen receptor alpha
SUMO
small ubiquitin-like modifier
TRIM3
Tripartite motif-containing 3
UBC9
ubiquitin-conjugating enzyme 9
SD
stable disease
PD
progressive disease
CR
complete remission
PR
partial remission
OS
overall survival
RFS
relapse-free survival
DMSF
distant metastasis-free survival Acknowledgements： Not applicable. Ethics approval and consent to participate: Page 10/30 All animal experiments in this study were under the guideline of National Institutes of Health Guide for
Care and Use of Laboratory Animals. Prior patient consent and approval from the Institutional Research
Ethics Committee were obtained. Consent for publication: Not applicable. Competing interests: The authors declare that they have no competing interests. Funding: This study was supported by National Natural Science Foundation of China (Grants：81772800
and 82072945). Authors' contributions: RY and XK designed this study, analyzed data and wrote the manuscript. HZ and
NS performed the vitro experiments. RY and YL performed the vivo experiments. RY and XK contributed to
collection of clinical samples. PL and SW provided important comments for the manuscript. All authors
read and approved the final manuscript. Availability of data and materials: All data generated or analyzed during this study are included in this published article and its
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2677-2683. Figures Page 14/30 Page 14/30 Figure 1 TRIM3 overexpression promotes tamoxifen resistance in breast cancer. (a) A boxplot representing TRIM3
expression levels in ER+ breast cancer with tamocifen treatment [SD/PD versus CR/PR, P=0.012, stable
disease (SD), progressive disease (PD), complete remission (CR), partial remission (PR)]. (b)
Representative images of TRIM3 expression in recurrence- and recurrence+ ER+ breast cancer tissues
with tamoxifen treatment (left panel). Scale bars: 200μm (upper panel) and 100μm (lower panel). Statistical significance of TRIM3 levels was assessed in right panel. P<0.05. (c) Analysis of the effect of
TRIM3 expression level on Overall Survival (OS), Relapse-Free Survival (RFS) and Distant Metastasis Free
suvival (DMFS) of ER+ bresat cancer with tamoxifen therapy. Page 15/30 Figure 1 TRIM3 overexpression promotes tamoxifen resistance in breast cancer. (a) A boxplot representing TRIM3
expression levels in ER+ breast cancer with tamocifen treatment [SD/PD versus CR/PR, P=0.012, stable
disease (SD), progressive disease (PD), complete remission (CR), partial remission (PR)]. (b)
Representative images of TRIM3 expression in recurrence- and recurrence+ ER+ breast cancer tissues
with tamoxifen treatment (left panel). Scale bars: 200μm (upper panel) and 100μm (lower panel). Statistical significance of TRIM3 levels was assessed in right panel. P<0.05. (c) Analysis of the effect of
TRIM3 expression level on Overall Survival (OS), Relapse-Free Survival (RFS) and Distant Metastasis Free
suvival (DMFS) of ER+ bresat cancer with tamoxifen therapy. Page 16/30 Figure 2
The effect of TRIM3 on tamoxifen response in breast cancer. (a) mRNA and protein levels of TRIM3 in
ER+ breast cancer cell lines. Data are presented as mean ± SD. (b-c) Cell biability was measured by MTT
(b) and colony formation (c) in the indicated cell lines treated with E2 (10 nM) and/or TAM (1μM). Statistical data are presented as mean ± SD. Figure 2 Figure 2 The effect of TRIM3 on tamoxifen response in breast cancer. (a) mRNA and protein levels of TRIM3 in
ER+ breast cancer cell lines. Data are presented as mean ± SD. (b-c) Cell biability was measured by MTT
(b) and colony formation (c) in the indicated cell lines treated with E2 (10 nM) and/or TAM (1μM). Statistical data are presented as mean ± SD. Page 17/30 Figure 2
The effect of TRIM3 on tamoxifen response in breast cancer. (a) mRNA and protein levels of TRIM3 in
ER+ breast cancer cell lines. Data are presented as mean ± SD. (b-c) Cell biability was measured by MTT
(b) and colony formation (c) in the indicated cell lines treated with E2 (10 nM) and/or TAM (1μM). Statistical data are presented as mean ± SD. Figure 2 Figure 2 The effect of TRIM3 on tamoxifen response in breast cancer. (a) mRNA and protein levels of TRIM3 in
ER+ breast cancer cell lines. Data are presented as mean ± SD. (b-c) Cell biability was measured by MTT
(b) and colony formation (c) in the indicated cell lines treated with E2 (10 nM) and/or TAM (1μM). Statistical data are presented as mean ± SD. Page 18/30 Figure 3
TRIM3 contributes to tamoxifen resistance in breast cancer. Figure 1 (a) Western blot analysis of TRIM3
expression in indicated breast cancer cell lines stably overexpression TRIM3 and silencing TRIM3. (b-c)
Cell biability was measured by MTT (b) and colony formation (c) in the indicated cell lines treated with
(10 nM) and TAM (1μM). Statistical data are presented as mean ± SD. Figure 3 Figure 3 Figure 3 TRIM3 contributes to tamoxifen resistance in breast cancer. (a) Western blot analysis of TRIM3
expression in indicated breast cancer cell lines stably overexpression TRIM3 and silencing TRIM3. (b-c)
Cell biability was measured by MTT (b) and colony formation (c) in the indicated cell lines treated with E2
(10 nM) and TAM (1μM). Statistical data are presented as mean ± SD. Page 19/30 Figure 3
TRIM3 contributes to tamoxifen resistance in breast cancer. (a) Western blot analysis of TRIM3
expression in indicated breast cancer cell lines stably overexpression TRIM3 and silencing TRIM3. (b-c)
Cell biability was measured by MTT (b) and colony formation (c) in the indicated cell lines treated with
(10 nM) and TAM (1μM). Statistical data are presented as mean ± SD. Figure 3 Figure 3 Figure 3 Page 22/30 Figure 5 Figure 5
TRIM3 induced estrogen receptor (ER)-dependent transcription in reponse to tamoxifen therapy. (a) GSEA
showed that TRIM3 expression positively correlated with ESR1-activated gene signatures and negatively
with ESR1-suppressed gene signatures in ER+ breast cancer profiles (TCGA, n=400). (b) Estrogen
response element (ERE) luciferase reporter activity was analyzed in indicated cells treated with E2 (10 Figure 5
TRIM3 induced estrogen receptor (ER)-dependent transcription in reponse to tamoxifen Figure 3 TRIM3 contributes to tamoxifen resistance in breast cancer. (a) Western blot analysis of TRIM3
expression in indicated breast cancer cell lines stably overexpression TRIM3 and silencing TRIM3. (b-c)
Cell biability was measured by MTT (b) and colony formation (c) in the indicated cell lines treated with E2
(10 nM) and TAM (1μM). Statistical data are presented as mean ± SD. Page 20/30 Figure 4
upregulation of TRIM3 promotes tamoxifen resistance in vivo. (a) Soft agar of indicated cells treated with
E2 (10 nM) and TAM (1μM) (left panel) and statistical significance of colony formation was assessed in
right panel. data are presented as mean ± SD. P<0.05. (b) Representative images of the tumors in the
xenografts (left panel). Tumor growth curves (middle panel) and tumor weight (right panel) of the
indicated xenograft xenograft tumors (n=6 /group). Data are presented as mean ± SD. *P<0.05, **P<0.01. Figure 4 upregulation of TRIM3 promotes tamoxifen resistance in vivo. (a) Soft agar of indicated cells treated with
E2 (10 nM) and TAM (1μM) (left panel) and statistical significance of colony formation was assessed in
right panel. data are presented as mean ± SD. P<0.05. (b) Representative images of the tumors in the
xenografts (left panel). Tumor growth curves (middle panel) and tumor weight (right panel) of the
indicated xenograft xenograft tumors (n=6 /group). Data are presented as mean ± SD. *P<0.05, **P<0.01. Page 21/30 Figure 4
upregulation of TRIM3 promotes tamoxifen resistance in vivo. (a) Soft agar of indicated cells treated with
E2 (10 nM) and TAM (1μM) (left panel) and statistical significance of colony formation was assessed in
right panel. data are presented as mean ± SD. P<0.05. (b) Representative images of the tumors in the
xenografts (left panel). Tumor growth curves (middle panel) and tumor weight (right panel) of the
indicated xenograft xenograft tumors (n=6 /group). Data are presented as mean ± SD. *P<0.05, **P<0.01. Figure 4 upregulation of TRIM3 promotes tamoxifen resistance in vivo. (a) Soft agar of indicated cells treated with
E2 (10 nM) and TAM (1μM) (left panel) and statistical significance of colony formation was assessed in
right panel. data are presented as mean ± SD. P<0.05. (b) Representative images of the tumors in the
xenografts (left panel). Tumor growth curves (middle panel) and tumor weight (right panel) of the
indicated xenograft xenograft tumors (n=6 /group). Data are presented as mean ± SD. *P<0.05, **P<0.01. nM) and/or TAM (1μM). Data are presented as mean ± SD. *P<0.05. (c) Heatmap generated of ER-
regulated genes expression in indicated cells with E2 and TAM treatment. Page 24/30
Figure 5
TRIM3 induced estrogen receptor (ER)-dependent transcription in reponse to tamoxifen thera
showed that TRIM3 expression positively correlated with ESR1-activated gene signatures an
with ESR1-suppressed gene signatures in ER+ breast cancer profiles (TCGA, n=400). (b) Estr Figure 5 Figure 5 Figure 5 TRIM3 induced estrogen receptor (ER)-dependent transcription in reponse to tamoxifen therapy. (a) GSEA
showed that TRIM3 expression positively correlated with ESR1-activated gene signatures and negatively
with ESR1-suppressed gene signatures in ER+ breast cancer profiles (TCGA, n=400). (b) Estrogen
response element (ERE) luciferase reporter activity was analyzed in indicated cells treated with E2 (10 Page 23/30 Page 23/30 nM) and/or TAM (1μM). Data are presented as mean ± SD. *P<0.05. (c) Heatmap generated of ER-
regulated genes expression in indicated cells with E2 and TAM treatment. nM) and/or TAM (1μM). Data are presented as mean ± SD. *P<0.05. (c) Heatmap generated of ER-
regulated genes expression in indicated cells with E2 and TAM treatment. Figure 5 TRIM3 induced estrogen receptor (ER)-dependent transcription in reponse to tamoxifen therapy. (a) GSEA
showed that TRIM3 expression positively correlated with ESR1-activated gene signatures and negatively
with ESR1-suppressed gene signatures in ER+ breast cancer profiles (TCGA, n=400). (b) Estrogen TRIM3 induced estrogen receptor (ER)-dependent transcription in reponse to tamoxifen therapy. (a) GSEA
showed that TRIM3 expression positively correlated with ESR1-activated gene signatures and negatively
with ESR1-suppressed gene signatures in ER+ breast cancer profiles (TCGA, n=400). (b) Estrogen Page 24/30 Page 24/30 response element (ERE) luciferase reporter activity was analyzed in indicated cells treated with E2 (10
nM) and/or TAM (1μM). Data are presented as mean ± SD. *P<0.05. (c) Heatmap generated of ER-
regulated genes expression in indicated cells with E2 and TAM treatment. response element (ERE) luciferase reporter activity was analyzed in indicated cells treated with E2 (10
nM) and/or TAM (1μM). Data are presented as mean ± SD. *P<0.05. (c) Heatmap generated of ER-
regulated genes expression in indicated cells with E2 and TAM treatment. Figure 6
TRIM3 induced SUMOylation of ESR1 via binding with UBC9. (a-b) Western blot of ESR1 (a-b) and SENP1
(b) expression in indicated breast cancer cell lines. GAPDH was used as loading control. Star represented
SUMOylation of ESR1. (c) Estrogen response element (ERE) luciferase reporter activity was analyzed in
indicated cells treated with E2 (10 nM) and/or TAM (1μM). Data are presented as mean ± SD. *P<0.05. (d)
Immunoprecipitation assay reveal TRIM3 interacted with ESR1, UBC9 and SAE1 in indicated cells. (e)
Schematic illustration of wile-type ESR1 and truncated ESR1. (f) Immunoprecipitation assay reveal regulated genes expression in indicated cells with E2 and TAM treatment. ced SUMOylation of ESR1 via binding with UBC9. (a-b) Western blot of ESR1 (a-b) and SENP1
on in indicated breast cancer cell lines. GAPDH was used as loading control. Star represented
n of ESR1. (c) Estrogen response element (ERE) luciferase reporter activity was analyzed in
ls treated with E2 (10 nM) and/or TAM (1μM). Data are presented as mean ± SD. *P<0.05. (d)
cipitation assay reveal TRIM3 interacted with ESR1, UBC9 and SAE1 in indicated cells. (e)
lustration of wile-type ESR1 and truncated ESR1. (f) Immunoprecipitation assay reveal Fi
6 Figure 6 TRIM3 induced SUMOylation of ESR1 via binding with UBC9. (a-b) Western blot of ESR1 (a-b) and SENP1
(b) expression in indicated breast cancer cell lines. GAPDH was used as loading control. Star represented
SUMOylation of ESR1. (c) Estrogen response element (ERE) luciferase reporter activity was analyzed in
indicated cells treated with E2 (10 nM) and/or TAM (1μM). Data are presented as mean ± SD. *P<0.05. (d)
Immunoprecipitation assay reveal TRIM3 interacted with ESR1, UBC9 and SAE1 in indicated cells. (e)
Schematic illustration of wile-type ESR1 and truncated ESR1. (f) Immunoprecipitation assay reveal Page 25/30 Page 25/30 Page 25/30 TRIM3 interacted with ER2 domain of ESR1. (g) Far western blot reveal TRIM3 directly interacted with
Flag-ESR1 cell lysate. TRIM3 interacted with ER2 domain of ESR1. (g) Far western blot reveal TRIM3 directly interacted with
Flag-ESR1 cell lysate. TRIM3 interacted with ER2 domain of ESR1. (g) Far western blot reveal TRIM3 directly interacted with
Flag-ESR1 cell lysate. Figure 6
TRIM3 induced SUMOylation of ESR1 via binding with UBC9. (a-b) Western blot of ESR1 (a-b) and SENP1
(b) expression in indicated breast cancer cell lines. GAPDH was used as loading control. Star represented
SUMOylation of ESR1. (c) Estrogen response element (ERE) luciferase reporter activity was analyzed in
indicated cells treated with E2 (10 nM) and/or TAM (1μM). Data are presented as mean ± SD. *P<0.05. (d)
Immunoprecipitation assay reveal TRIM3 interacted with ESR1, UBC9 and SAE1 in indicated cells. (e)
Schematic illustration of wile-type ESR1 and truncated ESR1. (f) Immunoprecipitation assay reveal ed SUMOylation of ESR1 via binding with UBC9. (a-b) Western blot of ESR1 (a-b) and SENP1
n in indicated breast cancer cell lines. GAPDH was used as loading control. Star represented
of ESR1. (c) Estrogen response element (ERE) luciferase reporter activity was analyzed in
s treated with E2 (10 nM) and/or TAM (1μM). Data are presented as mean ± SD. *P<0.05. (d)
pitation assay reveal TRIM3 interacted with ESR1, UBC9 and SAE1 in indicated cells. (e)
ustration of wile-type ESR1 and truncated ESR1. (f) Immunoprecipitation assay reveal Fi
6 Figure 6 TRIM3 induced SUMOylation of ESR1 via binding with UBC9. (a-b) Western blot of ESR1 (a-b) and SENP1
(b) expression in indicated breast cancer cell lines. GAPDH was used as loading control. Star represented
SUMOylation of ESR1. (c) Estrogen response element (ERE) luciferase reporter activity was analyzed in
indicated cells treated with E2 (10 nM) and/or TAM (1μM). Data are presented as mean ± SD. *P<0.05. (d)
Immunoprecipitation assay reveal TRIM3 interacted with ESR1, UBC9 and SAE1 in indicated cells. (e)
Schematic illustration of wile-type ESR1 and truncated ESR1. (f) Immunoprecipitation assay reveal Page 26/30 TRIM3 interacted with ER2 domain of ESR1. (g) Far western blot reveal TRIM3 directly interacted with
Flag-ESR1 cell lysate. TRIM3 interacted with ER2 domain of ESR1. (g) Far western blot reveal TRIM3 directly interacted with
Flag-ESR1 cell lysate. Flag-ESR1 cell lysate. Page 27/30
Figure 7
silencing UBC9 reverses the tamoxifen resistance effect of TRIM3. (a-b) Representative images of
formation (a) and MTT assay (b) of indicated cells treated with E2 (10 nM) and TAM (1μM). Histo
showing colony formed by indicated cells. Data are presented as mean ± SD. *P<0.05. (c) Represe Figure 7 Figure 7 Page 27/30
silencing UBC9 reverses the tamoxifen resistance effect of TRIM3. (a-b) Representative images of colong
formation (a) and MTT assay (b) of indicated cells treated with E2 (10 nM) and TAM (1μM). Histograms
showing colony formed by indicated cells. Data are presented as mean ± SD. *P<0.05. (c) Representative Page 27/30 images of the tumors in the xenografts (left panel). Tumor growth curves (middle panel) and tumor
weight (right panel) of the indicated xenograft xenograft tumors (n=6/group). Data are presented as
mean ± SD. *P<0.05, **P<0.01. Page 28/30
mean ± SD. P<0.05,
P<0.01. Figure 7
silencing UBC9 reverses the tamoxifen resistance effect of TRIM3. (a-b) Representative images of c
formation (a) and MTT assay (b) of indicated cells treated with E2 (10 nM) and TAM (1μM). Histogr Fi
7 Figure 7 Figure 8 TRIM3 promotes transcription of ER-target genes in clinical breast cancer. (a-b) Western blot of TRIM3
and ESR1 (a), qPCR of ER-targeted genes (b) in 10 cases freshly ER+ breast cancer with tamoxifen
therapy, including tamoxifen resistant and tamoxifen sensitive. GAPDH was used as loading control. Star
represented SUMOylation of ESR1. Figure 8 TRIM3 promotes transcription of ER-target genes in clinical breast cancer. (a-b) Western blot of TRIM3
and ESR1 (a), qPCR of ER-targeted genes (b) in 10 cases freshly ER+ breast cancer with tamoxifen
therapy, including tamoxifen resistant and tamoxifen sensitive. GAPDH was used as loading control. Star
represented SUMOylation of ESR1. Figure 7 silencing UBC9 reverses the tamoxifen resistance effect of TRIM3. (a-b) Representative images of colong
formation (a) and MTT assay (b) of indicated cells treated with E2 (10 nM) and TAM (1μM). Histograms showing colony formed by indicated cells. Data are presented as mean ± SD. *P<0.05. (c) Representative
images of the tumors in the xenografts (left panel). Tumor growth curves (middle panel) and tumor
weight (right panel) of the indicated xenograft xenograft tumors (n=6/group). Data are presented as
mean ± SD. *P<0.05, **P<0.01. showing colony formed by indicated cells. Data are presented as mean ± SD. *P<0.05. (c) Representative
images of the tumors in the xenografts (left panel). Tumor growth curves (middle panel) and tumor
weight (right panel) of the indicated xenograft xenograft tumors (n=6/group). Data are presented as
mean ± SD. *P<0.05, **P<0.01. Supplementary Files Page 29/30 Page 29/30 This is a list of supplementary files associated with this preprint. Click to download. This is a list of supplementary files associated with this preprint. Click to download. This is a list of supplementary files associated with this preprint. Click to download. SMFigure1.tif
SMFigure1.tif
SMFigure2.tif
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English
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Model‐Free Rheo‐AFM Probes the Viscoelasticity of Tunable DNA Soft Colloids
|
Small
| 2,019
|
cc-by
| 11,909
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1. Introduction Several materials of daily use are made
of colloidal particles dispersed in a con-
tinuous medium, including among many
others paints, cosmetics, foodstuff, fluids
for oil extraction, and ferrofluids.[1] Of
great importance for many applications is
the knowledge of their state diagram, i.e.,
whether they exist in a gas, fluid, or solid
state.[1,2] This is mainly governed by the
interactions between the colloidal particles
that can be of different nature and coexist at
the same time, and include for instance van
der Waals, electrostatic, steric, and mag-
netic forces.[1,3] The relative contribution
of these forces depend on several factors,
such as the constituent materials of the col-
loids, their shape, internal architecture, and
surface properties.[4] Moreover, recently it
has been proposed that the degree of soft-
ness, or compressibility, of the colloidal
particles influences substantially their bulk
phase behavior.[5,6] In particular, it has been
shown that the transition point from a fluid
to a solid crystalline state occurs at grow-
ingly larger packing fractions for increas-
les; with a fluid-solid coexistence state that
pears.[6] The softness of the colloidal particles
rmation of crystalline states such as exotic lat-
and simple hexagonal,[7–10] which are usually
e case of hard colloidal spheres. 1. Introduction Atomic force microscopy rheological measurements (Rheo-AFM) of the linear
viscoelastic properties of single, charged colloids having a star-like architecture
with a hard core and an extended, deformable double-stranded DNA (dsDNA)
corona dispersed in aqueous saline solutions are reported. This is achieved
by analyzing indentation and relaxation experiments performed on individual
colloidal particles by means of a novel model-free Fourier transform method
that allows a direct evaluation of the frequency-dependent linear viscoelastic
moduli of the system under investigation. The method provides results that
are consistent with those obtained via a conventional fitting procedure of the
force-relaxation curves based on a modified Maxwell model. The outcomes
show a pronounced softening of the dsDNA colloids, which is described by an
exponential decay of both the Young’s and the storage modulus as a function
of the salt concentration within the dispersing medium. The strong softening
is related to a critical reduction of the size of the dsDNA corona, down to ≈70%
of its size in a salt-free solution. This can be correlated to significant topological
changes of the dense star-like polyelectrolyte forming the corona, which are
induced by variations in the density profile of the counterions. Similarly, a
significant reduction of the stiffness is obtained by increasing the length of the
dsDNA chains, which we attribute to a reduction of the DNA density in the
outer region of the corona. José A. Moreno-Guerra, Ivany C. Romero-Sánchez, Alejandro Martinez-Borquez,
Manlio Tassieri, Emmanuel Stiakakis, and Marco Laurati* José A. Moreno-Guerra, Ivany C. Romero-Sánchez, Alejandro Martinez-Borquez,
Manlio Tassieri, Emmanuel Stiakakis, and Marco Laurati* FULL PAPER Rheo-AFM 1. Introduction Laurati
División de Ciencias e Ingenierías
Universidad de Guanajuato
Lomas del Bosque 103, 37150 León, Mexico
E-mail: mlaurati@fisica.ugto.mx
Dr. M. Tassieri
Division of Biomedical Engineering
School of Engineering
University of Glasgow
Glasgow G12 8LT, UK
Dr. E. Stiakakis
Forschungszentrum Jülich
Institute of Complex Systems 3
Leo-Brandt-Strasse, 52425 Jülich, Germany
The ORCID identification number(s) for the author(s) of this article
can be found under https://doi.org/10.1002/smll.201904136. DOI: 10.1002/smll.201904136
© 2019 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. 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. Small 2019, 15, 1904136 Over the past few years, several experimental techniques have
been proposed for measuring the particles’ softness; these include
centrifugation,[13,14] micropipette aspiration,[15,16] dynamic light
scattering combined with variations of the osmotic pressure,[17]
microdrop confinement,[18] microfluidic flow,[19] and atomic force
microscopy (AFM).[20–22] Interestingly, all these methods provide
a measure of the “elasticity” of the particles by means of their bulk
or Young’s modulus. However, it must be noted that in most cases
colloids are made of materials like polymers, biopolymers, or gels, The ORCID identification number(s) for the author(s) of this article
can be found under https://doi.org/10.1002/smll.201904136. © 2019 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. 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. © 2019 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. 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. © 2019 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 1. Introduction Furthermore,
onequilibrium solids such as glasses and jams
er packing fraction values.[5,6] In addition, in
uch as ionic microgels, the softness is affected
erplay between packing and counterions’ dis-
ignificant deswelling can be observed at large
due to the osmotic pressure generated by the
ns.[11,12]
l
l
h
h ike architecture
DNA (dsDNA)
is achieved
on individual
orm method
r viscoelastic
results that
cedure of the
e outcomes
escribed by an
as a function
ong softening
down to ≈70% i
cant topological
a, which are
imilarly, a
e length of the
nsity in the
Several materials of daily use are made
of colloidal particles dispersed in a con-
tinuous medium, including among many
others paints, cosmetics, foodstuff, fluids
for oil extraction, and ferrofluids.[1] Of
great importance for many applications is
the knowledge of their state diagram, i.e.,
whether they exist in a gas, fluid, or solid
state.[1,2] This is mainly governed by the
interactions between the colloidal particles
that can be of different nature and coexist at
the same time, and include for instance van
der Waals, electrostatic, steric, and mag-
netic forces.[1,3] The relative contribution
of these forces depend on several factors,
such as the constituent materials of the col-
loids, their shape, internal architecture, and
surface properties.[4] Moreover, recently it
has been proposed that the degree of soft-
ness, or compressibility, of the colloidal
particles influences substantially their bulk
phase behavior.[5,6] In particular, it has been
shown that the transition point from a fluid
to a solid crystalline state occurs at grow-
ingly larger packing fractions for increas-
ingly softer particles; with a fluid-solid coexistence state that
progressively disappears.[6] The softness of the colloidal particles
also governs the formation of crystalline states such as exotic lat-
tices like σ, A15, and simple hexagonal,[7–10] which are usually
not observed in the case of hard colloidal spheres. Furthermore,
the formation of nonequilibrium solids such as glasses and jams
also shifts to higher packing fraction values.[5,6] In addition, in
charged systems such as ionic microgels, the softness is affected
by the complex interplay between packing and counterions’ dis-
tribution, so that significant deswelling can be observed at large
packing fractions due to the osmotic pressure generated by the
expelled counterions.[11,12] 1904136 (1 of 10)
J. A. Moreno-Guerra, I. C. Romero-Sánchez, Dr. A. Martinez-Borquez,
Dr. M. www.small-journal.com www.small-journal.com FULL PAPER Model-Free Rheo-AFM Probes the Viscoelasticity of Tunable
DNA Soft Colloids José A. Moreno-Guerra, Ivany C. Romero-Sánchez, Alejandro Martinez-Borquez,
Manlio Tassieri, Emmanuel Stiakakis, and Marco Laurati* In these cases, the mate-
rials’ viscoelastic properties are usually obtained by modeling the
force relaxation curve with either a generalized Maxwell model[24]
or more complex fractional models,[26] from which parametric
quantities such as the bulk modulus and the effective viscosity are
educed. An alternative, but time-consuming and limited in the
range of explored frequencies, AFM method is based on the meas-
urement of the frequency-dependent amplitudes and phase-shift
between an imposed oscillatory indentation and the relative force
response of the material detected via the cantilever deflection.[27–31]
In this work we exploit a novel and simple broadband method,[25]
in which the time-dependent force relaxation curve is Fourier
transformed to obtain the frequency-dependent linear viscoelastic
properties of the system under investigation without the adoption
of any viscoelastic model to interpret the data, as described below. We anticipate that our records reveal a pronounced softening
of the dsDNA corona with increasing of salt concentration. This can be mainly attributed to the conformational changes of
the polyelectrolyte corona, with an abrupt softening at a critical
size of the DNA corona (or salt concentration). Moreover, we
observe a similar softening of the particles when the DNA
chain length is increased (i.e., doubled) and we attribute this
to the reduced chain density in the outer layer of the corona. Finally, we shall highlight that the results reported in this man-
uscript are of particular interest to all those studies aimed at
gaining a better understanding of the mechanical properties of
a broad range of soft colloidal particles, such as polyelectrolyte
brushes and microgels[36–40] and microgel particles.[5,16,17] Amongst a great variety of available soft colloids, star-like
double-stranded DNA (dsDNA) colloids have been recently
introduced as a novel class of polyelectrolyte grafted colloids.[32]
Indeed, in contrast to solid charged colloids, the complex archi-
tecture of their corona allows to accommodate a large number of
counterions that provide unique properties. For instance, under
salt-free conditions and for relatively large grafting densities the
corona incorporates all counterions, which induce an osmotic
swelling and a rod-like conformation of the arms (i.e., they are
completely stretched). In particular, the DNA stretching, while
costing in terms of entropy, maximizes the available space for
the mobile counterions, overall resulting in an increase of
entropy. This configuration leads to an osmotic imbalance
between the interior of the corona and the bulk. With the addi-
tion of NaCl the osmotic imbalance is progressively reduced until
balance is reached and the corona deswells and shrinks, with a
salt-dependent size in agreement with a modified blob model for
dense polyelectrolyte brushes.[32,33] Moreover, star-like dsDNA
colloids show interesting behavior in overcrowded conditions,
where for example the formation of crystalline states through
particle faceting without corona interpenetration has been
observed in salt-free dispersions; a process that is novel for hairy
colloids, but often observed in microgels.[12,34] Interpenetration
of the dsDNA coronas occurs instead with the addition of salt. Despite these interesting phenomena, a relationship between
the mechanical properties of a single hairy colloid and either the
salt-induced changes of its corona conformation or their phase
behavior is still missing from the literature. which have an inherent viscoelastic nature. This indeed plays an
important role in systems with dense particle packings (e.g., crys-
tals, glasses, and jams) by affecting their dynamics and structural
relaxations, and its importance has been already recognized for
biological tissues[23] and cells, especially under pathological con-
ditions.[24,25] Most of these studies have been performed by using
time-dependent AFM indentation measurements, also known as
“indentation-relaxation” experiments. In these cases, the mate-
rials’ viscoelastic properties are usually obtained by modeling the
force relaxation curve with either a generalized Maxwell model[24]
or more complex fractional models,[26] from which parametric
quantities such as the bulk modulus and the effective viscosity are
educed. An alternative, but time-consuming and limited in the
range of explored frequencies, AFM method is based on the meas-
urement of the frequency-dependent amplitudes and phase-shift
between an imposed oscillatory indentation and the relative force
response of the material detected via the cantilever deflection.[27–31]
In this work we exploit a novel and simple broadband method,[25]
in which the time-dependent force relaxation curve is Fourier
transformed to obtain the frequency-dependent linear viscoelastic
properties of the system under investigation without the adoption
of any viscoelastic model to interpret the data, as described below. and II) by Fourier transforming the same curves by means of a
novel method recently proposed by Tassieri and co-workers.[25]
We investigate the viscoelastic nature of single-colloidal particles
as a function of salt concentration and dsDNA chain length for
a fixed number of grafted DNA chains (functionality f = 2 × 105
chains per particle). For the arm length and the f values inves-
tigated here, the DNA coated colloids behave as osmotic stars
under salt-free conditions, i.e., the size of the dsDNA corona
equals the contour length of the grafted chains.[35] which have an inherent viscoelastic nature. This indeed plays an
important role in systems with dense particle packings (e.g., crys-
tals, glasses, and jams) by affecting their dynamics and structural
relaxations, and its importance has been already recognized for
biological tissues[23] and cells, especially under pathological con-
ditions.[24,25] Most of these studies have been performed by using
time-dependent AFM indentation measurements, also known as
“indentation-relaxation” experiments. Small 2019, 15, 1904136 DOI: 10.1002/smll.201904136 DOI: 10.1002/smll.201904136 1904136 (1 of 10) 1904136 (1 of 10) © 2019 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Small 2019, 15, 1904136 Small 2019, 15, 1904136 www.small-journal.com 2.2. Force Relaxation Curves: Elastic and Viscous Contributions ( )
8
3(1
)
1
1
( )
0
3/2
1/3
2
1/3
3/2
F t
R
R
G t
δ
ν
=
−
+
∗
−
(2) (2) Due to the small applied indentation depth, the regime of
deformation can be described by means of a modified version
of the Hertz model for a spherical indenter.[41,42] The relaxa-
tion of the force during step (III) was sampled at a frequency
of 100 Hz. This is the upper frequency limit of the Piezo stage
(controlling the cantilever height), above which it moves out
from its linear operational regime. The probe approaching
speed was chosen to be equal to 20 µm/s in all the experi-
ments. At this loading rate no overshoot of the loading
force[43] were observed; corroborating the adoption of the
Hertz model. Therefore, the time-dependent relaxation modulus G(t) of the
viscoelastic colloid is simply proportional to the measured force
and ready to be modeled.[24,26,46] Notably, once the experimental
conditions have been defined, the constant of proportionality
between F(t) and G(t) in Equation (2) is fully determined. In
this work the parameters are R1 = 4 µm, δ0 = 0.1–0.4 µm,
depending on sample, and ν = 0.5 according to the values
reported in the literature.[47] Notice that, small variations of ν
would only shift the magnitude of the outcomes, but not their
frequency behavior. In order to calculate the radius of the col-
loids as a function of cs, we have used the following relation:
R2 = Rps + L. Where Rps = 0.49 µm is the radius of the polysty-
rene inner core, which is independent by cs, and L(cs) is the size
of the dsDNA corona. The latter has been determined by using
the following scaling law presented by others in a previous
work,[32]
s
0.11
L
c
≈
−
. Measurements were performed on dsDNA hairy colloids
suspended in water solutions at seven different concentra-
tions of NaCl, i.e., cs = 0.0, 0.005, 0.025, 0.050, 0.100, 0.250,
1.000 m. The synthesis of the colloidal particles follows the
protocol introduced in a previous work[32] and here reported
in the experimental section for the convenience of the reader. 2.2. Force Relaxation Curves: Elastic and Viscous Contributions In the case of a relatively small indentation of a viscoelastic
sphere of radius R2 by means of a (solid) spherical tip of radius
R1 (which is to a first approximation a good representation of
our system), the time-dependent force relaxation can be written
as[44,45] ( )
8
3(1
)
1
1
(
) d
( )
d
d
1/3
2
1/3
3/2
3/2
F t
R
R
G t
t
t∫
ν
τ
δ
τ
τ
=
−
+
−
∗
−
−∞
(1) (1) where 1/R* = 1/R1 + 1/R2, ν is the Poisson’s ratio, G(t) and
δ(t) are the time-dependent relaxation modulus and indenta-
tion depth, respectively. Notably, in the limit of R2 → ∞, which
corresponds to the indentation of a flat surface by means of a
spherical tip, the pre-factor in Equation (1) only depends on
R1 and ν, returning the well-known expression of the Hertz
model. In the specific case of a finite step-indentation, for
which the time derivative of δ(t) is equal to zero before and
after the time interval required to perform the indentation of
amplitude δ0 (step II, in Figure 2), Equation (1) simplifies as it
follows (please see the Supporting Information for an extended
derivation) Figure 2. Force versus time curve of a typical indentation and relaxa-
tion experiment, which consists of three sequential steps as shown in
the figure: I) probe equilibration; II) probe approach and indentation;
III) probe at constant depth into the sample. between the probe and the colloidal particle was previously
determined from a force–distance curve. The force versus time
curve corresponding to steps (I to III) of a typical experiment is
shown in Figure 2. During step (I), in which the tip lies above
the sample, the registered force is zero. When the tip starts to
indent the sample (step II), the force rapidly increases, reaching
a maximum value at the deepest indentation point; then the
force starts to relax, potentially revealing the viscoelastic proper-
ties of the material (step III). 2.1. Indentation and Relaxation Experiments length the size of the corona is double the size for 10 kbp, i.e.,
Lc ≈ 7.8 µm in salt-free water. © 2019 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Small 2019, 15, 1904136 2.1. Indentation and Relaxation Experiments Figure 1 shows a schematic representation of the AFM setup
adopted to perform indentation and relaxation experiments. A colloidal AFM tip was used to indent a single colloidal par-
ticle attached to a glass substrate and dispersed in an open
liquid cell environment (see the Experimental Section for more
details). By following the experimental procedure introduced by
Tassieri and co-workers,[25] the measurement protocol consisted
of the following steps: I) the colloidal probe was equilibrated
above the sample surface for 5 s; II) the probe was successively
indented into the sample to a predefined indentation depth
δ0 < 10% of the sample thickness, i.e., the particle diameter; III)
the probe was maintained at constant height within the sample;
IV) the probe was retracted from the sample. The contact point Figure 1. A schematic representation of the experimental setup. A spher-
ical tip of an AFM cantilever is brought into contact with a dsDNA star-
like colloid suspended in water within an open liquid cell environment. The colloid is attached to a glass substrate. In this work we address this lack of knowledge by exploiting
the effectiveness of a novel Rheo-AFM technique. In particular,
we measure the yet unexplored viscoelastic properties of single
dsDNA star-like colloids as function of salt concentration, size,
number, and length of the grafted DNA chains. Notably, all these
properties are rationally tunable and can be effectively character-
ized by means of Rheo-AFM measurements. The data are ana-
lyzed by using two complementary approaches: I) by modeling
the force relaxation-curves with a modified fractional Hertz model Figure 1. A schematic representation of the experimental setup. A spher-
ical tip of an AFM cantilever is brought into contact with a dsDNA star-
like colloid suspended in water within an open liquid cell environment. The colloid is attached to a glass substrate. 1904136 (2 of 10) © 2019 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Small 2019, 15, 1904136 www.small-journal.com www.small-journal.com Figure 2. Force versus time curve of a typical indentation and relaxa-
tion experiment, which consists of three sequential steps as shown in
the figure: I) probe equilibration; II) probe approach and indentation;
III) probe at constant depth into the sample. length the size of the corona is double the size for 10 kbp, i.e.,
Lc ≈ 7.8 µm in salt-free water. www.small-journal.com www.small-journal.com www.advancedsciencenews.com Figure 3. a) Shear relaxation modulus (G(t)) versus time for dsDNA hairy
colloids dispersed in water at different concentrations of salt cs: 0 m (⦁),
0.005 m (◾), 0.025 m (▸), 0.05 m (▴), 0.1 m (▾), 0.25 m (◂), and 1 m (♦). Lines are the data best fits of Equation (3). b) The best-fit curves as in (a),
but normalized by their value at the longest measured time, GL. Figure 3 shows the shear relaxation modulus curves (i.e., the
force relaxation (F(t)) via Equation (2)) obtained from the inden-
tation measurements on the dsDNA hairy colloids suspended
in water at different salt concentrations cs. The absolute value
of G(t) decreases with increasing cs, implying that a smaller
force is required to reach the same relative indentation depth
δ0 ≈ 5% and therefore inferring a softening of the dsDNA par-
ticle’s corona with increasing the salt content in the solution. For most of the measurements the decay of the shear relaxa-
tion modulus with time is evident as well as the existence of a
finite plateau value. Moreover, it can be seen that the addition
of salt to the solution (i.e., at cs = 0.005 m) induces first a slow-
down of the relaxation process (when compared with the salt-
free case) and then a faster decay for larger values of cs. This
is highlighted in Figure 3b, where we report the best fits of the
experimental data by means of Equation (3), but normalized by
their values at long times (i.e., GR). In Figures 4 and 5 are reported the parameter values of the
best fits of Equation (3) as a function of salt concentration cs. From these diagrams it can be seen that both the total modulus
GT = G0 + GR and the residual modulus GR show a reduction
with increasing of cs (Figure 4a, which indicates a progressive
softening of the colloidal particles). Notice that the presence
of a residual modulus indicates a kind of glassy behavior of
the corona. Interestingly, the salt concentration dependence
of both GT and GR can be described with an exponential decay
having the following expression:
exp(
/
)
0
s
s
0
G
G
c
c
B
i
i
=
−
+ ; where
i = T, R and B is a baseline value. We find that for both the
moduli
0.026
s
0c ≈
m. www.small-journal.com Figure 4b shows the values of GT plotted
against the salt-dependent corona thickness L relative to its
value in the absence of salt, L0. It can be seen that the par-
ticle stiffness (i.e., GT) shows a sharp transition by almost an
order of magnitude at L/L0 ≈ 0.72 (which corresponds to a salt
concentration of cs = 0.025 m) between the two well-defined
regimes, within which only a moderate reduction of the mod-
ulus is observed as a function of L (or equivalently of cs). This
is a riveting finding that suggests the existence of a critical
size of the corona, below which a significant softening of the
colloidal particles is observed. This critical size of the corona
corresponds to the end of the osmotic regime. The inset
of Figure 5 shows the ratio GR/GT, which reveals an initial
decrease at salt concentration values of cs = 0.005 and 0.25 m,
followed by an increase at cs = 0.05 and 0.1 m, and then the
ratio remains almost constant for cs = 0.25 and 1 m. Given
that for viscoelastic fluids GR should tend to 0, an increase of
the ratio GR/GT indicates a tendency of the system to behave
like a viscoelastic solid (e.g., gels and rub-
bers). Therefore, the initial reduction of
GR/GT can be seen as a transition to a more
fluid behavior of the corona, followed by a
transition to a behavior closer to a viscoe-
lastic solid; however, with a continuously
decreasing modulus as shown by the mono-
tonic decrease of GT. The inset of Figure 4
shows the salt concentration functionality
of the model viscosity η0, which exhibits an
initial increase followed by a pronounced
decrease of almost two orders of magni-
tude. A similar behavior is observed for
the characteristic average relaxation time
he force relaxation
tion obtained from
β
0.41 ± 0.01
0.72 ± 0.01
0.35 ± 0.02
0.81 ± 0.03
0.37 ± 0.06
0.51 ± 0.01
0.39 ± 0.06 Figure 3. a) Shear relaxation modulus (G(t)) versus time for dsDNA hairy
colloids dispersed in water at different concentrations of salt cs: 0 m (⦁),
0.005 m (◾), 0.025 m (▸), 0.05 m (▴), 0.1 m (▾), 0.25 m (◂), and 1 m (♦). Lines are the data best fits of Equation (3). b) The best-fit curves as in (a),
but normalized by their value at the longest measured time, GL. © 2019 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim © 2019 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 2.2. Force Relaxation Curves: Elastic and Viscous Contributions The experiments were performed on colloidal particles having a
streptavidin-coated polystyrene sphere of radius Rps = 0.49 µm
as core, on the surface of which biotin terminated dsDNA
chains were grafted. The dsDNA chain length was equal to
10 kbp, which corresponds to a contour length of Lc ≈ 3.4 µm
in salt-free water.[32] The functionality f = 2 × 105 chains per
particle. In addition, as a means of comparison, dsDNA hairy
colloids with 20 kbp chain length having the same function-
ality f and core radius of those described above, were tested to
investigate the effect of chain length on particle softness. Note
that due to the osmotic nature of the corona, for 20 kbp chain The simplest analytical expression for describing the tem-
poral behavior of G(t) is a single mode Maxwell model, which
is a single exponential decay with a characteristic relaxation
time[48] determined by the relative contribution of the elastic
(G0) and the viscous (η0) components of the model; i.e.,
G(t) = G0 exp (−t/τ), with τ = η0/G0. However, it must be noted
that, none of our measurements would have been adequately
modeled by means of this simple expression, which implies
that i) the force fully decays to zero at long times ii) via a single 1904136 (3 of 10) © 2019 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Small 2019, 15, 1904136 www.small-journal.com relaxation process, which is not the case of our experiments as
we shall demonstrate below. Therefore, in order to account for
the existence of a residual (rubber-like) modulus GR and a dis-
tribution of relaxation times,[26] we have adopted the following
stretched exponential expression of G(t): ( )
exp[ ( / ) ]
R
0
G t
G
G
t τ
=
+
−
β
(3) (3) with an average relaxation time determined as 〈τ〉 = (η0/G0)1/β. The best fits of the experimental data of Equation (3) are shown
in Figure 3 and the output parameters G0, GR, η0, and β are
reported in Table 1 together with the relative errors, which
correspond to one standard deviation, as obtained from the fits. with an average relaxation time determined as 〈τ〉 = (η0/G0)1/β. The best fits of the experimental data of Equation (3) are shown
in Figure 3 and the output parameters G0, GR, η0, and β are
reported in Table 1 together with the relative errors, which
correspond to one standard deviation, as obtained from the fits. Table 1. Fitting parameters and relative errors obtained by interpreting the force relaxation
data in Figure 3 via Equation (3). Errors correspond to one standard deviation obtained from
the fits. Table 1. Fitting parameters and relative errors obtained by interpreting the force relaxation
data in Figure 3 via Equation (3). Errors correspond to one standard deviation obtained from
the fits 1904136 (4 of 10)
© 2019 The Authors. P
Small 2019 15 1904136
cs [m]
G0 [105 Pa]
GR [105 Pa]
η0 [105 Pa s]
β
0
3.86 ± 0.07
3.29 ± 0.03
5.9 ± 0.1
0.41 ± 0.01
0.005
3.71 ± 0.03
2.35 ± 0.03
17.1 ± 0.4
0.72 ± 0.01
0.025
3.9 ± 0.2
1.62 ± 0.1
8.4 ± 0.05
0.35 ± 0.02
0.05
0.84 ± 0.02
1.14 ± 0.01
0.41 ± 0.01
0.81 ± 0.03
0.10
0.28 ± 0.05
0.70 ± 0.005
0.09 ± 0.02
0.37 ± 0.06
0.25
0.29 ± 0.01
0.68 ± 0.004
0.08 ± 0.005
0.51 ± 0.01
1.00
0.23 ± 0.04
0.65 ± 0.005
0.06 ± 0.015
0.39 ± 0.06 www.advancedsciencenews.com
Figure 4. Best-fit parameters as a function of cs, obtained by modeling
the force-relaxation curves of Figure 3 by means of Equation (3). a) Total
modulus GT = G0 + GR (◽) and residual modulus GR (⚪). www.small-journal.com The lines are the
best fits of the data of the following expression: G
G
c
c
B
i
i
=
−
+
exp(
/
)
0
s
s0
,
with i = T, R. Inset: the viscosity η0, same x-axis as in the main plot. b) GT as a function of relative corona thickness L/L0. The top axis reports
the corresponding salt concentration cs. Error bars correspond to one
standard deviation obtained from the fits. © 2019 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 5. Mean relaxation time 〈τ〉 = η0/G0. Inset: ratio between the
residual and the total modulus, GR/GT, same x-axis as in the main plot. Error bars for 〈τ〉 and GR/GT were obtained from standard error propaga-
tion using the uncertainties of η0, G0, GR, and GT. 〈τ〉 (Figure 5), which suggests that while the static stiffness
of the particles decreases monotonically with increasing the
salt concentration, the dynamic response of these colloidal
systems is more complex; hence the need of measuring the
frequency-dependent viscoelastic moduli for a better under-
standing of their dynamics, as described below. www.small-journal.com An advantage of the direct Fourier transform of the
experimental data relies on the absence of any preconceived
model for describing the viscoelastic behavior of the material. A similar approach has been successfully applied in different
studies[50,51] and recently adopted for the evaluation of the viscoe-
lastic properties of single cells, after testing the effectiveness of
the analytical procedure on different complex soft materials.[25] the force–relaxation curves were smoothed using a Savitzky-
Goyal filter to reduce the disruptive contribution of the noises,
given the integral nature of the Fourier transform. Notice that
the use of such a filter does not alter the shape of the relaxa-
tion curves. An advantage of the direct Fourier transform of the
experimental data relies on the absence of any preconceived
model for describing the viscoelastic behavior of the material. A similar approach has been successfully applied in different
studies[50,51] and recently adopted for the evaluation of the viscoe-
lastic properties of single cells, after testing the effectiveness of
the analytical procedure on different complex soft materials.[25] Figure 6 shows the frequency-dependent viscoelastic
moduli of the colloidal particles dispersed in water at dif-
ferent salt concentrations. The storage modulus G′(ω) shows
a reduction with the increase of cs, mirroring the reduction
of the stiffness deduced from the best fits of G(t) described
in the previous section. This can be further corroborated by
extracting the high-frequency value of the storage modulus,
h
G′
as a function of cs as shown in Figure 7a. The dependence of
h
G′ on cs can be described by means of an exponential decay,
exp[ ( /
)]
h
s
s
0
G
A
B
c c
′ ∝
+
−
with
0.026
s
0c ≈
m, in close agreement
with the results obtained from the analysis of GT and GR, and
therefore educing the same conclusions. The frequency dependence of the moduli and their rela-
tive variation provide additional crucial information on the
viscoelastic nature of the colloidal particles. Indeed, the DNA
corona is a dense polyelectrolyte solution, which is intrinsi-
cally viscoelastic. In particular, while the storage modulus G′(ω) Figure 4. Best-fit parameters as a function of cs, obtained by modeling
the force-relaxation curves of Figure 3 by means of Equation (3). a) Total
modulus GT = G0 + GR (◽) and residual modulus GR (⚪). © 2019 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.small-journal.com The lines are the
best fits of the data of the following expression: G
G
c
c
B
i
i
=
−
+
exp(
/
)
0
s
s0
,
with i = T, R. Inset: the viscosity η0, same x-axis as in the main plot. b) GT as a function of relative corona thickness L/L0. The top axis reports
the corresponding salt concentration cs. Error bars correspond to one
standard deviation obtained from the fits. www.small-journal.com www.advancedsciencenews.com www.advancedsciencenews.com (4) 3(1
)
8
1
1
0
3/2
1/3
2
1/3
3/2
C
R
R
ν
δ
=
−
+
∗
(5) (5) where G′(ω) and G″(ω) are the materials’ viscoelastic moduli
describing the frequency-dependent elastic and viscous char-
acter of the system, respectively. The Fourier transform is
performed by using the analytical method proposed by Evans
and Tassieri.[25,49,50] Before performing the Fourier transform, where G′(ω) and G″(ω) are the materials’ viscoelastic moduli
describing the frequency-dependent elastic and viscous char-
acter of the system, respectively. The Fourier transform is
performed by using the analytical method proposed by Evans
and Tassieri.[25,49,50] Before performing the Fourier transform,
the force–relaxation curves were smoothed using a Savitzky-
Goyal filter to reduce the disruptive contribution of the noises,
given the integral nature of the Fourier transform. Notice that
the use of such a filter does not alter the shape of the relaxa-
tion curves. An advantage of the direct Fourier transform of the
experimental data relies on the absence of any preconceived
model for describing the viscoelastic behavior of the material. A similar approach has been successfully applied in different
studies[50,51] and recently adopted for the evaluation of the viscoe-
lastic properties of single cells, after testing the effectiveness of
the analytical procedure on different complex soft materials.[25] where G′(ω) and G″(ω) are the materials’ viscoelastic moduli
describing the frequency-dependent elastic and viscous char-
acter of the system, respectively. The Fourier transform is
performed by using the analytical method proposed by Evans
and Tassieri.[25,49,50] Before performing the Fourier transform,
the force–relaxation curves were smoothed using a Savitzky-
Goyal filter to reduce the disruptive contribution of the noises,
given the integral nature of the Fourier transform. Notice that
the use of such a filter does not alter the shape of the relaxa-
tion curves. 1904136 (5 of 10)
Small 2019, 15, 1904136 2.3. Indentation and Relaxation Experiments: Frequency-
Dependent Viscoelastic Moduli The dissipat
tially almost quadratically with frequency (
cs = 0.1 m and higher), then at intermediat
almost linearly proportional to ω up to circa
which it increases again quadratically, Wd(ω
ency behavior of the storage G′(ω) (solid symbols)
ω) (open symbols) moduli and b) of the average
n a cycle, Wd, obtained from indentation and relaxa-
or different salt concentrations, as indicated in the
) are guides for power laws with exponents 1 and 2. nt viscoelastic moduli in (a) were obtained by Fourier
data in Figure 3a according to Equations (4) and (5). Figure 7. a) High frequency storage modulus
h
G′ as
centration cs. The solid line represents a fit to an exp
time τmax at which the maximum of G″ is observed
b) Value of the energy dissipated in a cycle, Wd, calc
(Wd
L, left axis) and highest (Wd
h, right axis) frequenci Figure 7. a) High frequency storage modulus
h
G′ as a function of salt con-
centration cs. The solid line represents a fit to an exponential decay. Inset:
time τmax at which the maximum of G″ is observed, as a function of cs. b) Value of the energy dissipated in a cycle, Wd, calculated for the lowest
(Wd
L, left axis) and highest (Wd
h, right axis) frequencies, as a function of cs. Figure 6. a) Frequency behavior of the storage G′(ω) (solid symbols)
and the loss G″(ω) (open symbols) moduli and b) of the average
energy dissipated in a cycle, Wd, obtained from indentation and relaxa-
tion experiments for different salt concentrations, as indicated in the
legend. Lines in (b) are guides for power laws with exponents 1 and 2. Frequency-dependent viscoelastic moduli in (a) were obtained by Fourier
transforming G(t) data in Figure 3a according to Equations (4) and (5). the direct Fourier transform of the force–relaxation curve, as
already stressed, does not need the assumption of any precon-
ceived model describing the viscoelastic nature of the system
under investigation. Addi i
l i f
i
b
b i
d b
l
l i g
Additional information can be obtained by calculating
the average energy dissipated within a cycle at a specific
frequency[52]:
( )
(1/2)
( )
d
0
2
W
G
ω
ω
ω δ
=
′′
(see Figure 6b). 2.3. Indentation and Relaxation Experiments: Frequency-
Dependent Viscoelastic Moduli At
cs = 0 and at relatively low frequencies (i.e., below 1 Hz), we
observe an almost linear proportionality of Wd(ω) with ω;
whereas, at higher frequencies the slope is smaller than 1,
indicating a reduction of dissipation and a more pronounced
solid-like behavior. At a salt concentration of cs = 0.005 m, a
similar behavior is observed at relatively low frequencies, but
a pronounced change of the slope of Wd(ω) is observed at
a frequency value of ω = 0.5 Hz; then the gradient remains
approximately equal to zero, indicating a solid-like behavior of
the colloidal particle. At a salt concentration of cs = 0.025 m the
colloids show a similar behavior to those in salt-free solution
up to a frequency value of circa ω = 20 Hz, then they also show
a solid-like behavior. At salt concentrations of cs = 0.05 m and
higher, the dissipated energy shows again a marked change
in the frequency dependence. The dissipation increases ini-
tially almost quadratically with frequency (this is clearer for
cs = 0.1 m and higher), then at intermediate frequencies it is
almost linearly proportional to ω up to circa ω ≈ 20 Hz, after
which it increases again quadratically, Wd(ω)∝ω2. In order to shows a weak frequency dependence for all the explored salt
concentrations, indicating a solid-like behavior, G″(ω) in the
absence of salt shows an almost constant value up to a max-
imum frequency of ω ≈ 1.5 Hz, after which the loss modulus
starts to decrease. This indicates a maximum of energy dis-
sipation that can be associated with dsDNA chains’ structural
relaxation. Moreover, it is interesting to notice that the region
of maximum dissipation shifts to slightly lower frequencies for
cs = 0.005 m, and then it moves back to almost the same value
obtained in the salt-free solution at cs = 0.025 m. At relatively
larger values of cs, the G″(ω) maximum shifts toward the higher
end of the explored frequencies, implying a substantial accel-
eration of the relaxation processes occurring at relatively small
length scales. To quantify these changes we plot in the inset of
Figure 7a the inverse of the frequency at which the maximum
of G″(ω) is observed; i.e., a characteristic relaxation time τmax of
the topological changes of the dsDNA chains as a function of cs. 2.3. Indentation and Relaxation Experiments: Frequency-
Dependent Viscoelastic Moduli An alternative approach to determine the viscoelastic properties
of the colloidal particles is via the Fourier transform of the time-
dependent force relaxation measurement F(t). This is possible
because the material’s complex shear modulus G*(ω) = G′(ω) +
iG″(ω) is defined as the Fourier transform of the time derivative
of G(t) and therefore by considering Equation (2) one obtains Figure 5. Mean relaxation time 〈τ〉 = η0/G0. Inset: ratio between the
residual and the total modulus, GR/GT, same x-axis as in the main plot. Error bars for 〈τ〉 and GR/GT were obtained from standard error propaga-
tion using the uncertainties of η0, G0, GR, and GT. 1904136 (5 of 10)
Small 2019, 15, 1904136 1904136 (5 of 10) © 2019 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Small 2019, 15, 1904136 www.advancedsciencenews.com
www.small-journal.com
shows a weak frequency dependence for all the explored salt
concentrations, indicating a solid-like behavior, G″(ω) in the
absence of salt shows an almost constant value up to a max-
imum frequency of ω ≈ 1.5 Hz, after which the loss modulus
starts to decrease. This indicates a maximum of energy dis-
sipation that can be associated with dsDNA chains’ structural
relaxation. Moreover, it is interesting to notice that the region
of maximum dissipation shifts to slightly lower frequencies for
cs = 0.005 m, and then it moves back to almost the same value
obtained in the salt-free solution at cs = 0.025 m. At relatively
larger values of cs, the G″(ω) maximum shifts toward the higher
end of the explored frequencies, implying a substantial accel-
eration of the relaxation processes occurring at relatively small
length scales. To quantify these changes we plot in the inset of
Figure 7a the inverse of the frequency at which the maximum
of G″(ω) is observed; i.e., a characteristic relaxation time τmax of
the direct Fourier transform of the force–relaxation curve, as
already stressed, does not need the assumption of any precon-
ceived model describing the viscoelastic nature of the system
under investigation. Additional information can be obtained by calculating
the average energy dissipated within a cycle at a specific
frequency[52]:
( )
(1/2)
( )
d
0
2
W
G
ω
ω
ω δ
=
′′
(see Figure 6b). 2.3. Indentation and Relaxation Experiments: Frequency-
Dependent Viscoelastic Moduli At
cs = 0 and at relatively low frequencies (i.e., below 1 Hz), we
observe an almost linear proportionality of Wd(ω) with ω;
whereas, at higher frequencies the slope is smaller than 1,
indicating a reduction of dissipation and a more pronounced
solid-like behavior. At a salt concentration of cs = 0.005 m, a
similar behavior is observed at relatively low frequencies, but
a pronounced change of the slope of Wd(ω) is observed at
a frequency value of ω = 0.5 Hz; then the gradient remains
approximately equal to zero, indicating a solid-like behavior of
the colloidal particle. At a salt concentration of cs = 0.025 m the
colloids show a similar behavior to those in salt-free solution
up to a frequency value of circa ω = 20 Hz, then they also show
a solid-like behavior. At salt concentrations of cs = 0.05 m and
Figure 6. a) Frequency behavior of the storage G′(ω) (solid symbols)
and the loss G″(ω) (open symbols) moduli and b) of the average
energy dissipated in a cycle, Wd, obtained from indentation and relaxa-
tion experiments for different salt concentrations, as indicated in the
legend. Lines in (b) are guides for power laws with exponents 1 and 2. Frequency-dependent viscoelastic moduli in (a) were obtained by Fourier
transforming G(t) data in Figure 3a according to Equations (4) and (5). Figure 7. a) High frequency storage modulus
h
G′ as a function of salt con-
centration cs. The solid line represents a fit to an exponential decay. Inset:
time τmax at which the maximum of G″ is observed, as a function of cs. b) Value of the energy dissipated in a cycle, Wd, calculated for the lowest
(Wd
L, left axis) and highest (Wd
h, right axis) frequencies, as a function of cs. www.small-journal.com
Figure 7. a) High frequency storage modulus
h
G′ as a function of salt con-
centration cs. The solid line represents a fit to an exponential decay. Inset:
time τmax at which the maximum of G″ is observed, as a function of cs. b) Value of the energy dissipated in a cycle, Wd, calculated for the lowest
(Wd
L, left axis) and highest (Wd
h, right axis) frequencies, as a function of cs. 2.3. Indentation and Relaxation Experiments: Frequency-
Dependent Viscoelastic Moduli www.small-journal.com www.advancedsciencenews.com equency dependence for all the explored salt
ndicating a solid-like behavior, G″(ω) in the
hows an almost constant value up to a max-
of ω ≈ 1.5 Hz, after which the loss modulus
e. This indicates a maximum of energy dis-
be associated with dsDNA chains’ structural
over, it is interesting to notice that the region
sipation shifts to slightly lower frequencies for
d then it moves back to almost the same value
salt-free solution at cs = 0.025 m. At relatively
s, the G″(ω) maximum shifts toward the higher
red frequencies, implying a substantial accel-
axation processes occurring at relatively small
quantify these changes we plot in the inset of
verse of the frequency at which the maximum
ved; i.e., a characteristic relaxation time τmax of
hanges of the dsDNA chains as a function of cs. ight have already anticipated, not only both the
τmax show the same behavior of GT (or GR) and
but also they show very similar values. There-
speculate that the two analytical approaches
and quantitatively congruent. Nonetheless,
the direct Fourier transform of the force–re
already stressed, does not need the assumpt
ceived model describing the viscoelastic na
under investigation. Additional information can be obtaine
the average energy dissipated within a c
frequency[52]:
( )
(1/2)
( )
d
0
2
W
G
ω
ω
ω δ
=
′′
(see
cs = 0 and at relatively low frequencies (i.e. observe an almost linear proportionality o
whereas, at higher frequencies the slope i
indicating a reduction of dissipation and a
solid-like behavior. At a salt concentration
similar behavior is observed at relatively low
a pronounced change of the slope of Wd(
a frequency value of ω = 0.5 Hz; then the
approximately equal to zero, indicating a sol
the colloidal particle. At a salt concentration
colloids show a similar behavior to those in
up to a frequency value of circa ω = 20 Hz, th
a solid-like behavior. At salt concentrations
higher, the dissipated energy shows again
in the frequency dependence. © 2019 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 2.5. Discussion The reduction of the particle stiffness with increasing of the
amount of salt in solution can be interpreted in terms of the
morphological variations of the dsDNA corona, which mimics
in general those of brushes formed by long polyelectrolyte
chains. At very low salt concentration the corona absorbs almost
all counterions and the chains are completely stretched in order
to maximize the available volume for the mobile counterions. With addition of salt the osmotic imbalance between the inside
of the corona and the bulk is progressively removed and the
chains become more flexible, thus their configuration becomes
increasingly random reducing the effective size of the corona,
until they collapse at large salt concentrations. The softening of
the particles shows a pronounced exponential dependence on
salt concentration and an abrupt transition at a critical corona
size Ls ≈ 0.72 L0, which are dictated by the changes in the
counterion distribution. It should be noticed that the observed
softening of the dsDNA corona is much more pronounced
than the increased flexibility of isolated dsDNA chains with
increasing salt concentration, which has been determined by
existing studies on the persistence length (or equivalently the
Young’s modulus) of single dsDNA chains.[53–55] Indeed, in the better elucidate these results, the values of the energy dissipa-
tion determined at the extremes of the explored frequencies
(i.e.,
d
L
W at a frequency of ω = 0.1 Hz and
d
h
W at a frequency
ω = 100 Hz) are reported in Figure 7b. From the latter, one can
observe that there is a moderate increase of
d
L
W from a salt-free
condition to cs = 0.005 m, followed by a monotonic decrease for
higher values of cs. This behavior closely resemble those of the
relaxation times 〈τ〉 and τmax. On the other hand, the behavior
of
d
h
W is more complex, showing an initial decrease with a
relative minimum value at cs = 0.005 m, followed by a relative
maximum at cs = 0.05 m and further smaller variations at larger
values of cs. The similarities between
d
L
W , 〈τ〉 and τmax induce us
to infer that small dissipation values are associated with the top-
ological changes of the corona, possibly related to the change in
stiffness of the single DNA chains (i.e., from rod-like to coil-
like, as cs increases). 2.4. Effect of dsDNA Chain Length on Viscoelasticity Frequency-dependent viscoelastic moduli in (b) were obtained by
Fourier transforming G(t) data in (a) according to Equations (4) and (5). 2.3. Indentation and Relaxation Experiments: Frequency-
Dependent Viscoelastic Moduli Notably, as we might have already anticipated, not only both the
terms of
h
G′ and τmax show the same behavior of GT (or GR) and
〈τ〉, respectively, but also they show very similar values. There-
fore, we could speculate that the two analytical approaches
are qualitatively and quantitatively congruent. Nonetheless, 1904136 (6 of 10) © 2019 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Small 2019, 15, 1904136 www.small-journal.com www.advancedsciencenews.com www.advancedsciencenews.com
Figure 8. a) Time-dependent shear relaxation modulus and b) frequency-
dependent viscoelastic moduli, G′(ω) (solid symbols) and G″(ω) (open
symbols), obtained from indentation and relaxation experiments on PS
particles decorated with 10 kbp (circles) or 20 kbp (pentagons) dsDNA
chains. Frequency-dependent viscoelastic moduli in (b) were obtained by
Fourier transforming G(t) data in (a) according to Equations (4) and (5). high salt concentrations than for rigid (charged) chains at small
salt concentration. high salt concentrations than for rigid (charged) chains at small
salt concentration. 2.4. Effect of dsDNA Chain Length on Viscoelasticity To test the influence of the length of the dsDNA chains on the
viscoelastic behavior of the colloidal particles, we performed
indentation and relaxation experiments on particles with longer
grafted DNA chains (i.e., 20kbp long) and in salt-free solu-
tion. Both the colloids had the same grafting density (200 000
chains) and size of the PS core (RPS = 0.49 µm). We observed
that the magnitude of the modulus G(t) of the colloidal parti-
cles covered with 10kbp chains is about three times bigger
than the particles covered with 20kbp chains (Figure 8a), and
with a longer relaxation process. Indeed, by fitting the experi-
mental data of the 20kbp system with Equation (3) we obtain
GR ≈ 1.1 × 105 ± 0.03 Pa, GT ≈ 2.1 × 105 ± 0.05 Pa, and τ ≈ 0.04
± 0.005 s. The first two values are comparable to those obtained
for the smaller colloidal particles in a 0.05 m NaCl solution,
whereas the value of τ is closer and even slightly smaller than
those obtained for cs ⩾ 0.1 m. This suggests that a longer chain
length induces changes similar to the addition of salt, how-
ever, with a different balance between the viscous and elastic
contribution to the final mechanical properties of the colloid. Figure 8b shows the viscoelastic moduli obtained by Fourier
transforming the time-dependent shear relaxation modulus. The storage modulus decreases in magnitude while keeping a
similar frequency behavior, whereas the loss modulus shows
the qualitative change discussed in the previous section for the
largest values of cs, i.e., the maximum moves to larger frequen-
cies indicating a faster relaxation of the system. We can there-
fore conclude that a thicker corona also leads to a significant
softening of the particles. Figure 8. a) Time-dependent shear relaxation modulus and b) frequency-
dependent viscoelastic moduli, G′(ω) (solid symbols) and G″(ω) (open
symbols), obtained from indentation and relaxation experiments on PS
particles decorated with 10 kbp (circles) or 20 kbp (pentagons) dsDNA
chains. Frequency-dependent viscoelastic moduli in (b) were obtained by
Fourier transforming G(t) data in (a) according to Equations (4) and (5). Figure 8. a) Time-dependent shear relaxation modulus and b) frequency-
dependent viscoelastic moduli, G′(ω) (solid symbols) and G″(ω) (open
symbols), obtained from indentation and relaxation experiments on PS
particles decorated with 10 kbp (circles) or 20 kbp (pentagons) dsDNA
chains. Small 2019, 15, 1904136 © 2019 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim We interpret these results as the manifesta-
tion of the changes in the counterion distribution in the poly-
electrolyte brush with increasing ionic strength, which strongly
affect the chain conformations and chain-chain interactions. Indeed, the moduli of the dsDNA brush show a much stronger
dependence on salt concentration than the stiffness of isolated
dsDNA chains.[53–55] Our results are in agreement with recent
numerical studies on films of shorter dsDNA chains grafted
on substrates.[56,57] A closer look at the frequency-dependent
viscoelastic properties suggests that the energy dissipation at
low frequencies (i.e., long times) is dominated by the collective
force relaxation in the corona, whereas at high frequencies (i.e.,
short times) chain dynamics might be more important. Finally,
we report that particles with a thicker DNA corona are softer,
which can be associated with a lower DNA density in the outer
side of the corona and/or a weaker contribution of the rigid
core of the colloid. The reduction of stiffness with increasing cs is consistent
with recent model systems used to calculate the elastic prop-
erties of dense films of shorter (few tenths of nm) dsDNA
chains on a substrate as a function of salt concentration.[56,57] The reduction of stiffness with increasing cs is consistent
with recent model systems used to calculate the elastic prop-
erties of dense films of shorter (few tenths of nm) dsDNA
chains on a substrate as a function of salt concentration.[56,57]
These models are static, i.e., they only predict the elasticity of
the films via its Young’s modulus (E). These films are typically
used as coatings for AFM microcantilevers for biodetection.[58]
In particular, Wu and co-workers[56] have presented a bead-
chain model where the DNA chain is divided into spherical
fragments connected by elastic rods, which interact with the
fragments of other chains through a Yukawa potential. By
solving numerically the model, Wu and co-workers obtained
the values of the Young’s modulus as a function of salt con-
centration, which is in good agreement with our findings and
shows an exponential decay similar to those found in this work
for Gh′, GT or GR. In particular, the data of Wu and co-workers[56]
for the Young’s modulus can de described through the relation:
exp[ (
/
)]
s
s
0
E
A
B
c
c
≈
+
−
(see the Supporting Information), with
0.01
s
0c ≈
m in their case. 4. Experimental Section Synthesis of DNA Star Polyelectrolyte Colloids and Dispersions’
Preparation: The procedure to obtain DNA star polyelectrolyte colloids
was described in detail before.[32] Here, the main steps of the procedure
are recalled that can be summarized as follows: i) Double-stranded DNA
synthesis through the amplification to 20Kbp and 10Kbp end-biotinylated
fragments via Polymerase Chain Reaction (PCR). ii) Grafting of DNA
chains to the streptavidin functionalized surface of polystyrene beads
(Rps = 0.49 µm). The particles are then dispersed in deionized water
and different saline buffer solutions containing 5 × 10−3, 2.5 × 10−2, 5 ×
10−2, 10−1, 2.5 × 10−1, and 1 m of NaCl. For a fraction of the particles,
the DNA was prelabeled with YOYO-1 (Thermofisher) at a dye—DNA
base pair ratio of 1:25 and the colloids were characterized by confocal
microscopy revealing a particle size R ≈ 8 µm in pure water solution. Dispersions with a particle volume fraction ϕ = 0.01 were prepared for
the experiments. Finally, it must be noted that the decrease of the particle stiff-
ness with the increase of chain length should not be attributed
to variations of the dsDNA flexibility. This is because in the low-
salt limit the persistence length of sufficiently long dsDNA does
not depend on the chain length.[61] We could speculate that the
reduced elasticity of the colloidal particles having longer chain
lengths is related to a lower DNA density at the external surface
of the corona for longer chains than shorter ones. Such behavior of the Young’s modulus
as function of cs has been explained by Wu and co-workers[56] in
terms of increased flexibility of the dsDNA chain together with
the contribution of the complex interactions between different
dsDNA chains in the brush. We show here that the counterion
distribution governing the configurations of the dsDNA chains
in the corona is the dominant contribution to the softening. The growth of DNA flexibility with increasing cs is consistent
with the faster relaxation process of the force and the increase
of the frequency at which the maximum of G″ is observed. Indeed, the dynamics of the semi-flexible dsDNA chains are
expected to become faster with decreasing their persistence
length.[59,60] At the same time, the non-monotonic behavior of
the ratio of the viscous to the elastic moduli as a function of salt
concentration cannot be directly related to any existing predic-
tion and will need further investigation. To conclude, we would like to highlight that both the results
and the method presented in this work open the way to a sys-
tematic investigation of the viscoelastic properties of colloidal
particles of different nature, including microgel particles, soft
composite particles, micelles, and others. These properties
are of great importance for the understanding of the phase
behavior of these systems. In particular, we stress that the vis-
coelastic properties of colloidal particles can be determined
through a model-free method, based on the Fourier transform
of force relaxation curves. strength of the solution. This occurs exponentially as a function
of the salt concentration, with a sharp decrease at cs ≲ 0.026 m,
which in terms of the brush size corresponds to a critical size
of Ls ≈ 0.72 L0. The stiffness reduction of the colloids as func-
tion of the salt concentration is also manifested with a faster
relaxation of the force, which relates to a shift of the maximum
of G″(ω) (i.e., the point of maximum dissipation) to a higher
frequency value. We interpret these results as the manifesta-
tion of the changes in the counterion distribution in the poly-
electrolyte brush with increasing ionic strength, which strongly
affect the chain conformations and chain-chain interactions. Indeed, the moduli of the dsDNA brush show a much stronger
dependence on salt concentration than the stiffness of isolated
dsDNA chains.[53–55] Our results are in agreement with recent
numerical studies on films of shorter dsDNA chains grafted
on substrates.[56,57] A closer look at the frequency-dependent
viscoelastic properties suggests that the energy dissipation at
low frequencies (i.e., long times) is dominated by the collective
force relaxation in the corona, whereas at high frequencies (i.e.,
short times) chain dynamics might be more important. Finally,
we report that particles with a thicker DNA corona are softer,
which can be associated with a lower DNA density in the outer
side of the corona and/or a weaker contribution of the rigid
core of the colloid. latter studies the dependence of E on cs is milder; i.e., roughly
inversely proportional to cs. This is indicative that the entropic
effects of the counterions in a dense dsDNA brush are domi-
nating its mechanical properties. latter studies the dependence of E on cs is milder; i.e., roughly
inversely proportional to cs. This is indicative that the entropic
effects of the counterions in a dense dsDNA brush are domi-
nating its mechanical properties. strength of the solution. This occurs exponentially as a function
of the salt concentration, with a sharp decrease at cs ≲ 0.026 m,
which in terms of the brush size corresponds to a critical size
of Ls ≈ 0.72 L0. The stiffness reduction of the colloids as func-
tion of the salt concentration is also manifested with a faster
relaxation of the force, which relates to a shift of the maximum
of G″(ω) (i.e., the point of maximum dissipation) to a higher
frequency value. © 2019 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Small 2019, 15, 1904136 2.5. Discussion At high frequencies,
d
h
W indicates that the
energy is dissipated more efficiently by the samples with a col-
lapsed corona and showing lower values of GT . Here we specu-
late that this phenomena might be governed by the single DNA
chain relaxation, which is faster for flexible (screened) chains at better elucidate these results, the values of the energy dissipa-
tion determined at the extremes of the explored frequencies
(i.e.,
d
L
W at a frequency of ω = 0.1 Hz and
d
h
W at a frequency
ω = 100 Hz) are reported in Figure 7b. From the latter, one can
observe that there is a moderate increase of
d
L
W from a salt-free
condition to cs = 0.005 m, followed by a monotonic decrease for
higher values of cs. This behavior closely resemble those of the
relaxation times 〈τ〉 and τmax. On the other hand, the behavior
of
d
h
W is more complex, showing an initial decrease with a
relative minimum value at cs = 0.005 m, followed by a relative
maximum at cs = 0.05 m and further smaller variations at larger
values of cs. The similarities between
d
L
W , 〈τ〉 and τmax induce us
to infer that small dissipation values are associated with the top-
ological changes of the corona, possibly related to the change in
stiffness of the single DNA chains (i.e., from rod-like to coil-
like, as cs increases). At high frequencies,
d
h
W indicates that the
energy is dissipated more efficiently by the samples with a col-
lapsed corona and showing lower values of GT . Here we specu-
late that this phenomena might be governed by the single DNA
chain relaxation, which is faster for flexible (screened) chains at 1904136 (7 of 10) © 2019 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Small 2019, 15, 1904136 www.small-journal.com 1904136 (8 of 10)
Small 2019, 15, 1904136 The authors declare no conflict of interest. The authors declare no conflict of interest. p
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The Functions of Geography in the Elementary School: A Study in Educational Values
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public-domain
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Journal of Geography To cite this article: William Chandler Bagley (1904) The Functions of Geography in
the Elementary School: A Study in Educational Values, Journal of Geography, 3:5,
222-233, DOI: 10.1080/00221340408985504 To cite this article: William Chandler Bagley (1904) The Functions of Geography in
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The Functions of Geography in
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in Educational Values
William Chandler Bagley a
a The Montana State Normal College
Published online: 12 May 2008.
To cite this article: William Chandler Bagley (1904) The Functions of Geography in
the Elementary School: A Study in Educational Values, Journal of Geography, 3:5,
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The Functions of Geography in
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William Chandler Bagley a
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Published online: 12 May 2008. To cite this article: William Chandler Bagley (1904) The Functions of Geography in
the Elementary School: A Study in Educational Values, Journal of Geography, 3:5,
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y
p
Probably the sun is moving in a sensibly straight line, for the stars
are so extremely remote that their attractive influences are quite
inappreciable. It is not necessary to assume, as is sometimes done,
that its motion is due to the attraction of other bodies, for this implies
that it was originally at rest, an assumption which is by no means
necessary, and not even probable. One possible consequence of the sun’s motion remains to be men-
tioned, and that is that it may some time encounter meteoric matter
or even collide with a star. In fact, this outcome seems to be almost
inevitable, ultimately. If a collision should occur, it would result in
the destruction of the present system by the enormous amount of heat
generated in the impact. The combined mass would become nebulous,
after which it would undergo an evolution of cooling and shrinking. Indeed, it may be that our present system has evolved from a nebulous
mass generated by collisions of earlier and smaller bodies than the
sun. It is fairly probable that temporary stars owe their sudden
intense luminosity to the heat generated by the impact of collisions
of some sort. THE FUNCTIONS OF GEOGRAPHY I N THE
ELEMENTARY SCHOOL: A STUDY IN
EDUCATIONAL VALUES BY WILLIAM CHANDLER BAGLEY
Of the Montana State Normal College expressly forbidden. Terms & Conditions of access and use can be found at
http://www.tandfonline.com/page/terms-and-conditions expressly forbidden. Terms & Conditions of access and use can be found at
http://www.tandfonline.com/page/terms-and-conditions THE JOURNAL OF GEOGRAPHY May 2 2 2 per second, or more than 300,000,000 miles annually. Although this
has been going on indefinitely no observable change in the appearance of
the star has taken place since scientific observations of it have been
made. The reason is that this distance, great as it may be, is but an
extremely small part of the vast distance between the star and us. Although Sinus is comparatively near us, as the distances to the stars
go, it will be more than 800 years before a velocity of twenty miles
per second will increase its distance by one per cent. Vega, the brightest
star in Lyra and quite near the apex of the sun’s way, and the sun,
are approaching each other at the rate of about fifty miles per second. BY WILLIAM CHANDLER BAGLEY
Of the Montana State Normal College K
E K
EDUCED to its lowest terms, education consists in giving to
the individual experiences which shall modify his future adjust-
‘ments with reference to certain social or moral ends. Such
experiences may be imparted eitller directly (through the individual’s
personal contact with the environment) or indirectly (through lan-
guage or some other symbolic n.edium for the transmission of experi- THE FUNCTIONS OF GEOGRAPHY 2 2 3 ‘904 ence). Such experiences may function either automatically (as habit]
or consciously (as judgment or reason). The problem of the science of
educational values is to determine the part which the various items
of the curriculum play in this process. Given any subject of instruc-
tion, for example, the question must be answered: In what different
ways will this knowledge be likely to function in future adjustments? This question answered, the detailed problems of method can then
be attacked: How shall we teach this subject in order that it may
efficiently fulfill its function? How much time and energy shall be
allotted to this subject in comparison with other departments of instruc-
tion? Downloaded by [Carnegie Mellon University] at 09:25 02 February 2015 The increasing importance of geography in all stages of education
renders the question of values especially important. Why has this
subject so suddenly assumed a position of great importance, not only
in the elementary schools, but also in the high schools, the colleges,
and the universities? What has caused so marvelous a change in the
status of a discipline which, only a few years since, was derisively
termed “the sick man of the curriculum”? An answer to these ques-
tions immediately suggests itself: A knowledge of geography has been
found to be of service to its possessor. What this service is, and how
the ‘‘new” geography has come to render it efficiently, when the ‘‘old”
geography was inadequate for this purpose, are questions that certainly
merit careful consideration. It is obvious that one’s estimation of the value of geographical facts
depends entirely upon one’s connotation of the term l1 geographical.”
If we include under this term only those facts that were commoiily
made the subjects of geographical instruction twenty years ago, we
must say in all candor that such facts have but a minimum of utility
for their possessor-unless,
indeed, he chance to be a sailor. If, how-
ever, we mean by geographical facts all the knowledge which man
has accumulated concerning his environment and its relation to his
life, then the question obviously assumes a different aspect. Thus
defined, geography becomes the nucleus of all the sciences that deal
with natural phenomena. But geography is something more and
something less than a mere blending of these various departments of
knowledge. It is both more and less than astronomy plus geology
plus botany plus zoology plus anthropology. Twenty years ago this
statement would not have been true, at least in so far as the geography
of the schools was concerned. Geography at that time was but a mosaic
of materials borrowed from the various sciences of nature. This
mosaic was not in itself a science, because it lacked a unifying principle. THE JOURNAL OF GEOGRAPHY May May 2 24 The very looseness of the old definition betrays this weakness:
“Geography is a description of the surface of the earth and of its
countries and their inhabitants.” The unifying principle which has made geography a science is Man. Not all the facts of botany are important in geography, but only those
that are directly connected with man’s welfare. * J. W. Redway: Proceedings, N. E. A., 1903, p. 155. Downloaded by [Carnegie Mellon University] at 09:25 02 February 2015 This does not imply
that a complete treatment of geography would not perhaps involve
all the facts of botany, but these would be arranged and classified with
reference to this unifying principle. The pure science of botany, on
the other hand, takes no account whatsoever of this principle. To
all intents and purposes, a pure science is a closed system. Downloaded by [Carnegie Mellon University] at 09:25 02 February 2015 p
p
p
y
This distinctively human view of geography was first clearly enun-
ciated by the great German geographer, Karl Ritter, who died in 1859. It has only been within recent years, however, that the principle has
come to be generally adopted, and even to-day there are some author-
ities who refuse to recognize the limitations which it imposes; but even
the latter would probably agree that, for educational purposes, this
view of geography is the most satisfactory. Mr. Redway has summed
up the matter in the following words: “The question of the nature of
geography is gradually settling itself into one that inquires into the
proper basis and scope of the subject. During the past twenty years
we have seen public opinion throw aside the notion . . . of geography
as a ‘description of the earth’s surface,’ and substitute therefore a very
broad idea, ‘the study of the earth as the home of man.’ . . . If I
felt called upon still further to add to the literature of definition, I
should put it as ‘ the study of man and his environment,’ or, perhaps,
‘life and its environment.’ ” * If, then, we look upon geography as a study of the environment in
its relation to the life of man, the utilitarian value that attaches to
this subject is obvious from the outset. Broadly speaking, all life is
adjustment to an environment. Anything that tends to render this
adjustment more efficient is of value from the standpoint of utility. Whatever reduces waste, whatever saves time, energy, labor, whatever
increases wealth and material prosperity may be looked upon as utili-
tarian in its value. That the facts of geography, as we now understand
that term, possess such utilitarian value in a degree sufficient to war-
rant their wide dissemination is easily demonstrated. A few concrete
instances will suffice to illustrate this point. The process of distribution that is continually going on, tending to
relieve the congested areas of the earth’s surface and to populate the * J. W. Redway: Proceedings, N. E. A., 1903, p. 155. THE FUNCTIONS OF GEOGRAPHY 2 2 j 19"4 undeveloped areas, can take place either blindly or intelligently. Downloaded by [Carnegie Mellon University] at 09:25 02 February 2015 In
the former case, lack of accurate information concerning the condi-
ti ons of different regions-their
relative productivity, healthfulness,
etc.-leads
to a chance or fortuitous selection of favorable environ-
inents; that is, under conditions of geographical ignorance, migratory
movements frequently entail a tremendous material waste, to say
nothing of human suffering. Inadequate knowledge of climatic con-
ditions, for example, led to the misfortunes that followed the wild rush
into the semi-arid regions of Kansas and Nebraska in the early 80's. To-day the work of the various scientific bureaus of the national Govern-
ment is devoted to the gathering of accurate information regarding the
temperature, rainfall, fertility, and salubrity of various parts of the
country. Annually a vast mass of information is collected, digested,
and published-information
which is, in its very essence, geographical
knowledge. The pupil in the upper grades of the elementary school
could and certainly should be made acquainted with the sources of this
information and trained in its interpretation. The expense which is
involved in the collecting of this data would be repaid in a generation, if
the schools would see to it that their pupils know where to get at it
and how to use it. In fact, a more intimate connection between the
Department of Agriculture and the public schools is earnestly to be
desired. In some instances a start in this direction has been made
through the medium of the State agricultural colleges, but, as yet, it
is only a start. The only possible objection that could be urged against
such a correlation would come from wild-cat land companies that
attempt to colonize unproductive regions. An examination of the
advertising pages of recent magazines will demonstrate that such com-
panies exist even to-day Downloaded by [Carnegie Mellon University] at 09:25 02 February 2015 The merchant engaged in the export trade has no longer to send
his vessels to distant shores on the chance that a market may tbere be
found for his goods. The Consular Reports published by the Govern-
ment give accurate information concerning the commercial geography
of foreign countries-what
goods are in demand, at what profit they
can be sold, what duty must be paid for their importation, what com-
modities will not find a sale, and a host of other valuable facts which
operate to reduce losses and increase profits. All this geographical
knowledge is important, from a utilitarian point of view, to many differ-
ent classes of people. It is knowledge which the merchant, the farmer,
the manufacturer, and the legislator may frequently use to their advan-
tage. And the laborer seeking a market for his labor may be just as
materially benefited by such knowledge as the manufacturer seeking THE JOURNAL OF GEOGRAPHY 226 May May a market for his products. Here, again, is a suggestion toward the
making of geography practical in the elementary school. The writer
once proposed this question to an eighth grade class that had been
exceptionally well prepared in commercial geography: The Great
Northern Railroad recently sent a representative to Asiatic Russia to
study the Trans-Siberian Railroad; what motives led the management
to take this step? A variety of answers were obtained, nearly all
showing commendable acumen of thought. These were criticised by
the class with the aid of suggestive questions, and finally the conclusion
was reached that the Great Northern directors were anxious to know
whether they could compete with Russia in supplying wheat for the
oriental market. It is obvious that such a question of commercial
geography is of vital interest not only to the stockholders of the Great
Northern Railroad but also to the entire population of the northwestern
States. Downloaded by [Carnegie Mellon University] at 09:25 02 February 2015 We have spoken so far only of the utilitarian vaIue of detailed facts
of geography. But the new geography, like all true sciences, renders
deductive processes possible. From the facts are induced great prin-
ciples which can, in turn, be applied to particular instances with rea-
sonable certainty that the conclusions will be justified by actual facts. “All knowledge,” says Professor Ostwald, “is prescience ” ; that is,
the ultimate value of knowIedge, as such, lies in the fact that with it one
can forecast the future on the basis of the past. The value of the prin-
ciples of geography, from a utilitarian standpoint, is as unmistakable
as the value of detailed geographical facts. To-day we not only know,
as a matter of direct observation, that certain regions are unsuitable
for agricultural pursuits, but, given the contour of a certain region,
given other facts of its topography, given a few hints as to its geological
history, given the prevailing winds, and its distance from the sea, we
can determine a priori its suitability for agriculture. There is, of
course, a possibility of error. Actual test may overthrow the results of
our theoretical considerations; but the chances are greatly in favor of
their validity. An interesting example of a gigantic enterprise, based
upon a priori reasoning from geographical generalizations, is furnished
by the recent exploitation of the water power of Sault Ste. Marie. All
precedents seemed to justify the assumption that a great manu-
facturing and commercial center should grow up at this point. Its
situation near the wheat fields of the Northwest, near the immense
virgin forests of Ontario, near the unrivaled deposits of iron ore in the
Lake Superior region; its facilities for water communication with the
most populous centers of the continent; its proximity to the labor market THE FUNCTIONS OF GEOGRAPHY 2 2 7 =904 -all
these conditions supplemented the extensive water power devel-
oped by the rapids of St. Marys River in warranting the rosiest pictures
of the future. A large corporation was organized for the purpose of
developing these resources. A canal was dug on the Canadian side-
cut through the solid rock-to
bring the water to the turbines a t a
convenient point. Factories were constructed on a scale hitherto
unheard of in the business world. Downloaded by [Carnegie Mellon University] at 09:25 02 February 2015 Steamer lines were operated on
the lakes, and a railroad was pushed north into the forest to bring
down pulp wood and iron ore. For a time everything went as antici-
pated, but a few months ago the entire organization collapsed. Some
important factor, no one seems to know just what, had been overlooked. Possibly the results were expected too quickly; time was not allowed
for natural development. But that the fundamental conclusion was
valid-that
a great city will sometime grow up at this point-no
one
seems to doubt, even after this disastrous failure. * H. B. George: Thx Relations of Geography and History, Oxford, 1901, p. 1. Downloaded by [Carnegie Mellon University] at 09:25 02 February 2015 Does the utilitarian value of geography justify the importance
which this subject has assumed in education? Is it of value to special
classes rather than to the average citizen? Should the study of geog-
raphy be left, in the main, to the higher institutions? Every one must
know how to read and write, how to compute, how to express himself
effectively; could not the time of the elementary school be spent more
profitably upon such subjects as these? Upon the basis of the above
considerations, and bearing in mind the fact that the majority of chil-
dren never get beyond the elementary school, these questions must
be answered in favor of geography. The utilitarian value of geography,
however, would not justify its presminence in the elementary school
to the neglect of these other branches. Nor is the utilitarian value the
only value that accrues to its study. It adds an increment, and a
large increment, to the total value of the subject, but very few disciplines
rest upon utility alone. It is only necessary here to point out that the
utilitarian value of geography is extremely important, and that our
methods of teaching must be modified in some degree by this fact. To what extent they should be modified can be determined only by
a comparison of the utilitarian value with the other values which geog-
raphy may possess. We know that the prominence of certain items of the curriculum is
justified, not by the utility of their facts and principles in actual appli-
cation to the problems of life, but rather by the condition that ignorance
of these facts and principles brands a person as uneducated, and hence
serves to militate against his maximal efficiency in society. The study of
grammar is, perhaps, the best instance of a subject of formal instruction, THE JOURNAL OF GEOGRAPHY 2 2 8 May the main value of which is conventional. A sentence that is gram-
matically incorrect may express one’s thought just as clearly as a
sentence that is grammatically correct, yet habitual use of incorrect
forms-disregard of conventional requirements-will distract the atten-
tion of one’s auditors from the thought to the form, and hence will
militate against the maximal efficiency of one’s expression. The
question now arises: In what degree will conventional requirements
justify the teaching of geography in the elementary schools? Downloaded by [Carnegie Mellon University] at 09:25 02 February 2015 Geographical knowledge is certainly “assumed” as part of the
intellectual equipment of every one who would claim for his thoughts
and opinions the consideration of the average man. The man who
does not know that the earth is round will surely be handjcapped in his
dealings with others; for, in social intercourse, men and women generalize
upon slight bases, and the man who has proved himself to be ignorant
upon so common a branch of knowledge as geography will receive scant
attention upon other matters. The elementary school owes it to the
individual to furnish him with those geographical facts and concepts
that (‘every one must know.” In this day, when “learning by heart” has
been practically banished from orthodox pedagogy, it is especially
necessary to emphasize this point. A number of facts must certainly
be memorized for this reason if for no other. In addition to the value of its facts in direct application to the
needs of life, and in addition to its conventional value, geography
has a peculiar value as a preparation for other subjects. A knowledge
of geography is especially important in the successful study of (1) his-
tory and current events, (2) literature, and (3) natural science. (1) “History is not intelligible without geography,’’ says a recent
writer.”
‘(This is obviously true in the sense that the reader of history
must learn where the frontiers of States are, where battles are fought
out, whither colonies were dispatched. It is equally if less obviously
true that geographical facts very largely influence the course of history. Even the constitutional and social developments within a settled
region are scarcely independent of them, since geographical position
affects the nature and extent of intercourse with other nations, and
therefore of the influence exerted by foreign ideas. All external relations,
hostile and peaceful, are based largely on geography, while industrial
progress depends primarily, though not exclusively, on matters described
in every geography book-the
natural products of a country and the
facilities which its structure affords for trade, both domestic and for-
eign. ” 229 THE FUNCTIONS OF GEOGRAPHY ‘904 It should not be overlooked, however, that the relation of geography
to history is, in some measure, reciprocal-that,
while geography is
essential to the understanding of history, history is sometimes no less
important in the rational study of geography. Downloaded by [Carnegie Mellon University] at 09:25 02 February 2015 It is perfectly obvious
that the significance of many geographical facts depends in no small
measure upon historical conditions. Boundaries between countries,
for example, are important geographically, yet they frequently have
no adequate geographical explanation and must be interpreted entirely
from historical bases. This point is also illustrated by the location of
certain cities, although here a geographical influence may often be traced
through historical media. For example, the capitals of the South
American republics in the Andes region (including also Venezuela) are
all situated away from the seacoast in all but inaccessible mountain
regions. The cause of this phenomenon must be sought, not directly
in geographical, but rather in historical conditions. For generations
the neighboring seas were infested with pirates, and cities on the coast
were constantly subject to pillage and sack at the hands of these out-
laws. Nevertheless, hhat this historical condition shouId have arisen
is undoubtedly due to the operation of geographical causes. Downloaded by [Carnegie Mellon University] at 09:25 02 February 2015 Admitting the reciprocal nature of geography and history, however,
it is plainly apparent that geography is the more fundamental, hence
its greater preparatory value in connection with history. The study of geography is also essential to the rational understand-
ing of “current events.”
Not to evaluate current tendencies with some
degree of intelligence is certainly not to prove oneself efficient in society. In this day when an occurrence on the other side of the globe may
immediately and directly influence the humblest citizen on this side,
the ability to read newspapers intelligently needs no elaborate argu-
ment for its defense. And the ability to read newspapers intelligently
certainly involves not a superficial but a thorough knowledge of geog-
raphy, as the contemporary happenings in the Orient abundantly
testify. (2) Geography stands in an intimate relation to the study of litera-
ture. The classics commonly read in the elementary schools-Robinson
Crusoe and Evangeline, to name only two-could
not be adequately
appreciated without a prior knowledge of geographical facts. Just
what weight should be attached to geographical study upon this ground
is necessarily indeterminate, but this factor certainly adds an increment,
and a large increment, to its total value. (2) Geography stands in an intimate relation to the study of litera-
ture. The classics commonly read in the elementary schools-Robinson
Crusoe and Evangeline, to name only two-could
not be adequately
appreciated without a prior knowledge of geographical facts. Just
what weight should be attached to geographical study upon this ground
is necessarily indeterminate, but this factor certainly adds an increment,
and a large increment, to its total value. (3) Rather more tangible is the relation of geography to the natural
sciences. As pointed out above, geography borrows many of its facts (3) Rather more tangible is the relation of geography to the natural
sciences. As pointed out above, geography borrows many of its facts THE JOURNAL OF GEOGRAPHY 2 30 May from different fields of natural science-from
geology, meteorology,
astronomy, botany, zoology, etc. In the high school and college each
of these sciences is treated in and for itself as a pure science-that
is,
without explicit reference to its economic or human relations. It is
obvious, however, that the initial study of a science should be from
the human side. The child should be introduced to facts and principles
in their relation to his life. This is what geography attempts to do. “This would seem to indicate the fallacy of President Hall’s proposals for
Cf. The Ideal School
replacing geography in the grades with elementary science.
Proceedings, N. E. A., 1901. Downloaded by [Carnegie Mellon University] at 09:25 02 February 2015 In a sense it might be looked upon as an introduction to all the sciences
of nature. It is here that the child must get that first large view which
should precede all detailed and abstract study-abstract
in the sense
of being considered apart from its human relations. Educators are
now coming to believe that the curriculum should include geography
not only as a preparation for the sciences, but also as a culmination
of all scientific study; that is, an advanced course in geography
should form the capstone of the science work in high school or college. The student should bring together the facts and principles that lie has
acquired in the detailed study of the various sciences, and discover
their relations to human life. This is only a consistent application
of the general principle that mind begins with large wholes, passes
from these to detailed parts and then back again to the wholes-
analysis followed by synthesis, differentiation followed by integration. In any case, however, we can not doubt that geography has great value
as preparatory to the study of science, and that, if the student is to
get the most from the study of science in high school or college, he
must be thoroughly grounded in geography in the elementary school. Here, however, we are speaking for the few rather than for the many. To the majority of our pupils the initial study of geography forms
the sum total of their scientific instruction. Therefore the preparatory
value of geography can not be unduly pressed as a justification for its
preeminence in the elementary school.” Downloaded by [Carnegie Mellon University] at 09:25 02 February 2015 The dividing line between practical and cultural values is indeter-
minate. What we have discussed under the head of utility is beyond
doubt “practical” in the narrowest sense of the word. But conven-
tional values become practical when we look at them from a certain
standpoint-when
we remember that conventionally valuable facts aid
one in one’s social adjustments. The preparatory values are practical
ultimately, provided that the subjects which they look forward to are
in themselves practical. The values which we have now to discuss THE FUNCTIONS OF GEOGRAPHY 2 3 1 1904 will doubtless appeal to one as ultimately practical, although with
more intermediate steps than is the case with those considered above. Because of this distinction we may class the following as cultural
values, remembering, however, that the distinction between practical
and cultural is one of degree rather than of kind. g
Like all instincts it owes its
existence to the forces of natural selection working upon fortuitous
variations in nerve structure. It has been good for man to be curious
about his environment, to study his environment, and to determine
the laws that govern it. Primitive man did not realize, probably,
that his inordinate curiosity was good for him. In his own rude way
he investigated things for the mere “fun of it”-for
the pleasure which
it afforded him. Later in his development he came to find out that
many of the facts which he discovered and many of the laws which he
worked out were “good” for him-that
the knowledge which he had
gained helped him in solving the problems of his life. But this appre-
ciation of the value of. acquisitiveness came only after a long lapse of
time. Acquisitiveness in man is an instinct. Downloaded by [Carnegie Mellon University] at 09:25 02 February 2015 The desire to satisfy curiosity is thus seen to be at the basis of
knowledge. The child evinces this desire. His curiosity is boundless,
and upon this native instinct the educator may build. It is clear,
however, that he can not trust to it entirely, for the very fact that it
is an instinct means that it runs its course in passive attention. It is
not sustained, directed, organized. All these things mean active
attention, mean work. Curiosity soon tires, but any measurable addi-
tion to knowledge involves persistent effort. It is the problem of the educator, then, to replace this instinctive
curiosity with a higher mental process. The desire to obtain knowledge
for the sake of knowledge is not to be discouraged, but it is to be held
to a definite line until results follow. Wherever possible, the child’s
curiosity should be directed along lines that will help him most in his
future adjustments. There are times, however, when this curiosity
may be directed toward ends the practical significance of which is not
once apparent, and it is these cases that we must discuss under the
head of cultural values. In the first place, some children may be curious in certain special
directions. They may evince a desire, perhaps, to learn all that they
can about Arctic exploration. The facts that they obtain from various
sources may not be applicable to the problems that they must solve
in later life, yet no sensible teacher would attempt for a moment to
curtail this interest. He has here the opportunity to replace instinctive THE JOURNAL OF GEOGIWPHY 2 3 2 May curiosity with a higher sentiment, namely, intellectual interest. This
is closely akin to other forms of sentiment, such as appreciation of art,
music, and literature. None of these is, in itself, “practical,” yet
each subserves a very practical end. Without some form of pleasure,
life would be impossible. If the higher forms of pleasure-the
senti-
ments-are
not developed, the individual will be thrown back upon
the primitive pleasures. He will follow the instincts, the lines of least
resistance. In our school work to-day we are trying to develop the
aesthetic sentiments-to
cultivate an appreciation for art, music, and
literature. We should certainly not neglect the intellectual senti-
ment-the
pleasure that comes from knowing. * The importance of “thought-connections” in recall has been demonstrated:
Cf.
The work of
time and again, by the methods of experimental psychology.
Ebbinghaus, Steffens, and Lobsien. Downloaded by [Carnegie Mellon University] at 09:25 02 February 2015 It is for this reason that
the wise teacher would never think for a moment of curtailing interest
in such a subject as Arctic exploration. The opportunity is too
valuable to be lost. With a little trouble he may lead the child to take
delight in an intellectual pursuit, just as with a little trouble he may
lead the child to see the beauty in a great picture, or a classical musical
composition, or a world epic. Downloaded by [Carnegie Mellon University] at 09:25 02 February 2015 y p
g
p
If the foregoing analysis of the aims and functions of geography
as an integral part of the elementary school curriculum is valid, it
follows that our methods of teaching must be organized with these
points in view. If possible, the various aims should be classified with
reference to their relative importance in fulfilling the general end of
education, namely, the production of the socially efficient individual. We should know with approximate accuracy just what facts and prin-
ciples are to be impressed because of their utilitarian value, what are
essential from the conventional standpoint, what from the preparatory,
and so on. In many instances the groups will, of course, cut across
one another, but it seems tolerably clear that methods of impressing
facts and developing principles will vary according to the function
which the facts and principles are to subserve. These are problems which it must be left for educational research
to solve. From the standpoint of the practitioner, at least, this suggests
a field of investigation infinitely more promising than those which con-
temporary educational experts are attempting to exploit. Downloaded by [Carnegie Mellon University] at 09:25 02 February 2015 p
p
In the second place, items of knowledge which have little or no
significance in the practical affairs of life may nevertheless be necessary
to a system of knowledge. It is a well-known fact that systematic
arrangement or organization is an extremely important factor in the
“efficient recall” of items of experience at times when they are needed.*
Very frequently in making a system of knowledge-in
arranging the
items of experience in an orderly fashion-it
is necessary to insert
many facts and principles which have in themselves little practical
value. Thus the individual may never be called upon to apply his
knowledge of the Arctic regions, but such knowledge is necessary in
order to make his world view comprehensive. Without it there would
be a distracting gap. The briefest examination of the curricula of the secondary schools
and colleges will serve to demonstrate the importance of the “cultural ”
values which we have discussed in the two preceding paragraphs. The
larger part of these curricula is made up of subjects which subserve
one or the other of these two functions: tending either to develop
intellectual interests or to make more comprehensive and complete
the body of knowledge. The science, the mathematics, the language
and literature, which occupy so prominent a place in the higher education, THE FUNCTIONS OF GEOGRAPHY 233 1904 can be justified only upon these grounds. In the elementary school,
on the other hand, the cultural values are not so prominent. The
bulk of the time is given over to the study of arithmetic and language,
the latter including reading, writing, composition, and grammar. Literature and geography divide most of the remaining time between
them. Arithmetic and language are justified principally because of
their utilitarian values. Literature is prominent chiefly because of its
conventional and cultural values. Geography might be said to occupy
a midway position, being important from all sides. GEOGRAPHICAL NOTES Wind-Blown Trees.-The
communication on Wind Effects, by Prof. M. S. W. Jefferson, in your January number, interests me much, as it
concerns a subject to which I have given some attention for several
years. Observations of the kind to which your contributor refers are
easily made, and add much to the interest of the study of meteorol-
ogy, because they give it life. If any of your readers cares to pursue
this subject further, he will find an instructive discussion of it in a recent
paper by Prof. J. Friih, entitled Die Abbildung der vorherrschenden
Winde (lurch die Pflanzeniuelt (Jahresber, Geogr. Ethnogr. Gesells., Ziirich,
1901-02,97 pp.). In this study Professor Frah classifies the effects
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https://openalex.org/W4365935322
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https://jurnalsosiologi.fisip.unila.ac.id/index.php/jurnal/article/download/352/134
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Indonesian
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PARADIPLOMASI LAMPUNG: Harapan dan Realitas
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Sosiologi
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cc-by
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ABSTRACT The Lampung provincial government, in accordance with Lampung Province regulation
No. 14 of 2013 concerning regional cooperation, can establish foreign cooperation. Provincial involvement in this international dimension has also been supported by the
foreign cooperation guidelines issued by the Ministry of Foreign Affairs of Indonesia in
2006 and the Ministry of Home Affairs in 2008. Although the legal and technical
provisions for implementation already exist, exploration of how regional foreign
cooperation decisions are designed and taking into account the global-regional
intersection dimension is an important matter that must be understood. Using a
descriptive qualitative approach with interviews and literature methods, the primary and
secondary data obtained were analyzed with the framework of the concept of regional
foreign cooperation and paradiplomacy. The study shows that the initiation of
paradiplomacy at the regional level has not been designed with the same understanding of
the international and domestic dimensions. Although pioneering foreign cooperation has
been carried out, the absence of policy integration has made the sustainability of these
initiatives not very successful. Key Words: Paradiplomacy, International Regional Cooperation, Province of Lampung ABSTRAK Pemerintah provinsi Lampung, sesuai dengan peraturan Provinsi Lampung No.14 Tahun
2013 mengenai kerjasama daerah, dapat menjalin kerjasama luar negeri. Keterlibatan
provinsi dalam dimensi internasional ini juga sudah didukung oleh panduan kerjasama
luar negeri yang dikeluarkan Kementerian Luar Negeri di tahun 2006 dan Kementerian
Dalam Negeri di tahun 2008. Meskipun ketentuan hukum dan teknis pelaksanaan sudah
ada namun eksplorasi bagaimana keputusan kerjasama luar negeri daerah dirancang dan
mempertimbangkan dimensi interseksi global-daerah merupakan hal penting yang harus
dipahami. Penggunaan pendekatan kualitatif deskriptif menjadi pilihan dengan metode
wawancara dan literatur, data primer dan sekunder yang didapat dianalisis dengan
kerangka konsep kerjasama luar negeri daerah dan paradiplomasi. Hasil penelitian
menyajikan bahwa inisiasi paradiplomasi dilevel daerah belum dirancang dengan
pemahaman yang sama mengenai dimensi internasional dan domestik. Meskipun sudah
dilakukan rintisan kerjasama luar negeri namun tidak adanya integrasi kebijakan
membuat keberlanjutan inisiasi tersebut tidak banyak berjalan. Kata Kunci: Paradiplomasi, Kerjasama Luar Negeri Daerah, Provinsi Lampung PARADIPLOMASI LAMPUNG: Harapan dan Realitas Hasbi Sidik1)
1) Jurusan Hubungan Internasional Fakultas Ilmu Sosial dan Ilmu Politik Universitas Lampung SOSIOLOGI: Jurnal Ilmiah Kajian Ilmu Sosial dan Budaya, Vol. 21, No. 2 September 2019: 201-216 SOSIOLOGI: Jurnal Ilmiah Kajian Ilmu Sosial dan Budaya, Vol. 21, No. 2 September 2019: 201-216 PENDAHULUAN Otonomi daerah dan desentralisasi memberi peluang atau kesempatan bagi
pemerintah daerah di Indonesia untuk secara aktif membuat perancangan
kebijakan dan kegiatan internasional yang mendukung pertumbuhan daerah. Situasi ini bertolak belakang dengan konteks dan peluang pada era sebelum
reformasi. Lebih jauh, pemerintah daerah juga seringkali dipersepsikan sebagai
aktor yang cenderung pasif, korban kebijakan desentralisasi dan menjadi korban
persinggungan arus globalisasi serta persaingan antar-negara (Simanjuntak, 2015). Disisi lain, konsekuensi perkembangan kewenangan hubungan luar negeri
pemerintah daerah ini juga seharusnya mendorong pemerintah daerah untuk
mempertimbangkan konteks luar negeri dalam formulasi kerangka kebijakan
kerjasama daerahnya. Selanjutnya, realitas model kebijakan kerjasama pemerintah daerah yang
dipergunakan selama ini cenderung membahas aspek domestik tanpa
menghubungankan dengan konteks internasional. Hal ini menyebabkan kebijakan
pembangunan daerah khususnya dalam hal keterlibatan mitra dan bentuk
kerjasama masih berorientasi ke dalam negeri. Dinamika internasional,
kemunculan kesepakatan Masyarakat Ekonomi ASEAN di kawasan Asia
Tenggara serta percepatan perkembangan informasi dan teknologi menuntut aktor
pemerintah daerah untuk berinovasi dan tanggap mengambil keuntungan dari
kondisi saat ini. Pemerintah Provinsi Lampung melalui peraturan nomor 14 tahun 2013
mengenai kerjasama daerah telah menyatakan mengenai adanya peluang
kerjasama provinsi kembar (sister province) dengan negara lain. Selain itu,
regulasi ini juga menyajikan peluang pemerintah daerah untuk dapat melakukan
kerjasama dengan badan dan lembaga/organisasi internasional; Lembaga Swadaya
Masyarakat internasional serta BUMN/Negara Bagian/daerah dan pihak ketiga. Kerjasama lainnya adalah kerjasama teknik, kebudayaan, sosial dan sebagainya. Selanjutnya, kerjasama ini memungkinkan dilakukan oleh semua Satuan Kerja
Perangkat Daerah (SKPD) di bawah naungan Tim Kerja Kerjasama Daerah
(TKKD). Penelitian mengenai keterlibatan pemerintah daerah di Indonesia dalam
konteks internasional atau dikenal dengan terminologi paradiplomasi telah banyak
dilakukan. Beberapa peneliti mengeksplorasi paradiplomasi sebagai strategi
pembangunan, perdagangan dan pariwisata Indonesia, (Fathun, 2016; Fhadly et
al., 2018; Harakan, 2018; Isnarti & Fhadly, 2018), pemanfaatan teknologi informasi sebagai pendukung kebijakan internasional daerah dan kota pintar
(Adibowo & Putri, 2016; Fathun, 2016), Bentuk sister-city (Anggraini, 2017;
Irdayanti, 2015) serta penguatan komunitas ASEAN (Damayanti, 2012). Meskipun
begitu
sedikit
penelitian
paradiplomasi
di
Indonesia
yang
mengeksplorasi tantangan provinsi dalam inisiasi paradiplomasi. Penelitian ini
kemudian bertujuan mengeksplorasi bagaimana persinggungan dimensi domestik
dan internasional dalam perancangan kegiatan luar negeri daerah, khususnya
provinsi Lampung. SOSIOLOGI: Jurnal Ilmiah Kajian Ilmu Sosial dan Budaya, Vol. 21, No. 2 September 2019: 201-216 Paradiplomasi Konsep adanya hubungan-hubungan, koalisi dan interaksi lintas batas
negara yang tidak dikontrol oleh badan luar negeri pemerintah pusat telah di kenal
sebagai hubungan transnasional atau transnational relations (Nye & Keohane,
1971). Konsep ini kemudian menjadi pemicu sejumlah pemerhati kajian hubungan
internasional ketika mengeksplorasi peran aktor non-negara dan keterlibatannya
dalam isu-isu hubungan internasional. Beberapa aktor tersebut diantaranya adalah
korporasi, organisasi non-pemerintah, komunitas dan individu dan hal ini tidak
bisa lepas dari semakin meningkatnya jumlah, ukuran, dan ragam aktivitas aktor-
aktor tersebut. Disisi lain, kajian keterlibatan subnasional atau daerah dalam
hubungan internasional yang dikenal dengan term paradiplomasi telah dimulai
pada tahun 1970an. Diperkenalkan oleh Soldatos (Michelmann & Soldatos, 1990),
paradiplomasi merujuk pada aktor baru negara dibawah pemerintah pusat yaitu
pemerintah daerah. Selanjutnya, perbedaan paling jelas antara transnasionalisme
dan paradiplomasi diletakkan pada subjek aktor yang terlibat dalam melakukan
hubungan internasional. Selama perkembangannya, konsep paradiplomasi memiliki rentang
perdebatan akademis yang cukup panjang. Paradiplomasi sebagai kontraksi
Parallel diplomacy / Diplomasi paralel (Michelmann & Soldatos, 1990) dikritik
sebagai kata yang tidak memiliki definisi yang jelas baik batasan-batasan maupun
dasar penggunaanya meskipun sangat sukses dalam literature (Aguirre, 1999). Kritik lainnya melihat paradiplomasi sebagai konsep yang melemahkan gagasan penguatan pemerintah federal karena kata paralel dalam konsep paradiplomasi
hanya mendukung gagasan kepentingan pemerintah pusat atau turunan diplomasi
aktor negara. Selanjutnya, constituent paradiplomacy dianggap lebih tepat dalam
menggambarkan partisipasi pemerintah daerah dalam hubungan luar negeri
(Kincaid, 1990). Hal ini dikarenakan aktivitas diplomasi pemerintah daerah
dikonstruksi dari suara atau keinginan pemilih/konstituen di sebuah daerah. Term
keterlibatan daerah dalam hubungan internasional juga dituangkan dalam konsep
multilayered-diplomacy. Sebuah konsep yang mengusulkan proposisi bahwa
pemerintah daerah juga memiliki kapasitas dan ragam peran dalam sebuah proses
negosiasi. Ide ini juga menyatakan bahwa memungkinkan jika pemerintah daerah
berseberangan dengan tujuan nasional namun juga bisa menjadi aliansi ataupun
agen dalam mencapai kepentingan negara (Hocking, 1993). Selanjutnya,
kemunculan praktik paradilomasi didorong oleh 2 hal utama yaitu faktor internal
yaitu dalam negeri serta dimensi eksternal yaitu luar negeri (Kuznetsov, 2014). Dimensi eksternal adalah faktor-faktor yang distimulus oleh hal-hal yang berasal
dari luar sebuah negara diantaranya globalisasi, regionalisasi, demokratisasi,
domestifikasi kebijakan luar negeri dan internasionalisasi kebijakan domestik. Disisi lain faktor internal adalah hal-hal yang diasumsikan berasal dari dalam
sebuah negara yaitu federalisasi dan desentralisasi, proses nation-building,
minimnya efektivitas hubungan luar negeri pemerintah pusat, ketidaksamaan
rerata Pendapatan Domestik Bruto (PDB), stimulus wilayah, peran pemimpin
daerah atau partai politik dan peran di perbatasan. SOSIOLOGI: Jurnal Ilmiah Kajian Ilmu Sosial dan Budaya, Vol. 21, No. 2 September 2019: 201-216 Kerjasama Luar Negeri Daerah Melalui Undang-undang No.22 Tahun 1999 ditegaskan bahwa daerah
memungkinkan melakukan kerjasama namun dengan kewenangan tidak wajib. Lebih jauh, pasal 88 ayat 1 menegaskan bahwa kerjasama menguntungkan bisa
dilakukan oleh daerah dengan pihak luar negeri hanya berdasarkan keputusan
bersama. Penguatan hubungan luar negeri daerah ini kembali ditegaskan dalam
UU No.32 Tahun 2004 dalam pasal 24 ayat 1 bahwa persetujuan kerjasama luar
pemerintah daerah juga bagian dari tugas dan kewenangan DPRD. Selanjutnya
juga dinyatakan bahwa bentuk kerjasama memungkinkan dalam hal teknik, kemanusiaan, pinjaman/hibah, pemodalan dan bentuk kerjasama lainnya dengan
merujuk ketentuan yang berlaku. Lebih jauh, untuk memperkuat UU No.32 Tahun 2004 tersebut, juga
disediakan panduan kerjasama internasional yang dikeluarkan Kementerian Luar
Negeri dan Kementerian Dalam Negeri. Peraturan Menteri Luar Negeri RI
No.09/A/KP/XII/2006/01 bertujuan memberi arah dan memfasilitasi daerah untuk
pembangunan. Selain itu peraturan ini juga kendali pemerintah pusat untuk
kebijakan ‘one door policy’ Indonesia dan bagian dari upaya pencegahan
terjadinya masalah pelaksanaan kerjasama luar negeri (Kementerian Luar Negeri,
2006). Sedangkan Kementerian Dalam Negeri melalui Peraturan Menteri Dalam
Negeri No.3 Tahun 2008 memuat Pedoman Pelaksanaan Kerjasama Pemerintah
Daerah dengan Pihak Luar Negeri yaitu prinsip-prinsip kerjasama, bentuk
kerjasama, perselisihan hingga pelaporan kerjasama (KEMENDAGRI, 2008). METODE Penelitian ini adalah penelitian dengan pendekatan kualitatif deskriptif. Logika induktif dipilih dengan mendeskripsikan ragam regulasi provinsi Lampung
dalam kerjasama luar negeri serta irisannya dengan konteks luar negeri. Langkah
berikutnya adalah mengidentifikasi aspek domestik yang menjadi landasan dalam
arah perubahan kebijakan khususnya kerjasama internasional. Lebih jauh, metode
atau instrumen riset yang digunakan adalah wawancara, observasi dan literature. Selanjutnya analisis data dilakukan melalui kondensasi yaitu pemadatan
keseluruhan data terkumpul dengan merujuk pada tujuan penelitian. Lebih jauh,
sajian data dilakukan dengan melakukan koding berdasarkan kategori yang dibuat
dan terakhir kesimpulan ditarik berdasarkan interpretasi atas keterhubungan data-
data tersebut dengan konsep paradiplomasi dan kerjasama luar negeri daerah
(Creswell & Creswell, 2018; Neuman, 2014). SOSIOLOGI: Jurnal Ilmiah Kajian Ilmu Sosial dan Budaya, Vol. 21, No. 2 September 2019: 201-216 Perancangan Kerjasama Luar Negeri Pemprov Lampun Perancangan Kerjasama Luar Negeri Pemprov Lampung Upaya pemerintah provinsi Lampung untuk kerjasama luar negeri diawali
dengan Pembentukan Tim Fasilitasi dan koordinasi Pelaksanaan Kerjasama Luar
Negeri dengan Pihak Ketiga dan Lembaga Internasional Tahun 2009. Dasar pembentukan ini adalah Keputusan Gubernur Lampung No: G/160/BII/HK/ 2009. Adapun pemaknaan kerjasama daerah menurut Perda Lampung No.14 tahun 2013
adalah kesepakatan antara gubernur/walikota/bupati dengan pejabat pemerintah
lainnya (gubernur/walikota/bupati) atau dengan ekstenal atau pihak ketiga. Selanjutnya pihak luar negeri yang dimaksud adalah pemerintah negara
bagian/daerah di luar Indonesia, lembaga-lembaga internasional, organisasi
internasional, serta Badan Usaha Milik Negara atau daerah luar negeri. Pada tahun 2015, terbentuk Tim Koordinasi Kerja Sama Daerah (TKKSD)
Lampung sebagai upaya teknis dan operasional kesiapan melakukan kerja sama. Adapun mitra kerjasamanya adalah pemerintah daerah lainnya, pihak swasta,
BUMN dan BUMD, serta ragam mitra lainnya. APW (Kepala Bagian Pejabat
Negara dan Kerjasama Biro Pemerintah dan Otonomi Daerah) pada wawancara
tanggal 10 Oktober 2018 di ruang kerjanya menjelaskan bahwa TKKSD bertugas
menginventarisir, memetakan potensi dan prioritas objek kerjasama, evaluasi
proposal kelayakan, persiapan materi kesepakatan, rekomendasi penandatangan
dan perjanjian serta pengawasan pelaksanaan kerjasama daerah kabupaten/kota. Keanggotan pada TKKSD terdiri dari 2 kategori anggota, yaitu anggota tetap dan
tidak tetap. Anggota tetap adalah Kepala Satuan Kerja Perangkat Daerah (SKPD)
bidang pemerintahan, keuangan dan pengelolaan asset. Sedangkan anggota tidak
tetap terdiri dari Kepala SKPD pelaksana kerjasama, Kepala SKPD terkait
kegiatan kerjasama dan tenaga ahli/pakar. “Pembentukan TKKSD diharapkan dapat memacu kinerja proses-proses
pembangunan di Provinsi Lampung dan mewujudkan percepatan
keberhasilan Lampung yang maju dan sejahtera.” (APW, Kepala Bagian
Pejabat Negara dan Kerjasama Biro Pemerintah dan Otonomi Daerah. Wawancara pada 10 Oktober 2018 di ruang kerjanya). “Pembentukan TKKSD diharapkan dapat memacu kinerja proses-proses
pembangunan di Provinsi Lampung dan mewujudkan percepatan
keberhasilan Lampung yang maju dan sejahtera.” (APW, Kepala Bagian
Pejabat Negara dan Kerjasama Biro Pemerintah dan Otonomi Daerah. Wawancara pada 10 Oktober 2018 di ruang kerjanya). SOSIOLOGI: Jurnal Ilmiah Kajian Ilmu Sosial dan Budaya, Vol. 21, No. 2 September 2019: 201-216 SOSIOLOGI: Jurnal Ilmiah Kajian Ilmu Sosial dan Budaya, Vol. 21, No. 2 September 2019: 201-216 Dinamika Inisiasi Paradiplomasi Provinsi Lampung Saat penelitian ini dilakukan tahun 2018, peneliti mendapatkan penjelasan
bahwa secara teknis kerjasama dapat dilakukan, berkoordinasi dengan Kepala Saat penelitian ini dilakukan tahun 2018, peneliti mendapatkan penjelasan
bahwa secara teknis kerjasama dapat dilakukan, berkoordinasi dengan Kepala
Bagian Pejabat Negara dan Kerjasama Biro Pemerintah dan Otonomi Daerah. Bagian Pejabat Negara dan Kerjasama Biro Pemerintah dan Otonomi Daerah. “Saat ini ide kerjasama dengan luar negeri seperti sister city dapat
disampaikan dari masing-masing SKPD kepada Kepala Bagian Pejabat
Negara dan Kerjasama Biro Pemerintah dan Otonomi Daerah. Sehingga
ide atau usulan tersebut dapat diteruskan ke pemerintah pusat. Pemerintah pusat
dapat
meneruskannya
dengan
mencarikan
peluang-peluang
kerjasama dengan negara-negara lain. Setelah kerjasama di level G to G
telah dibangun, kerjasama dapat dilanjutkan di level B to B.” (APW,
Kepala Bagian Pejabat Negara dan Kerjasama Biro Pemerintah dan
Otonomi Daerah. Wawancara pada 10 Oktober 2018 di ruang kerjanya). Tim Koordinasi Kerja Sama Daerah (TKKSD) Provinsi Lampung sudah terbentuk
tetapi tidak optimal dalam implementasi perda kerjasama daerah dan SK Tim
TKKSD. Tim Koordinasi Kerja Sama Daerah (TKKSD) Provinsi Lampung sudah terbentuk
tetapi tidak optimal dalam implementasi perda kerjasama daerah dan SK Tim
TKKSD. Tim Koordinasi Kerja Sama Daerah (TKKSD) Provinsi Lampung sudah terbentuk
tetapi tidak optimal dalam implementasi perda kerjasama daerah dan SK Tim
TKKSD. TKKSD. “TKKSD yang dibentuk tidak berjalan karena belum dianggap “urgent”,
masih ada hal lain yang prioritas. Inisiatif itu baru sedikit, karena berkaca
dengan sebelumnya, sudah penjajakan dan biaya banyak masih gagal. Kerjasama antarpemprov pun tidak jadi tentunya yang beda negara lebih
sulit.” (APW, Kepala Bagian Pejabat Negara dan Kerjasama Biro
Pemerintah dan Otonomi Daerah. Wawancara pada 10 Oktober 2018 di
ruang kerjanya). Menurut APW, muncul keraguan pada sebagian aparatur bahwa kerjasama
daerah akan berhasil. Kendala lainnya adalah perubahan struktur birokrasi/pejabat
setiap pergantian gubernur, dan anggaran. Pada tahun 2018 telah diusulkan pada
RAPBD tentang anggaran pelaksanaan kerjasama luar negeri, namun oleh DPRD
Provinsi Lampung dihapuskan. Inisiatif dari SKPD untuk mengajukan usul apa
saja potensi kerjasama masih minim. Demikian juga pada kerjasama level
kota/kabupaten,
kurangnya
koordinasi
antara
pemerintah
pemerintah
kota/kabupaten dengan provinsi. Lebih jauh, juga terjadi ide atau usulan
kerjasama di kabupaten/kota, hanya dilaksanakan oleh kabupaten itu sendiri, tanpa
berkoordinasi dengan pemerintah provinsi. Selanjutnya, APW juga menjelaskan
pemerintah daerah belum memiliki data base/referensi negara tujuan kerjasama. Sosialisasi mengenai prosedur kerjasama luar negeri masih minim, belum
dipahami oleh SKPD terkait. Penjajakan kerjasama antarpemerintah provinsi dengan pemerintah provinsi
negara lain telah dilakukan, namun menemukan sejumlah kegagalan sehingga
tidak sampai kepada LOI maupun MOU. Penjajakan tersebut diantaranya: 1. Sejak tahun 2015, Badan Penelitian dan Pengembangan Daerah Provinsi
Lampung telah melakukan dialog dan sharing dengan National University
Singapore dan lembaga di Jepang mengenai daya saing provinsi Lampung. 1. Sejak tahun 2015, Badan Penelitian dan Pengembangan Daerah Provinsi
Lampung telah melakukan dialog dan sharing dengan National University Singapore dan lembaga di Jepang mengenai daya saing provinsi Lampung. Ini menemukan kegagalan, karena keterbatasan anggaran (National
University of Singapore (NUS), 2018.) 2. Kerjasama sister province dengan Al Jazair (sister province Bouira). Menurut
APW kegagalan kerjasama g to g, karena perbedaan sistem hukum yang
berlaku di kedua pihak pemerintah daerah, dan anggaran provinsi yang
terbatas saat delegasi provinsi diminta hadir di Bouira, Aljazair (Lampung
Menjalin Kerja Sama Dengan Provinsi Bouira Aljazair - ANTARA News
Megapolitan, 2017.) 3. Upaya menjalin kerjasama dengan Australia untuk mengirimkan indukan sapi
ke Provinsi Lampung gagal. Menurut Argo Prasety Widi karena adanya
aturan pemerintah Australia yang melarang untuk mengirimkan indukan sapi
ke Lampung. 3. Upaya menjalin kerjasama dengan Australia untuk mengirimkan indukan sapi
ke Provinsi Lampung gagal. Menurut Argo Prasety Widi karena adanya
aturan pemerintah Australia yang melarang untuk mengirimkan indukan sapi
ke Lampung. 4. Penjajakan kerjasama antara provinsi Lampung dan Provinsi Los Rios,
Ekuador. SOSIOLOGI: Jurnal Ilmiah Kajian Ilmu Sosial dan Budaya, Vol. 21, No. 2 September 2019: 201-216 TKKSD. Tujuan
penjajakan
kerjasama
adalah
terjadinya
transfer
pengetahuan dan pembangunan kapasitas tiap provinsi di sektor perkebunan. Langkah ini juga berpeluang menyediakan target pasar pertanian dan
perkebunan yang lebih luas bagi provinsi Lampung. Adapun penjajakan
kerjasama ini dilakukan oleh Dubes RI untuk Ekuador Diennaryati
Tjokrosuprihatono pada saat melakukan kunjungan kerja dengan Gubernur
Lampung M. Ridho Ficardo pada 16 Mei 2017. Pembahasan juga
menyepakati kedatangan langsung investor Ekuador untuk melihat langsung
sektor pertanian dan perkebunan di provinsi Lampung (Lampung, 2017.) 4. Penjajakan kerjasama antara provinsi Lampung dan Provinsi Los Rios,
Ekuador. Tujuan
penjajakan
kerjasama
adalah
terjadinya
transfer
pengetahuan dan pembangunan kapasitas tiap provinsi di sektor perkebunan. Langkah ini juga berpeluang menyediakan target pasar pertanian dan
perkebunan yang lebih luas bagi provinsi Lampung. Adapun penjajakan
kerjasama ini dilakukan oleh Dubes RI untuk Ekuador Diennaryati
Tjokrosuprihatono pada saat melakukan kunjungan kerja dengan Gubernur
Lampung M. Ridho Ficardo pada 16 Mei 2017. Pembahasan juga
menyepakati kedatangan langsung investor Ekuador untuk melihat langsung
sektor pertanian dan perkebunan di provinsi Lampung (Lampung, 2017.) 5. Direktorat Eropa Tengah dan Timur, Kementerian Luar Negeri bekerja sama
dengan Pemprov Lampung di Lampung melakukan kegiatan “Temu Bisnis”
peningkatan ekspor Lampung. Acara ini dihadiri oleh wakil dari Kedutaan
Besar Serbia, Mr. Vukasin Todorovic dan Hongaria, Peter Farvi, kedua
pejabat negara juga menyampaikan sejumlah peluang dan potensi negara
mereka serta mengharapkan bentuk kerjasama yang lebih erat pada masa
mendatang (4 Produk Asal Lampung Berpeluang Masuk Eropa Tengah Dan
Timur, 2016.) 6. Delegasi Direktorat Amerika II Kementerian Luar Negeri RI melakukan
sejumlah kunjungan untuk penjajakan peluang kerjasama antara Indonesia khususnya Provinsi Lampung dengan Negara Amerika Latin seperti
Argentina, Brazil, dan Meksiko. Potensi yang dijajaki adalah ekspor hasil
pertanian dan pariwisata (Home et al., 2018.) 7. Duta Besar Luar Biasa dan Berkuasa Penuh Republik Indonesia Untuk
Republik Kroasia, Sjachroedin ZP melakukan kunjungan ke Pemerintah
provinsi Lampung pada 7 Februari. Ditegaskan juga bahwa ada potensi
kerjasama yang bisa dilakukan Lampung dan Kroasia khususnya komoditi
kakao dan kopi (Terkini, 2018) 8. Direktorat Timur Tengah, Kemenlu RI bekerjasama dengan Pemprov
Lampung menyelenggarakan acara “Familiarization Trip for Ambassadors of
Middle East and North African Countries". Kegiatan ini berupa kunjungan
Duta Besar negara-negara Timur Tengah dan Afrika Utara ke Provinsi
Lampung, pada 5-7 Maret 2018 (Tingkatkan Kerja Sama Antar Kawasan
Melalui Kunjungan Duta Besar Negara Timur Tengah Dan Afrika Utara Ke
Lampung — Nusakini, 2018.) 9. SOSIOLOGI: Jurnal Ilmiah Kajian Ilmu Sosial dan Budaya, Vol. 21, No. 2 September 2019: 201-216 TKKSD. Konsulat Jenderal Tiongkok di Medan Zhu Honghai merekomendasikan
Lampung kepada perusahaan-perusahaan di Tiongkok untuk berinvestasi di
bidang energi dan pelabuhan disebabkan potensi Lampung dalam
kepemilikan sumber daya (Home et al., 2016). Ragam penjajakan kerjasama luar negeri daerah diatas, menurut APW
belum menghasilkan MOU bahkan LOI yang berisi kesepakatan kerjasama antar-
pemerintah daerah. “Pemerintah provinsi belum memiliki kerjasama government to
government. Kerjasama yang terbangun baru sekedar berupa pelayanan
melalui Dinas Penanaman Modal Daerah berupa perijinan. Kerjasama luar
negeri yang sudah optimal adalah business to business, yang semata
bertujuan ekonomi.” (APW, Kepala Bagian Pejabat Negara dan Kerjasama
Biro Pemerintah dan Otonomi Daerah. Wawancara pada 10 Oktober 2018
di ruang kerjanya). Selain itu juga ditambahkan bahwa pemerintah daerah juga memiliki
tanggung jawab untuk menjadi fasilitator untuk kerja sama atau pelaksanaan
program-program yang mendukung pembangunan daerah. Kerjasama yang patut
dibentuk adalah bentuk kerjasama government-to-government (G to G). Kerjasama tersebut dapat saja antara provinsi Lampung dan provinsi lain di negara lain (sister province). Namun, provinsi Lampung belum secara optimal
melakukan kerja sama internasional. Peluang yang ada, masih berupa inisiasi yang
berbasis business-to-business, bukan berdasarkan inisiasi pemerintah (FD, Kabid
Perencanaan Pemerintahan dan Pembangunan Manusia, Bappeda Pemprov
Lampung. Wawancara pada 14 Oktober 2018). DM, Kepala Sub Bidang Bahan Promosi Dinas Penanaman Modal dan
Pelayanan Terpadu Satu Pintu Pemprov Lampung mengakui terdapat beberapa
kelemahan investasi asing di Lampung seperti ketidaksiapan lahan, birokrasi, dan
peraturan One Single Submission. Birokrasi, persyaratan administrasi yang harus
dilengkapi oleh investor cukup banyak dan terkadang dikeluhkan oleh para
investor. Biasanya para investor membutuhkan waktu berbulan-bulan untuk
melengkapi persyaratan administratif tersebut. Peraturan One Single Submission
(OSS) adalah pelayanan perizinan penanaman modal secara online. Penggunaan
OSS juga mengalami kendala karena hanya 4 sektor perizinan yang dilayani yaitu
Energi dan SDM, Pekerjaan Umum dan Perumahan Rakyat, Keuangan dan sektor
BKPM. Disisi lain sejumlah jenis usaha tidak diakomodir dalam OSS. Oleh
karena itu, banyak para investor merasa kesulitan sehingga harus terlebih dahulu
mengajukan jenis usaha kepada Kemenko Bidang Perekonomian. Pemindahan
izin dari DPMPTSP kepada Kemenko Bidang Perekonomian banyak menuai pro
dan kontra. Sehingga, OSS belum maksimal dijalankan, bahkan cenderung
membuat birokrasi lebih panjang sehingga membutuhkan waktu lama untuk
menyelesaikan persyaratan bagi investor asing. Secara umum, kerjasama yang dijalin oleh Pemerintah Lampung belum
berwujud formal cooperation. Jika melihat dari uraian upaya pembangunan
kerjasama dengan luar negeri di atas, pemerintah Lampung kesulitan mewujudkan
formal cooperation, pihak luar negeri seringkali mengharuskan kunjungan
langsung ke negara mereka. TKKSD. Berbagai masalah mulai dari birokrasi, waktu dan
masalah aturan, kemudian berujung pada masalah cost dan anggaran. SOSIOLOGI: Jurnal Ilmiah Kajian Ilmu Sosial dan Budaya, Vol. 21, No. 2 September 2019: 201-216 SOSIOLOGI: Jurnal Ilmiah Kajian Ilmu Sosial dan Budaya, Vol. 21, No. 2 September 2019: 201-216 Daya Dukung Paradiplomasi Provinsi Lampung DM, Kepala Sub Bidang Bahan Promosi Dinas Penanaman Modal dan
Pelayanan Terpadu Satu Pintu Pemprov menjelaskan bahwa Lampung memiliki
potensi yang cukup baik bagi investasi asing terutama bidang perkebunan. Pada tahun 2018, Tempo Inti Media bersama Frontier Consulting Grup memberikan
penghargaan Indonesia Attractiveness Award bagi Provinsi Lampung sebagai
provinsi potensial untuk berinvestasi. Selain itu, Lampung juga memiliki kondisi
daerah yang cukup stabil. Hal tersebut dibuktikan dengan diberikannya
penghargaan Penanganan Konflik Sosial Provinsi untuk Provinsi Lampung selama
tiga kali berturut-turut. Kondisi yang relatif stabil di wilayah Lampung ini dapat
membentuk iklim investasi yang baik sehingga menguntungkan bagi investor dan
masyarakat sekitar (wawancara pada 10 Oktober 2018). Sejumlah potensi sumber daya energi juga telah teridentifikasi mulai dari
panas bumi sampai bio mass namun belum ada investasi langsung yang
mengeksplorasi hal tersebut. Beberapa potensi tersebut diantaranya energi
geothermal mulai dari Way Umpu kecamatan Banjit hingga Fajar Bulan Lampung
Barat. Potensi ini juga dimiliki beberapa daerah lainnya yaitu Lampung Selatan
didaerah Natar, Padang Cermin dan Gunung Rajabasa. Dari sejumlah lokasi
tersebut, provinsi Lampung diasumsikan memiliki potensi energi panas mencapai
2900 Mw. (Lampung Punya Potensi Panas Bumi Cukup Tinggi - ANTARA News
Lampung, 2013) Pemerintah provinsi melalui Dinas Penanaman Modal Daerah berhasil
berpromosi sehingga mampu meningkatkan nilai investasi, termasuk kegiatan
ekspor dan impor. Pertumbuhan ekonomi Lampung triwulan II 2018 yaitu 5,35
%, didukung oleh kuatnya konsumsi rumah tangga, perbaikan net ekspor, dan
investasi yang cukup solid, bidang industri pengolahan, bidang konstruksi,
perdagangan eceran, dan reparasi kendaraan, melampaui Sumatera dan nasional,
yaitu 5,08% dan 5,27%. Angkatan kerja Lampung terus meningkat per Februari
2018 4,40 juta orang. Pangsa investasi dalam laporan triwulan dominan ditopang
oleh kegiatan penanaman modal asing (PMA) sebesar US$ 37,91 juta atau sebesar
59,72% dari total investasi PMA dan PMDN (Kanwil Dirjen Perbendaharaan
Lampung, 2018) Pada tahun 2018, Tempo Inti Media bersama Frontier Consulting Grup
memberikan penghargaan Indonesia Attractiveness Award bagi Provinsi
Lampung sebagai provinsi potensial untuk berinvestasi. Lampung juga memiliki
kondisi daerah yang stabil. Hal tersebut dibuktikan dengan diberikannya penghargaan Penanganan Konflik Sosial Provinsi untuk Provinsi Lampung selama
tiga kali berturut-turut (Ini Deretan Daerah Tempat Investasi Terbaik 2018 -
Nasional Tempo.Co, 2018.) Lebih jauh, negara importir utama Provinsi Lampung
adalah Tiongkok, India dan Amerika Serikat. Sementara itu, Lampung menjadi
importir dari negara-negara seperti Thailand, Tiongkok, Amerika Serikat dan
Australia. Daya Dukung Paradiplomasi Provinsi Lampung Produk gula rafinasi sebagai bahan input industri makanan dan
minuman di impor dari Thailand sedangkan impor dari Australia didominasi
adalah sapi bakalan untuk industri feedloter di Provinsi Lampung. Disisi aspek dukungan dari masyarakat, Lampung memiliki modal sosial
dan kultural yang luar biasa. Dari sisi jejaring di masyarakat, terdapat beberapa
Lembaga Swadaya Masyarkat (LSM) yang telah menjalankan program dengan
sejumlah Non-Governmental Organization seperti WWF, Green Peace, SNV dan
lainnya. Selain itu, pada tahun 2018 Provinsi Lampung juga telah bergabung
dalam United Cities and Local Government Asia Pacific (UCLG ASPAC)
Congress (terakhir adalah ke-7 pada 12-15 September 2018 di Surabaya). Provinsi
Lampung juga tergabung dalam kerangka kerja sama Indonesia Malaysia
Thailand Growth Triangle. SOSIOLOGI: Jurnal Ilmiah Kajian Ilmu Sosial dan Budaya, Vol. 21, No. 2 September 2019: 201-216 KESIMPULAN Inisiasi paradiplomasi provinsi Lampung dominan didorong regulasi yang
sudah dikeluarkan pemerintah pusat dan proses interaksi pejabat eksekutif daerah. Disisi lain, masih sangat minim stimulan muncul dari dimensi domestik. Selanjutnya perancangan kerjasama luar negeri daerah yang cenderung bersifat
elitis dan formalistik membuat isu dan topik usulan kerjasama tidak sepenuhnya
mewakili kebutuhan kerjasama luar negeri daerah Meskipun ada ketersediaan aturan hukum dan kelembagaan kerjasama
daerah, proses pelaksanaan kerjasama luar negeri daerah tidak selalu berjalan
lancar. Kompleksitas perancangan hingga pelaksanaan membuat inisiasi
kerjasama internasional jarang berlanjut pada nota kesepahaman Letter of
Intention (LoI) hingga Momerandum of Understanding (MoU). Kendala pelaksanaan kerjasama luar negeri bagi Pemprov Lampung
adalah, komitmen dan kemampuan manajemen pemerintahan, anggaran yang
terbatas, prosedur kerjasama yang membutuhkan waktu panjang, perubahan struktur organisasi pemerintah, dinilai belum urgent/prioritas, minimnya inisiatif
dari SKPD, sosialisasi pemahaman perkembangan konteks internasional,
pentingnya perluasan kerjasama, prosedur dan hukum internasional yang masih
lemah. SOSIOLOGI: Jurnal Ilmiah Kajian Ilmu Sosial dan Budaya, Vol. 21, No. 2 September 2019: 201-216 Adibowo, R., & Putri, S. O. (2016). PENERAPAN E-GOVERNMENT DALAM
PARADIPLOMASI PEMERINTAH KOTA BANDUNG. 2, 10. Transnational Relations and World Politics:
An
Introduction. International
Organization,
25(3,),
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Indonesia
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TAHUN 2008 TENTANG PEDOMAN PELAKSANAAN KERJASAMA
PEMERINTAH DAERAH DENGAN PIHAK LUAR NEGERI. Kementerian
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REPUBLIK INDONESIA NOMOR: 09/A/KP/XII/2006/01 TENTANG
PANDUAN UMUM TATA CARA HUBUNGAN DAN KERJASAMA LUAR
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509. Kuznetsov, A. (2014). Theory and Practice of Paradiplomacy (0 ed.). Routledge. https://doi.org/10.4324/9781315817088 Michelmann, H. J., & Soldatos, P. (Eds.). (1990). Federalism and international
relations: The role of subnational units. Clarendon Press Oxford
University Press. Neuman, W. L. (2014). Social research methods: Qualitative and quantitative
approaches (7. ed., Pearson new internat. ed). Pearson. Nye, J. S., & Keohane, R. O. (1971). Transnational Relations and World Politics:
An
Introduction. International
Organization,
25(3,),
329–349. http://www.jstor.org/stable/2706043 Simanjuntak, K. (2015). Implementasi Kebijakan Desentralisasi Pemerintahan di
Indonesia. Jurnal
Bina
Praja,
07(02),
111–130. https://doi.org/10.21787/JBP.07.2015.111-130 SOSIOLOGI: Jurnal Ilmiah Kajian Ilmu Sosial dan Budaya, Vol. 21, No. 2 September 2019: 201-216 Adibowo, R., & Putri, S. O. (2016). PENERAPAN E-GOVERNMENT DALAM
PARADIPLOMASI PEMERINTAH KOTA BANDUNG. 2, 10. Adibowo, R., & Putri, S. O. (2016). PENERAPAN E-GOVERNMENT DALAM
PARADIPLOMASI PEMERINTAH KOTA BANDUNG. 2, 10. Aguirre, I. (1999). Making sense of paradiplomacy? An intertextual enquiry about
a concept in search of a definition. Regional & Federal Studies, 9(1), 185–
209. https://doi.org/10.1080/13597569908421078 Anggraini, A. D. (2017). Kinerja Pemerintah DKI Jakarta Dalam Kerjasama Sister
City Dengan Seoul di Bidang Perdagangan. Insignia Journal of
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Quantitative, and Mixed Methods Approaches. Damayanti, C. (2012). POTENSI PARADIPLOMASI DALAM MENDUKUNG
KINERJA DIPLOMASI INDONESIA MENUJU KOMUNITAS ASEAN. 22, Damayanti, C. (2012). POTENSI PARADIPLOMASI DALAM MENDUKUNG
KINERJA DIPLOMASI INDONESIA MENUJU KOMUNITAS ASEAN. 22,
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9. Fathun, L. M. (2016). Paradiplomasi Menuju Kota Dunia: Studi Kasus Pemerintah Fathun, L. M. (2016). Paradiplomasi Menuju Kota Dunia: Studi Kasus Pemerintah
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BUKITTINGGI DENGAN MALAYSIA DALAM MENINGKATKAN
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A. (2018). Paradiplomasi
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KOTA SURABAYA-XIAMEN. Kutubkhanah, 17(1), 71–83. Isnarti, R., & Fhadly, M. (2018). PARADIPLOMASI KOTA BUKITTINGGI
DENGAN MALAYSIA DALAM MENINGKATKAN KUNJUNGAN
WISATA. Jurnal Hubungan Internasional, 16. Kanwil Dirjen Perbendaharaan Lampung. (2018). Kajian Fiskal Regional. Kementerian Keuangan. KEMENDAGRI. (2008). PERATURAN MENTERI DALAM NEGERI NOMOR 3
TAHUN 2008 TENTANG PEDOMAN PELAKSANAAN KERJASAMA
PEMERINTAH DAERAH DENGAN PIHAK LUAR NEGERI. Kementerian
Dalam Negeri. Kementerian Luar Negeri. (2006). PERATURAN MENTERI LUAR NEGERI
REPUBLIK INDONESIA NOMOR: 09/A/KP/XII/2006/01 TENTANG
PANDUAN UMUM TATA CARA HUBUNGAN DAN KERJASAMA LUAR
NEGERI OLEH PEMERINTAH DAERAH. Kementerian Luar Negeri. Kincaid, J. (1990). From Cooperative to Coersive Federalism. ANNALS AAPS,
509. Kuznetsov, A. (2014). Theory and Practice of Paradiplomacy (0 ed.). Routledge. https://doi.org/10.4324/9781315817088
Michelmann, H. J., & Soldatos, P. (Eds.). (1990). Federalism and international
relations: The role of subnational units. Clarendon Press Oxford
University Press. Neuman, W. L. (2014). Social research methods: Qualitative and quantitative
approaches (7. ed., Pearson new internat. ed). Pearson. Nye, J. S., & Keohane, R. O. (1971). SOSIOLOGI: Jurnal Ilmiah Kajian Ilmu Sosial dan Budaya, Vol. 21, No. 2 September 2019: 201-216 SOSIOLOGI: Jurnal Ilmiah Kajian Ilmu Sosial dan Budaya, Vol. 21, No. 2 September 2019: 201-216 SOSIOLOGI: Jurnal Ilmiah Kajian Ilmu Sosial dan Budaya, Vol. 21, No. 2 September 2019: 201-216 Internet 4 Produk Asal Lampung Berpeluang Masuk Eropa Tengah dan Timur. (n.d.). From https://ekonomi.bisnis.com/read/20160813/257/574813/4-produk-
asal-lampung-berpeluang-masuk-eropa-tengah-dan-timur Home, Terkini, News, T., Terpopuler, Nusantara, Nasional, Update, L., Wisata, B. D., Olahraga, Pendidikan, K. D., Hukum, P. D., Internasional, Khusus, L.,
Foto, Video, Penggunaan, K., Kami, T., Siber, P. M., & Privasi, K. (n.d.-
a). China Berminat Investasi Energi di Lampung. Antara News Lampung. Retrieved
from
https://lampung.antaranews.com/berita/289228/china-
berminat-investasi-energi-di-lampung Home, Terkini, News, T., Terpopuler, Nusantara, Nasional, Update, L., Wisata, B. D., Olahraga, Pendidikan, K. D., Hukum, P. D., Internasional, Khusus, L.,
Foto, Video, Penggunaan, K., Kami, T., Siber, P. M., & Privasi, K. (n.d.-
b). Lampung bidik pasar pertanian ke Amerika Latin. Antara News
Lampung.From https://lampung.antaranews.com/berita/303661/lampung-
bidik-pasar-pertanian-ke-amerika-latin Ini Deretan Daerah Tempat Investasi Terbaik 2018—Nasional Tempo.co. (n.d.). From
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investasi-terbaik-2018 Lampung, D. K. P. (n.d.). Lampung dan Ekuador Jajaki Kerjasama—Lampung
dan Ekuador Jajaki Kerjasama. Pemerintah Provinsi Lampung. From
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kerjasama Lampung Menjalin Kerja Sama Dengan Provinsi Bouira Aljazair—ANTARA
News
Megapolitan. (n.d.). From
https://megapolitan.antaranews.com/berita/30315/lampung-menjalin-kerja-
sama-dengan-provinsi-bouira-aljazair Lampung Punya Potensi Panas Bumi Cukup Tinggi—ANTARA News Lampung. (n.d.). From
https://lampung.antaranews.com/berita/270920/lampung-
punya-potensi-panas-bumi-cukup-tinggi National University of Singapore (NUS): Lampung Salah Satu Provinsi Paling
Berkembang. (n.d.). National University of Singapore (NUS): Lampung
Salah
Satu
Provinsi
Paling
Berkembang.From SOSIOLOGI: Jurnal Ilmiah Kajian Ilmu Sosial dan Budaya, Vol. 21, No. 2 September 2019: 201-216 https://www.kupastuntas.co/2018/05/03/national-university-of-singapore-
nus-lampung-salah-satu-provinsi-paling-berkembang https://www.kupastuntas.co/2018/05/03/national-university-of-singapore-
nus-lampung-salah-satu-provinsi-paling-berkembang Terkini, N. L. (2018, February 7). Duta Besar Indonesia untuk Republik Kroasia
Buka Peluang Kerjasama dengan Pemprov Lampung. News Lampung
Terkini. https://newslampungterkini.com/news/16153/duta-besar-
indonesia-untuk-republik-kroasia-buka-peluang-kerjasama-dengan-
pemprov-lampung.html Tingkatkan Kerja Sama Antar Kawasan Melalui Kunjungan Duta Besar Negara
Timur Tengah dan Afrika Utara ke Lampung—Nusakini. (n.d.) From
https://nusakini.com/news/tingkatkan-kerja-sama-antar-kawasan-melalui-
kunjungan-duta-besar-negara-timur-tengah-dan-afrika-utara-ke-lampung
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https://openalex.org/W2049903158
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https://pubs.rsc.org/en/content/articlepdf/2013/fd/c3fd00030c
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English
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Kinetic limitations in gas-particle reactions arising from slow diffusion in secondary organic aerosol
|
Faraday discussions
| 2,013
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cc-by
| 10,421
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aDepartment of Chemistry, University of Toronto, Ontario, ON M5S 3H6, Canada. E-mail: szhou@chem.
utoronto.ca; Fax: +1 416 946 7359; Tel: +1 416 946 7359
bDivision of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA91125, USA
cMultiphase Chemistry Department, Max Plank Institute for Chemistry, Mainz, D-55128, Germany
This journal is ª The Royal Society of Chemistry 2013
Faraday Discuss., 2013, 165, 391–406 | 391 Faraday Discussions Faraday Discussions Faraday Discussions Cite this: Faraday Discuss., 2013, 165, 391 View Article Online
View Journal | View Issue Open Access Article. Published on 17 May 2013. Downloaded on 10/24/2024 6:50:29 AM.
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Based on an equilibrium gas/particle partitioning theory,2,3 SOA has generally
been treated as a well-mixed liquid in atmospheric models3–5 in which the gas-
phase oxidation products are assumed to quickly adopt gas-particle equilibrium. However, a number of recent studies have shown that SOA behaves as a highly
viscous, amorphous solid or semi-solid. In this case the equilibration timescale of
SOA partitioning can be longer and the assumption of equilibrium partitioning
may be in question.6 Virtanen et al.7 investigated the bounce behavior of biogenic SOA formed from
ozone reaction with plant-emitted VOCs on smooth and hard surfaces and
demonstrated that the organic-rich atmospheric aerosol can exist in the amor-
phous solid state. Later work of Vaden et al.8 and Abramson et al.9 studied
evaporation of a-pinene SOA at room temperature and found evaporation kinetics
were orders of magnitude slower than those expected from well-mixed liquid
droplets. As well, Cappa and Wilson10 observed that the volatility of organic
aerosol was signicantly lower than that of liquid aerosols by studying the
thermal desorption properties of a-pinene SOA. Finally, Perraud et al.11 reported
that the partitioning of organic nitrates formed from the reaction of a-pinene
with NO3 radicals to a-pinene ozonolysis SOA can only be explained by a
non-equilibrium, kinetically limited condensation mechanism rather than
the equilibrium partitioning commonly assumed to occur between gas- and
liquid-particles. If SOA exists in an amorphous, semi-solid state, then its properties are
expected to be affected by environmental conditions. Saukko et al.12 showed that
a-pinene SOA remains solid or semi-solid at RH < 50% and a transition to more
liquid-like behavior was seen when RH is higher than 50%. Recent work of
Kuwata and Martin13 reported that gas-phase ammonia uptake by a-pinene SOA
under dry conditions (RH < 5%) was consistent with adsorption on highly viscous
semi-solid particles, whereas the adsorption/absorption of ammonia on SOA
particles was signicantly increased at RHs close to saturation, suggesting that
the SOA had become liquid. As evidence accumulates showing that SOA possesses solid or semi-solid
properties,14 it is important to understand how the fundamental physical prop-
erties of SOA-containing particles affect heterogeneous reactivity. In particular,
are all reactants available for surfaces reactions, implying rapid diffusion through
the particle? Or, does semi-solid SOA provide shielding from gas-phase oxidants? Kinetic limitations in gas-particle reactions
arising from slow diffusion in secondary
organic aerosol Shouming Zhou,*a Manabu Shiraiwa,bc Robert D. McWhinney,a
Ulrich P¨oschlc and Jonathan P. D. Abbatta Received 8th March 2013, Accepted 16th May 2013
DOI: 10.1039/c3fd00030c Received 8th March 2013, Accepted 16th May 2013
DOI: 10.1039/c3fd00030c The potential for aerosol physical properties, such as phase, morphology and viscosity/
diffusivity, to affect particle reactivity remains highly uncertain. We report here a study
of the effect of bulk diffusivity of polycyclic aromatic hydrocarbons (PAHs) in secondary
organic aerosol (SOA) on the kinetics of the heterogeneous reaction of particle-borne
benzo[a]pyrene (BaP) with ozone. The experiments were performed by coating BaP-
ammonium sulfate particles with multilayers of SOA formed from ozonolysis of a-
pinene, and by subsequently investigating the kinetics of BaP loss via reaction with
excess ozone using an aerosol flow tube coupled to an Aerodyne Aerosol Mass
Spectrometer (AMS). All reactions exhibit pseudo-first order kinetics and are empirically
well described by a Langmuir–Hinshelwood (L-H) mechanism. The results show that
under dry conditions (RH < 5%) diffusion through the SOA coating can lead to
significant mass transfer constraints on the kinetics, with behavior between that
previously observed by our group for solid and liquid organic coats. The reactivity of
BaP was enhanced at 50% relative humidity (RH) suggesting that water uptake
lowers the viscosity of the SOA, hence lifting the mass transfer constraint to some
degree. The kinetics for 70% RH were similar to results obtained without SOA coats,
indicating that the SOA had sufficiently low viscosity and was sufficiently liquid-like
that reactants could rapidly diffuse through the coat. A kinetic multi-layer model for
aerosol surface and bulk chemistry was applied to simulate the kinetics, yielding
estimates for the diffusion coefficients (in cm2 s1) for BaP in a-pinene SOA of 2
1014, 8 1014 and >1 1012 for dry (RH < 5%), 50% RH and 70% RH conditions,
respectively. These results clearly indicate that slow diffusion of reactants through SOA
coats under specific conditions can provide shielding from gas-phase oxidants, enabling
the long-range atmospheric transport of toxic trace species, such as PAHs and
persistent organic pollutants. Paper
View Article Online Paper
View Article Online Faraday Discussions Faraday Discussions 392 | Faraday Discuss., 2013, 165, 391–406
This journal is ª The Royal Society of Chemistry 2013 1
Introduction The question we address in this paper is whether coatings of secondary organic
aerosol (SOA) can provide a kinetic barrier to the rates of aerosol chemistry. In
particular, while it is known that SOA frequently contributes the majority of the
sub-micron aerosol mass,1 the physical properties of atmospheric SOA and their
impacts remain poorly understood. Open Access Article. Published on 17 May 2013. Downloaded on 10/24/2024 6:50:29 AM.
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Through the analysis of the kinetics of multi-component heterogeneous reac-
tions, we should be able to derive estimates for the diffusivity of reactants through
SOA, noting these are largely lacking for this hard-to-measure quantity. In particular, to the best of our knowledge, only one value of a bulk diffusion
coefficient of 2.5 1017 cm2 s1 for pyrene diffusion in a-pinene SOA has been
estimated to date.9 Faraday Discussions
View Article Online Faraday Discussions
View Article Online i
i
View Article Online To this end, we present a study on the effect of a-pinene SOA viscosity/diffu-
sivity on the kinetics of gas-phase ozone reacting heterogeneously with particle-
borne benzo[a]pyrene (BaP) that was initially buried under the SOA coat. The
effects of water-uptake induced changes in SOA on the heterogeneous reactivity
were also investigated. As a model heterogeneous reaction system, the kinetics of
particle-borne BaP reacting with gas-phase ozone have been studied previ-
ously.15–17 In particular, our group has demonstrated that this reaction can be
effectively shut down when a solid organic coating is deposited on top of adsor-
bed-BaP, whereas liquid coatings impose no mass transfer limitations on the
kinetics.17 Kinetic models for aerosol surface and bulk chemistry (K2-SURF18 and
KM-SUB19) are applied to simulate the BaP degradation kinetics and to estimate
the diffusivity of BaP in a-pinene SOA at room temperature and interpret the
effect of changing RH on the kinetics. Open Access Article. Published on 17 May 2013. Downloaded on 10/24/2024 6:50:29 AM.
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. A 1000 sccm dry nitrogen ow was added into a horizontally oriented
humidifying tube making a total particle ow of 1.9 slpm in the kinetics ow
tube (RH < 5%). A 50% RH in the kinetics ow tube was achieved by passing the
dry nitrogen ow through a bubbler containing deionized water, whereas 70%
RH was achieved by also adding 50 ml deionized water into the horizontally
oriented humidifying tube. The residence time for the particles and water vapor
in the humidifying tube was 60s. The dry or humidied AS-BaP-SOA particles
were then introduced into a vertically oriented kinetics ow tube (Fig. 1), where a
ow (100 sccm) containing ozone was added through a movable stainless steel
injector. The kinetics ow tube (6 cm i.d. and 96 cm in length) was operated at room
temperature and atmospheric pressure (296 3 K and 1 atm) under laminar ow
conditions. The reaction time (up to 66 s) between BaP and ozone was varied by
setting the injector to different positions along the ow tube. The total ow
exiting the kinetics ow tube was divided into three: 1) 100 sccm was introduced
into a UV photometric O3 analyzer (Thermo Model 49i) with a dilution ow of
1.3 slpm dry nitrogen; 2) 400 sccm passing through an ozone denuder, which
can remove more than 90% of ozone, was sampled by the SMPS and an Aerodyne
Aerosol Mass Spectrometer (C-ToF AMS) to measure particle mobility size
distributions and chemical composition, respectively; and 3) the remaining
aerosol ow was removed by a diaphragm pump (Fig. 1). The ozone was in large
excess to BaP in the kinetics ow tube and its mixing ratio was controlled by
varying the ratio of O2 to N2 passing the pen-ray lamp. 394 | Faraday Discuss., 2013, 165, 391–406
This journal is ª The Royal Society of Chemistry 2013 2.1
Particle generation, particle coating, and aerosol kinetics A schematic representation of the experimental set-up is shown in Fig. 1. The
particles were generated by nebulizing a 1 mM ammonium sulfate (AS) (solid,
$99.5%, Fluka) aqueous solution using an atomizer (TSI model 3076). A fraction
of the poly-disperse AS particles (300 sccm) was coated with sub-monolayer
benzo[a]pyrene (BaP) (solid, $98%, Sigma-Aldrich) in a manner analogous to our
previous work, by passing through a heated tube containing BaP.17 The BaP-
coated AS particles (AS-BaP) were then mixed with gas-phase a-pinene in a mixing
ow tube (Fig. 1). The gas-phase a-pinene was introduced into the tube by
passing 0.5–1 sccm nitrogen over the headspace of a-pinene liquid ($99%, liquid)
which had been placed in a bubbler. The bubbler was cooled to 15 C and
the a-pinene mixing ratio varied between 250–500 ppb, as measured by a Fig. 1
A schematic representation of the experimental set-up. This journal is ª The Royal Society of Chemistry 2013
Faraday Discuss., 2013, 165, 391–406 | 393 Fig. 1
A schematic representation of the experimental set-up. This journal is ª The Royal Society of Chemistry 2013
Faraday Discuss., 2013, 165, 391–406 | 393 This journal is ª The Royal Society of Chemistry 2013
Faraday Discuss., 2013, 165, 391–406 | 393 Paper
View Article Online Paper
View Article Online Faraday Discussions proton-transfer-reaction mass spectrometer. The AS-BaP particles and the gas-
phase a-pinene were then sent to a second coating tube, where ozone was added
and the AS-BaP particles were coated with SOA from the ozonolysis reaction with
a-pinene. Ozone (100 sccm, 2–3 ppm) was generated by ultraviolet irradiation of
a mixed ow of N2/O2 in a Pyrex glass chamber with a mercury pen-ray lamp (UVP
Inc.). The SOA coated AS-BaP particles (AS-BaP-SOA) then passed through two
charcoal denuders to remove the gas-phase reactants and products before
entering a humidifying tube. Using a scanning mobility particle sizer (SMPS)
consisting of a differential mobility analyzer (DMA, model 3080, TSI Inc.) and a
condensation particle counter (CPC 3025, TSI Inc.), no SOA formation was
observed with only ozone and a-pinene present in the coating ow tube, con-
rming that the SOA measured in this work all arises from the condensation of
low volatility products from ozonolysis of a-pinene onto AS-BaP particles, with
negligible new particle formation. 2.2
Aerosol composition measurement The aerosol chemical composition was measured by Aerodyne AMS. The instru-
ment operation and sample analysis have been detailed in our previous work.17
Briey, the C-ToF AMS was operated in either mass spectrum (MS) mode or
particle time-of-ight (PTOF) mode, with the former producing the mass spectra
of non-refractory components of submicron particles and the latter providing
size-resolved mass spectra. The molecular ion of m/z 252 was used to detect
particle-borne BaP and was normalized by the sulfate mass loading in the kinetics
studies. Test experiments showed that the SOA contributed between 5–20% to the
total m/z 252 signal intensities of the AS-BaP-SOA particles. This contribution is Faraday Discussions
View Article Online i
i
View Article Online Paper found to be proportional to the total organic mass loadings and was unaffected by
the presence of ozone in the ow tube. Therefore, the contribution of the SOA to
the m/z 252 signal is easily accounted for. In addition, as has been pointed out in
our previous work,17 a small amount of organics, arising either from the deionized
milli-Q water or laboratory contamination, have always been observed by the AMS
in the AS particles. However, the background organics can only account for a
maximum of 10% and 2% of the total organic mass loadings aer ‘thin’ and
‘thick’ SOA coatings, respectively, were applied. The amount of BaP coated onto
the AS particles was very small, representing well less than a monolayer coverage,
assuming uniform coverage. P
View Article Online Paper
View Article Online Faraday Discussions coating thickness increased to some degree with particle size, consistent with the
observation above that the larger particles are more organic rich. coating thickness increased to some degree with particle size, consistent with the
observation above that the larger particles are more organic rich. 396 | Faraday Discuss., 2013, 165, 391–406
This journal is ª The Royal Society of Chemistry 2013 3.1
Particle characterization Fig. 2 provides an example of the particle-size-resolved measurements from the
AMS before (a) and aer (b) the SOA coating is applied. The size distributions of
the three materials, i.e. organic, sulfate and m/z 252, are all shied to larger sizes
aer the AS-BaP particles are coated with SOA, with the mass loadings for organic
and sulfate increased while that for m/z 252 decreased (note the different scales
for organic mass loadings in Fig. 1 before (a) and aer (b) SOA is coated). The
increase in sulfate mass loading is likely due to the enhanced collection efficiency
of the AMS with SOA coated AS-BaP particles, implying that the SOA formed from
ozonolysis of a-pinene is not as rigid as solid AS-BaP particles. The decrease in m/z
252 mass loading is because of a small degree of reaction of surface-bound BaP
with ozone in the SOA coating tube. In addition, we note that the size distribu-
tions for these three materials are qualitatively similar before SOA is coated. However, aer the SOA coating is applied, sulfate and m/z 252 remain with
generally similar size distributions, whereas the larger particles are more organic
rich than the smaller particles. The SOA coating thickness is estimated using a method detailed in our
previous work (in supplementary information of Zhou et al.17) to be 5–10 nm and
20–80 nm in thickness for ‘thin’ and ‘thick’ coatings, respectively. In particular,
we use the AMS data for these estimates, assuming uniform spherical coats. The
estimated absolute thickness of the coated organic is almost independent of
particle size for the ‘thin’ coating experiments; for the ‘thick’ coats, however, the Fig. 2
Size-resolved mass distributions for chemical species obtained from the PTOF mode of the AMS
for the AS-BaP particles before (a) and after (b) SOA coating is applied. This journal is ª The Royal Society of Chemistry 2013
Faraday Discuss., 2013, 165, 391–406 | 395 Fig. 2
Size-resolved mass distributions for chemical species obtained from the PTOF mode of the AMS
for the AS-BaP particles before (a) and after (b) SOA coating is applied. This journal is ª The Royal Society of Chemistry 2013
Faraday Discuss., 2013, 165, 391–406 | 395 Open Access Article. Published on 17 May 2013. Downloaded on 10/24/2024 6:50:29 AM.
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 3 gives an example of the BaP concentrations plotted against reaction time for
thin SOA coating experiments. The uncertainty in each data point was taken as
the standard error of the measurement. Control experiments were conducted in a
manner analogous to the kinetic measurements without ozone present. The small
changes in the BaP concentrations as a function of reaction time have been
accounted for in the data analysis. The good linearity of the kinetic plots in Fig. 3 demonstrates that the reaction
of particle-borne BaP with ozone exhibits pseudo-rst order kinetics in terms of
BaP loss. The linear least-squares t of the data in Fig. 3 yields the rst-order rate
constant (k1) at a specic ozone concentration. All the k1 obtained in this work are
summarized in Table 1. The uncertainties in k1 are given as a combination of the
least-squares 1-s uncertainties in the kinetic measurements and control experi-
ments. The k1 in Table 1 are plotted as a function of the corresponding ozone
concentrations and presented in Fig. 4 and 5 for dry (<5% RH) and high RH
experiments, respectively. All the plots in Fig. 4 and 5 (except for the thick solid organic coating in Fig. 4
taken from our previous work17) show saturation of the kinetics at high ozone
concentrations. This is consistent with a Langmuir–Hinshelwood (L-H) mecha-
nism, indicating a surface reaction between particle-borne BaP and ozone (or a
reactive intermediate). Using the following L-H equation, two parameters, i.e. kmax and KO3, can be
obtained by tting the data given in Fig. 4 and 5: k1 ¼ kmaxKO3½O3
1 þ KO3½O3 k1 ¼ kmaxKO3½O3
1 þ KO3½O3 where kmax is the maximum rst-order rate constant, KO3 is the gas-to-particle
partition coefficient of ozone, and [O3] is the gas-phase ozone concentration in the
kinetics ow tube. Fig. 3
Examples of the kinetic plots for the reaction of gas-phase ozone with BaP-AS particles with thin
SOA coats. Symbols represent different ozone concentrations in the kinetics flow tube. 396 | Faraday Discuss., 2013, 165, 391–406
This journal is ª The Royal Society of Chemistry 2013 Fig. 3
Examples of the kinetic plots for the reaction of gas-phase ozone with BaP-AS particles with thin
SOA coats. Symbols represent different ozone concentrations in the kinetics flow tube. Open Access Article. Published on 17 May 2013. Downloaded on 10/24/2024 6:50:29 AM.
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Paper
Faraday Discussions
View Article Online Table 1
The pseudo-first-order rate constants (k1) for the heterogeneous reactions of gas-phase ozone
(in concentration units of 1014 molecules cm3) with BaP with different thickness of SOA coatings under
dry and humid conditions Table 1
The pseudo-first-order rate constants (k1) for the heterogeneous reactions of gas-phase ozone
(in concentration units of 1014 molecules cm3) with BaP with different thickness of SOA coatings under
dry and humid conditions Thin SOA dry
Thick SOA dry
Thick SOA 50% RH
Thick SOA 70% RH
[O3]
k1 (s1)
[O3]
k1 (s1)
[O3]
k1 (s1)
[O3]
k1 (s1)
0.37
0.008 0.002
1.94
0.007 0.003
0.74
0.010 0.004
0.77
0.013 0.003
0.68
0.010 0.003
3.18
0.009 0.003
1.24
0.016 0.005
1.05
0.014 0.002
1.52
0.018 0.003
4.60
0.013 0.003
2.26
0.015 0.003
1.38
0.023 0.003
2.95
0.024 0.004
4.98
0.012 0.004
2.57
0.016 0.002
2.13
0.019 0.004
3.22
0.028 0.005
5.91
0.014 0.004
3.20
0.017 0.003
2.55
0.021 0.003
3.98
0.028 0.004
4.09
0.017 0.002
2.55
0.022 0.003
4.60
0.017 0.002
4.34
0.028 0.005
4.65
0.022 0.004
5.44
0.034 0.004
4.79
0.022 0.004
6.15
0.034 0.005
5.09
0.023 0.003
6.83
0.028 0.003
5.98
0.021 0.003
6.03
0.019 0.004 Open Access Article. Published on 17 May 2013. Downloaded on 10/24/2024 6:50:29 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. 2.95
0.024 0.004
4.98
0.012 0.004
2.57
0.016 0.002
2.13
0.019 0.004
3.22
0.028 0.005
5.91
0.014 0.004
3.20
0.017 0.003
2.55
0.021 0.003
3.98
0.028 0.004
4.09
0.017 0.002
2.55
0.022 0.003
4.60
0.017 0.002
4.34
0.028 0.005
4.65
0.022 0.004
5.44
0.034 0.004
4.79
0.022 0.004
6.15
0.034 0.005
5.09
0.023 0.003
6.83
0.028 0.003
5.98
0.021 0.003
6.03
0.019 0.004
Fig. 4
Pseudo-first-order rate constants k1 as a function of gas-phase ozone concentrations for the
reaction of particle-borne BaP and ozone with different organic coatings under dry conditions. Note that
the “Solid thick” and “Liquid” coating data come from Zhou et al.,17 where a thick coating of a solid
alkane was deposited onto BaP-AS particles. Fig. This journal is ª The Royal Society of Chemistry 2013
Faraday Discuss., 2013, 165, 391–406 | 397 Open Access Article. Published on 17 May 2013. Downloaded on 10/24/2024 6:50:29 AM.
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. 396 | Faraday Discuss., 2013, 165, 391–406
This journal is ª The Royal Society of Chemistry 2013 Table 1
The pseudo-first-order rate constants (k1) for the heterogeneous reactions of gas-phase ozone
(in concentration units of 1014 molecules cm3) with BaP with different thickness of SOA coatings under
dry and humid conditions
Thin SOA dry
Thick SOA dry
Thick SOA 50% RH
Thick SOA 70% RH
[O3]
k1 (s1)
[O3]
k1 (s1)
[O3]
k1 (s1)
[O3]
k1 (s1)
0.37
0.008 0.002
1.94
0.007 0.003
0.74
0.010 0.004
0.77
0.013 0.003
0.68
0.010 0.003
3.18
0.009 0.003
1.24
0.016 0.005
1.05
0.014 0.002
1.52
0.018 0.003
4.60
0.013 0.003
2.26
0.015 0.003
1.38
0.023 0.003
2.95
0.024 0.004
4.98
0.012 0.004
2.57
0.016 0.002
2.13
0.019 0.004
3.22
0.028 0.005
5.91
0.014 0.004
3.20
0.017 0.003
2.55
0.021 0.003
3.98
0.028 0.004
4.09
0.017 0.002
2.55
0.022 0.003
4.60
0.017 0.002
4.34
0.028 0.005
4.65
0.022 0.004
5.44
0.034 0.004
4.79
0.022 0.004
6.15
0.034 0.005
5.09
0.023 0.003
6.83
0.028 0.003
5.98
0.021 0.003
6.03
0.019 0.004
Fig. 4
Pseudo-first-order rate constants k1 as a function of gas-phase ozone concentrations for the
reaction of particle-borne BaP and ozone with different organic coatings under dry conditions. Note that
the “Solid thick” and “Liquid” coating data come from Zhou et al.,17 where a thick coating of a solid
alkane was deposited onto BaP-AS particles. Open Access Article. Published on 17 May 2013. Downloaded on 10/24/2024 6:50:29 AM.
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. this work for the thin SOA dry and thick SOA 70% RH of 0.042 s1 and 0.039 s1
(Table 2), respectively, are in good agreement with those for uncoated and liquid-
organic coated BaP particles, as well as most of the literature data (Table 2). 398 | Faraday Discuss., 2013, 165, 391–406
This journal is ª The Royal Society of Chemistry 2013 Open Access Article. Published on 17 May 2013. Downloaded on 10/24/2024 6:50:29 AM.
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. The kmax values obtained in This journal is ª The Royal Society of Chemistry 2013
Faraday Discuss., 2013, 165, 391–406 | 397 Table 2
Comparison of results from the present work and previous studies of the heterogeneous
reaction of particle-borne BaP or anthracene with gas-phase ozone (TTTS ¼ 1,1,5,5-tetraphenylte-
tramethylthisiloxane, BES ¼ bis(2-ethylhexyl)sebacate, PSO ¼ phenylsiloxane oil)
Faraday Discussions
Paper
View Article Online Faraday Discussions Table 2
Comparison of results from the present work and previous studies of the heterogeneous
reaction of particle-borne BaP or anthracene with gas-phase ozone (TTTS ¼ 1,1,5,5-tetraphenylte-
tramethylthisiloxane, BES ¼ bis(2-ethylhexyl)sebacate, PSO ¼ phenylsiloxane oil) Table 2
Comparison of results from the present work and previous studies of the heterogeneous
reaction of particle-borne BaP or anthracene with gas-phase ozone (TTTS ¼ 1,1,5,5-tetraphenylte-
tramethylthisiloxane, BES ¼ bis(2-ethylhexyl)sebacate, PSO ¼ phenylsiloxane oil) PAH
Aerosol substrate
KO3 (1015 cm3)
kmax (s1)
References
BaP
Soot
280 20
0.015 0.001
15
BaP
Azelaic acid
1.2 0.4
0.048 0.008
16
Anthracene
TTTS
100 40
0.010 0.003
20
Anthracene
Azelaic acid
2.2 0.9
0.057 0.009
20
BaP
AS
14 4
0.034 0.002
17
BaP
AS-BES
4.1 0.1
0.051 0.001
17
BaP
AS-PSO
13 4
0.047 0.004
17
BaP
AS-thin SOA dry
5.1 0.9
0.042 0.004
This work
BaP
AS-thick SOA dry
2.3 0.4
0.022 0.003
This work
BaP
AS-SOA 50% RH
14 4.4
0.023 0.004
This work
BaP
AS-SOA 70% RH
6.2 1.1
0.039 0.004
This work Open Access Article. Published on 17 May 2013. Downloaded on 10/24/2024 6:50:29 AM.
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. 5
Pseudo-first-order rate constants k1 as a function of gas-phase ozone concentrations for the
reaction of particle-borne BaP and ozone with thick SOA coatings under different RH. Fig. 4
Pseudo-first-order rate constants k1 as a function of gas-phase ozone concentrations for the
reaction of particle-borne BaP and ozone with different organic coatings under dry conditions. Note that
the “Solid thick” and “Liquid” coating data come from Zhou et al.,17 where a thick coating of a solid
alkane was deposited onto BaP-AS particles. Fig. 4
Pseudo-first-order rate constants k1 as a function of gas-phase ozone concentrations for the
reaction of particle-borne BaP and ozone with different organic coatings under dry conditions. Note that
the “Solid thick” and “Liquid” coating data come from Zhou et al.,17 where a thick coating of a solid
alkane was deposited onto BaP-AS particles. Fig. 4
Pseudo-first-order rate constants k1 as a function of gas-phase ozone concentrations for the
reaction of particle-borne BaP and ozone with different organic coatings under dry conditions. Note that
the “Solid thick” and “Liquid” coating data come from Zhou et al.,17 where a thick coating of a solid
alkane was deposited onto BaP-AS particles. Fig. 5
Pseudo-first-order rate constants k1 as a function of gas-phase ozone concentrations for the
reaction of particle-borne BaP and ozone with thick SOA coatings under different RH. Fig. 5
Pseudo-first-order rate constants k1 as a function of gas-phase ozone concentrations for the
reaction of particle-borne BaP and ozone with thick SOA coatings under different RH. Table 2 summarizes the best-t kmax and KO3 values obtained in this work with
associated standard deviations and those from previous studies on the hetero-
geneous reaction of ozone with BaP or anthracene. 3.3
Effect of SOA phase on the kinetics From Fig. 4 it is clear that under dry conditions (RH < 5%) a thin SOA coating
provides little-to-no mass transfer limitation on the kinetics of particle-borne BaP
with ozone compared to the uncoated AS-BaP particles, whereas the reactivity of
BaP towards ozone is substantially reduced by a thick SOA coating. In our
previous work,17 where we investigated the effects of organic coatings of different
physical states, we found that the reactivity of BaP towards ozone was unaffected
by liquid oil coatings regardless of whether the coating was thin or thick (Fig. 4). By contrast, the reactivity of BaP was reduced signicantly when a thin solid
alkane coating was applied and the reaction was effectively shut offwith a thick
solid coating (Fig. 4).17 These observations were explained by the different diffusivity of the reactants
through the organic coats. For liquid organic coatings, the diffusion of the BaP
through the coated materials was estimated to be fast, on a timescale of nano- to
microseconds.17 As a result, the kinetics were not limited by mass transfer. For the
solid organic, the reactant diffusivity was likely to be orders of magnitude lower so
kinetics were controlled by solid phase diffusion.17 We interpret the present results in a similar manner. The negligible effect of
the thin SOA coating may arise because diffusion is sufficiently fast to replace BaP
being lost at the surface of the particle through an interfacial process. Note that
we cannot rule out the possibility that the BaP may not be fully buried under the
SOA material, leaving the BaP directly exposed to gas-phase ozone. However, in a
similar
coating
procedure
of
SOA
onto
ammonium
bisulfate
particles,
we observed that equally thin coats provided a dramatic reduction in the reactivity
of N2O5.21 Compared to the kinetic plots for the uncoated and liquid-organic-coated
(Fig. 4) particles,17 the reduction in BaP reactivity by a thick SOA coating under dry
conditions (<5% RH) is likely a result of the mass transfer constraints that the Faraday Discussions
View Article Online Faraday Discussions
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i
View Article Online View Article Online Paper Faraday Discussions SOA coats place on the kinetics, indicating that the a-pinene SOA is not a low
viscosity liquid under these conditions. Open Access Article. Published on 17 May 2013. Downloaded on 10/24/2024 6:50:29 AM.
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. The effects of organic coatings on heterogeneous reactions have been inves-
tigated by a number of research groups. For example, studies on the reactive
uptake of N2O5 on different surfaces, such as AS particles,24 aqueous H2SO4
solution/particles25–28 and sea salt aerosols,29–32 and the uptake of ozone on deli-
quesced potassium iodide (KI) particles33 and aqueous pyrene solutions,34 have
shown a decrease in reactivity in the presence of a monolayer surfactant coating. A
decrease of the uptake of N2O5 on ammonium bisulfate particles coated with SOA
has also been reported.21,35 We note, however, that this is a reaction with a
substantial bulk-phase component, so the results are interpreted by the effects of
the organic coating on the ability of N2O5 to access water in the bulk of the
particle. By contrast, several studies have shown an enhancement of PAH uptake
at air–water interface,34 gas-phase HCl uptake on aqueous H2SO4 solutions36–38 as
well as O3 reactivity on aqueous pyrene solutions39 in the presence of soluble
organic surfactants. These are likely related to the changes in mass accommo-
dation coefficients. Thus, there is a complexity that can arise in the effects of
coatings on heterogeneous reactions beyond that which is explored in this paper,
likely related to the phase states of the organic coats and their specic interac-
tions with the reactants. This journal is ª The Royal Society of Chemistry 2013
Faraday Discuss., 2013, 165, 391–406 | 399 3.3
Effect of SOA phase on the kinetics This is consistent with previous studies
suggesting that SOA can be solid or semi-solid.7,10,14 Moreover, the difference in
the kinetic plots for the thick SOA and solid organic coatings (Fig. 4), with the
latter entirely shutting offthe reaction, implies that the SOA under dry conditions
is less viscous than a rigid solid, allowing the reactants to diffuse through the
organic coats to some extent over the experimental timescale of the order of
minutes. This is consistent with previous work showing that the SOA particles
possess some degree of uidity.8,13,22,23 3.5
Kinetic model for degradation kinetics of BaP with ozone 3.5.1
Degradation kinetics of uncoated BaP. A kinetic double-layer model for
aerosol surface chemistry (K2-SURF)18 is applied to analyze the experimental data
(Fig. 6a). The observed nonlinear dependence of k1 on [O3]g can be well repro-
duced under the assumption of a simple L-H mechanism formalism, in which an
adsorbed O3 molecule reacts with BaP in a surface reaction,15,48 as shown in
Fig. 6c. The O3 surface-residence time or desorption lifetime (sd,O3) inferred from
kinetic data based on simple L-H mechanims is more than milliseconds. Fig. 6
Kinetic modeling of ozonolysis of BaP on ammonium sulfate aerosol particles. (a) Schematics of
kinetic double-layer model (K2-SURF)18 applied for ozonolysis of uncoated BaP (experimental data taken
from our previous work17). (b) Schematics of kinetic multi-layer model (KM-SUB)19 applied for ozonolysis
of BaP coated by a-pinene SOA. It consists of a sorption layer, a surface layer, and a number of n bulk
layers. Red arrows show chemical reactions and green arrows show mass transport. (c) Pseudo first-order
decay rate coefficient (k1) for uncoated BaP as a function of gas-phase O3 concentration. The red line is
from a model with ROI formation using a desorption lifetime for O3 (sd,O3) of 3 ns. The black dotted line is
modeled using a simple L-H formalism without ROI formation with sd,O3 of 0.9 s. (d) k1 for BaP coated by
a-pinene SOA thinly and thickly under dry conditions. The red lines are modeled by KM-SUB. (e) k1
for BaP with thick a-pinene SOA coats at different RH (dry, 50% and 70% RH). The lines are modeled
by KM-SUB. (f) Bulk diffusion coefficient (cm2 s1) of BaP in a-pinene SOA as a function of RH derived
by KM-SUB. Fig. 6
Kinetic modeling of ozonolysis of BaP on ammonium sulfate aerosol particles. (a) Schematics of
kinetic double-layer model (K2-SURF)18 applied for ozonolysis of uncoated BaP (experimental data taken
from our previous work17). (b) Schematics of kinetic multi-layer model (KM-SUB)19 applied for ozonolysis
of BaP coated by a-pinene SOA. It consists of a sorption layer, a surface layer, and a number of n bulk
layers. Red arrows show chemical reactions and green arrows show mass transport. (c) Pseudo first-order
decay rate coefficient (k1) for uncoated BaP as a function of gas-phase O3 concentration. The red line is
from a model with ROI formation using a desorption lifetime for O3 (sd,O3) of 3 ns. 400 | Faraday Discuss., 2013, 165, 391–406
This journal is ª The Royal Society of Chemistry 2013 3.4
Effect of humidity-induced viscosity/diffusivity changes Published on 17 May 2013. Downloaded on 10/24/2024 6:50:29 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. 3.4
Effect of humidity-induced viscosity/diffusivity changes It can be seen from Fig. 5 that the heterogeneous reactivity of BaP towards ozone
is enhanced when RH increases. The higher reactivity is likely due to water uptake
by SOA resulting in a lower viscosity of the SOA materials and hence increased
diffusivity of the reactants through the coats. The similarities in the kinetic plots
for 70% RH and uncoated BaP, as well as those for liquid organic coats (Fig. 4, 5
and Table 2), suggest a transition of the SOA from semi-solid to lower viscosity,
more-liquid-like particles between 50 to 70% RH. We note that the RH in the ow
tube was kept lower than the AS deliquescence RH (DRH) of 80%40 to maintain a
uniform aerosol substrate; it has been reported that AS mixed with a-pinene
ozonolysis SOA has a DRH which is only 4% lower than pure AS.41 This lends
condence that the AS in the particles remains in the solid state at 70% RH. These results are in accord with previous studies. As mentioned above,
humidity-induced modication of biogenic SOA particles has been investigated
by Saukko et al.12 by studying particle bounce, where it was concluded that
a-pinene photooxidation SOA remains solid or semi-solid at RH < 50%. A phase
change from solid to liquid-like substances was suggested in the RH range of
50–64% RH.12 A similar observation was made in previous work on the multiphase
oxidation kinetics of protein substrates with ozone,42 where the increased ozone
uptake with RH was explained by hygroscopic water uptake of amorphous P
View Article Online Paper
View Article Online Faraday Discussions Faraday Discussions Faraday Discussions organics leading to an increase of the diffusivity of the reactants through the
organic matrix. While a number of laboratory and modeling studies have reported that the
multi-component organic/inorganic particles may undergo liquid–liquid phase
separation at high RH conditions,43–47 it is impossible for us to determine if this is
the case for the high RH experiments in the present work. Nevertheless, the
kinetic results indicate that the enhanced RH lowers the viscosity of the SOA,
liing the mass transfer constraint on the heterogeneous reactivity of BaP with
ozone. Also, we note that the results that have been observed in this work are
specic to the form of SOA studied. Subsequent studies of other SOA types are
required to determine the extent to which the results are generalizable. Open Access Article. Open Access Article. Published on 17 May 2013. Downloaded on 10/24/2024 6:50:29 AM.
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. O3 (g) 4 O3 (s)
(R1)
O3 (s) 4 O (s) + O2 (s)
(R2)
O (s) + PAH (ss) / O–PAH (ss)
(R3) (R1) O (s) + PAH (ss) / O–PAH (ss)
(R3) O (s) + PAH (ss) / O–PAH (ss) (R3) where g, s and ss denote the gas phase, sorption and surface layer, respectively. The weakly bound physisorbed O3 molecule can be desorbed thermally to the gas
phase with a desorption lifetime of nanoseconds, or it can overcome an activation
barrier (Ea,pc), undergo dissociation and enter into a state of stronger binding to
the surface. The second activation barrier (Ea,ox) is the reaction between O atoms
(or the appropriate form for the ROI) and BaP to form stable oxidation products. With this formulation K2-SURF can reproduce the kinetics observations very well
with parameters listed in Table 3 (red line in Fig. 6c). The estimated activation
energies are very similar to those for the ozone reaction with BaP on soot15,51 and
consistent with that estimated from DFT calculations.49,50 3.5.2
Degradation kinetics of PAH coated by SOA. For modeling the degra-
dation kinetics of BaP coated by SOA the kinetic multi-layer model for aerosol
surface and bulk chemistry (KM-SUB)19 is applied. Fig. 6b shows the schematics of
KM-SUB, which consists of a sorption layer, a quasi-static surface layer, and a
number of bulk layers. The thickness of each bulk layer is set to be 0.5 nm, cor-
responding to the molecular diameter of O3.18 KM-SUB treats the following
processes explicitly: gas-phase diffusion and reversible adsorption of O3, bulk Table 3
Kinetic parameters used in the model simulation assuming a multi-step Langmuir–Hinshel-
wood mechanism for the ozonolysis of BaP on ammonium sulfatea
Parameter
Description
Value
as,0,O3
surface accommodation coefficient of O3
1
sd,O3 (s)
desorption lifetime of O3
3.2 109
Db,O3 (cm2 s1)
bulk diffusivity of O3 in SOA
1010
Db,PAH (cm2 s1)
bulk diffusivity of PAH in SOA
*
Ea,pc (kJ mol1)
activation energy from physisorbed
O3 to ROI
42
Ea,ox (kJ mol1)
activation energy from ROI to
oxidized PAH
80
kBR (cm3 s1)
second-order bulk reaction rate
coefficient between PAH and O3
5 1018
Ksol,cc (mol cm3 atm1)
Henry's law coefficient of O3
6 104
a * The values are shown in Fig. 6f depending on RH. 3.5
Kinetic model for degradation kinetics of BaP with ozone The black dotted line is
modeled using a simple L-H formalism without ROI formation with sd,O3 of 0.9 s. (d) k1 for BaP coated by
a-pinene SOA thinly and thickly under dry conditions. The red lines are modeled by KM-SUB. (e) k1
for BaP with thick a-pinene SOA coats at different RH (dry, 50% and 70% RH). The lines are modeled
by KM-SUB. (f) Bulk diffusion coefficient (cm2 s1) of BaP in a-pinene SOA as a function of RH derived
by KM-SUB. 400 400 | Faraday Discuss., 2013, 165, 391–406
This journal is ª The Royal Society of Chemistry 2013 i
i
View Article Online Paper Faraday Discussions Faraday Discussions However, according to quantum mechanical calculations based on density
functional theory (DFT), sd,O3 should be only on the order of nanoseconds,49 more
than six orders of magnitude less. This implies that the actual reaction mecha-
nism is a multi-step L-H mechanism involving the decomposition of surface
ozone and formation of long-lived reactive oxygen intermediates (ROI). The
dissociated products are molecular oxygen such as a chemisorbed oxygen atom
bound to the delocalized p-electrons of an aromatic surface.49–51 The chemical
equations of the multi-step L-H mechanism can be described as follows: Open Access Article. Published on 17 May 2013. Downloaded on 10/24/2024 6:50:29 AM.
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. The kinetic parameters required for simulations are listed in Table 3. as,0,O3,
sd,O3 and the activation energies are kept the same as in the uncoated PAH case. The Henry's law coefficient of ozone (Ksol,cc) in a-pinene SOA matrix is not known. Ksol,cc in organics generally lies in the range of 104–103 mol cm3 atm1.48,54,55
Thus, Ksol,cc are varied in this range and other parameters are also varied
systematically and iteratively to t to the experimental data. The sensitivity
studies revealed that the bulk diffusivity of PAH in SOA (Db,PAH) is the most
sensitive parameter and other parameters are not as critical. Db,O3 is assumed to
be 1010 cm2 s1 which is a typical value of small oxidants in amorphous solids, as
percolation theory would predict only a slight increase of Db,O3 up to 80% RH.42
kBR is estimated to have a low value of 5 1018 cm3 s1, which is in line with
previous studies reporting that the bulk reaction between O3 and PAH proceeds
slower than surface reaction whose reaction rate coefficient ranges from 1017–
1016 cm3 s1.20,56–58 It is important to note that if a larger value of kBR is used, the
modeled k1 do not show saturation behavior with increasing ozone concentra-
tions but rather exhibit a linear increase trend. Fig. 6d shows experimentally observed k1 values for BaP when coated by
a-pinene SOA under dry conditions. The SOA coating thicknesses are taken as 8
nm and 40 nm for thin and thick coatings, respectively. KM-SUB successfully ts
to both data sets by a single parameter set listed in Table 3. Db,PAH is estimated to
be 2 1014 cm2 s1 under dry conditions. k1 for BaP coated thickly at 50% and
70% RH were also well t with Db,PAH of 8 1014 cm2 s1 and 1011 cm2 s1,
respectively (Fig. 6e). Note that at 70% RH KM-SUB is not sensitive enough to
constrain Db,PAH well but gives more accurately a lower limit of 1012 cm2 s1, as
k1 is suppressed only slightly by SOA coating. The obtained Db,PAH are summa-
rized in Fig. 6f, showing the clear increasing trend as RH increases. 402 | Faraday Discuss., 2013, 165, 391–406
This journal is ª The Royal Society of Chemistry 2013 Open Access Article. Published on 17 May 2013. Downloaded on 10/24/2024 6:50:29 AM.
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. This journal is ª The Royal Society of Chemistry 2013
Faraday Discuss., 2013, 165, 391–406 | 401 Table 3
Kinetic parameters used in the model simulation assuming a multi-step Langmuir–Hinshel-
wood mechanism for the ozonolysis of BaP on ammonium sulfatea Paper
View Article Online Paper
View Article Online Faraday Discussions diffusion of O3 and PAH in SOA matrix, surface reaction including formation of
ROI and subsequent reaction with PAH, which diffuses through the SOA coating
to the surface, and a bulk reaction between O3 and PAH. The bulk reaction
between O3 and SOA is assumed to be negligible, as the C]C should have been
consumed by the gas-phase ozonolysis reaction, forming oxygenated products
that then adsorb/condense to form the SOA.52,53 Before particles are exposed to O3,
they are humidied for 60 s during which diffusion of BaP commences. To make
the most direct comparison between the measurements and the models, the
ozone exposure is turned on aer a delay period of 60 s. This time matches the
residence time in the humidifying tube in the experimental system. Kinetics
simulations without this delay time yield essentially the same results. diffusion of O3 and PAH in SOA matrix, surface reaction including formation of
ROI and subsequent reaction with PAH, which diffuses through the SOA coating
to the surface, and a bulk reaction between O3 and PAH. The bulk reaction
between O3 and SOA is assumed to be negligible, as the C]C should have been
consumed by the gas-phase ozonolysis reaction, forming oxygenated products
that then adsorb/condense to form the SOA.52,53 Before particles are exposed to O3,
they are humidied for 60 s during which diffusion of BaP commences. To make
the most direct comparison between the measurements and the models, the
ozone exposure is turned on aer a delay period of 60 s. This time matches the
residence time in the humidifying tube in the experimental system. Kinetics
simulations without this delay time yield essentially the same results. Open Access Article. Published on 17 May 2013. Downloaded on 10/24/2024 6:50:29 AM.
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 17 May 2013. Downloaded on 10/24/2024 6:50:29 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 7
Radial profile of bulk concentration (cm3) of (a) ozone and (b) BaP and (c) BaP degradation rate
(cm3 s1) simulated by KM-SUB with dry conditions and a gas phase ozone concentration of 2.5 1014
cm3. The left axis is radial position in SOA coating (0 nm ¼ ammonium sulfate; 40 nm ¼ particle
surface). Fig. 7c. The highest reaction rate is observed at the surface (>1018 cm3 s1),
clearly showing that reaction proceeds faster at the surface than in the bulk. The
slight radial gradient of reaction rate in the bulk reects the concentration
gradient of O3 and PAH. We note that these modeling estimates of the diffusion coefficient are
dependent upon the assumed mechanism whereby BaP diffuses to the surface to
react with ozone at the interface. Other mechanisms cannot be fully ruled out but
we note that if we assume a faster bulk phase reaction, whereby ozone diffusivity
could end up being rate limiting, the model results yielded a linear dependence of
the BaP loss rate constant on ozone concentration. As well, there will be uncer-
tainty arising in our inferred values from the thickness of the SOA coats, which we
assume to be uniform. This journal is ª The Royal Society of Chemistry 2013
Faraday Discuss., 2013, 165, 391–406 | 403 Open Access Article. Published on 17 May 2013. Downloaded on 10/24/2024 6:50:29 AM.
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. As discussed
previously, this trend corresponds to humidity-induced changes in viscosity/
diffusivity of a-pinene SOA.14,40,59 Note that Abramson et al.9 estimated the bulk
diffusivity of pyrene in a-pinene SOA to be 2.5 1017 cm2 s1 under dry
conditions (<5% RH) based on measurements of evaporation kinetics, which is
three orders of magnitude lower than our estimate. Fig. 7 shows the radial distribution and temporal evolution of the bulk
concentration of (a) O3 and (b) BaP with thick SOA coatings under dry conditions
with a gas phase O3 concentration of 2.5 1014 cm3. The leaxis indicates the
radial distance from the ammonium sulfate core. Due to low diffusivity and
reactive consumption both O3 and BaP exhibit a steep concentration gradient; the
O3 concentration is higher near the surface and the BaP concentration is higher in
the inner bulk. The prole of the degradation rate of BaP (cm3 s1) is shown in View Article Online Fig. 7
Radial profile of bulk concentration (cm3) of (a) ozone and (b) BaP and (c) BaP degradation rate
(cm3 s1) simulated by KM-SUB with dry conditions and a gas phase ozone concentration of 2.5 1014
cm3. The left axis is radial position in SOA coating (0 nm ¼ ammonium sulfate; 40 nm ¼ particle
surface). Paper
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View Article Online Faraday Discussions the diffusivity (in cm2 s1) of BaP in SOA was estimated to be 2 1014, 8 1014
and >1 1012 for dry (RH < 5%), 50% RH and 70% RH, respectively. the diffusivity (in cm2 s1) of BaP in SOA was estimated to be 2 1014, 8 1014
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)
It is expected that PAHs, such as BaP, will become coated with SOA aer their
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dence time of a week or so. However, this estimate is for one type of SOA only. As
well it is not for oxidatively aged SOA, and it has been reported that the viscosity of
aged a-pinene SOA is higher than fresh SOA by a factor of three.9 As well, it is
expected that the diffusivity will be markedly lower at lower temperatures, e.g. aer a particle is loed out of the boundary layer. Mapping out these depen-
dencies may allow for a more accurate modeling of the lifetime of such species in
the atmosphere. It is expected that PAHs, such as BaP, will become coated with SOA aer their
formation from combustion sources. Both modeling studies and other experi-
mental studies suggest that PAHs buried under multi-layer organic lms could be
protected from heterogeneous oxidation.17,60–62 It is quite likely that this provides a 4
Conclusions and atmospheric implications This paper illustrates that mass transfer limitations can arise in atmospheric
heterogeneous reactions involving SOA. In particular, the effects of viscosity/
diffusivity of a-pinene ozonolysis SOA on the heterogeneous reactivity of particle-
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2000, 254, 93–234.
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Crescimento e fitomassa da beterraba sob irrigação suplementar com água de diferentes concentrações salinas
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Revista Ceres
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Submetido em 05/05/2015 e aprovado em 04/04/2016.
1 Universidade Federal Rural de Pernambuco, Departamento de Engenharia Agrícola, Recife, Pernambuco, Brasil. daniellapsantos@hotmail.com; celia_agron@hotmail.com;
2Universidade Federal de Campina Grande, Departamento de Engenharia Agrícola, Campina Grande, Paraíba, Brasil. patriciafssilva@hotmail.com
3Universidade Estadual Paulista “Júlio de Mesquita Filho”, Departamento Ciências Agronômicas, Botucatu, São Paulo, Brasil. mirian-mp@hotmail.com
4Universidade Estadual Paulista “Júlio de Mesquita Filho”, Departamento Ciências Agronômicas, Jaboticabal, São Paulo, Brasil. jandir_jc@hotmail.com
*Autora para correspondência: daniellapsantos@hotmail.com RESUMO O uso da água salina na agricultura é uma alternativa viável, tendo-se em vista o aumento da demanda de água
doce. Objetivou-se, com este trabalho, avaliar o crescimento e a produção de fitomassa da beterraba, sob irrigação com
água de diferentes concentrações salinas, em experimento em condição de campo, no Campus da Universidade Federal
de Alagoas, em Arapiraca. Os tratamentos foram cinco níveis de condutividade elétrica (1,0; 2,0; 3,0; 4,0 e 5,0 dS m-1). O delineamento foi em blocos casualizados, com quatro repetições. O máximo rendimento da beterraba aos 27 dias após
aplicação dos tratamentos salinos foi obtido com uma salinidade de 3,0 dS m-1, para as variáveis altura de planta (AP),
diâmetro do caule (DC), comprimento da raiz (CR), fitomassa seca da parte aérea (FSPA) e fitomassa seca total (FST). Aos 42 dias após aplicação dos tratamentos salinos, as variáveis fitomassa fresca da parte aérea (FFPA), fitomassa
fresca da raiz (FFR), fitomassa fresca total (FFT), fitomassa seca da parte aérea (FSPA) e fitomassa seca total (FST)
aumentaram com o aumento da salinidade da água. A chuva pode ter influenciado os resultados obtidos para as
avaliações, realizadas aos 42 dias após aplicação dos tratamentos salinos. Palavras-chave: Beta vulgaris L.; condutividade elétrica; qualidade da água. Crescimento e fitomassa da beterraba sob irrigação suplementar
com água de diferentes concentrações salinas
Daniella Pereira dos Santos1*, Célia Silva dos Santos1, Patrícia Ferreira da Silva2,
Mírian Paula Medeiros André Pinheiro3, Jandir Cruz Santos4 10.1590/0034-737X201663040011 CEa = Condutividade elétrica da solução (dS m-1). Segundo Ayers & Westcot (1991), a beterraba apre-
senta valores de salinidade limiar (CE) de 7,0 dS m-1, tor-
nando-se mais tolerante ao excesso de sais em estádios
avançados de crescimento. Por essa razão, a utilização
dessa cultura pode servir como alternativa de renda ao
produtor rural com problemas de salinidade na água. O cultivar de beterraba utilizado foi o “Early Wonder. As mudas foram produzidas em bandejas de polietileno
expandido, de 128 células, preenchidas com substrato com-
posto de casca de arroz e húmus de minhoca, na propor-
ção de 1:1. O transplantio foi realizado, utilizando-se uma
muda por vaso, quando apresentava de quatro a cinco
folhas definitivas, o que ocorreu por volta dos 25 dias
após a semeadura. Apesar de sua importância econômica, existem pou-
cos trabalhos que estudam o efeito da salinidade sobre a
cultura, particularmente estudos do crescimento e do de-
senvolvimento inicial da beterraba sob estresse salino e
seu potencial para cultivo em condições de campo
salinizado por manejo incorreto da irrigação. Antes do plantio, foi efetuada uma irrigação, para ele-
var o solo à capacidade de campo, sendo coletado o solo
dos vasos, seguindo-se o método gravimétrico (padrão)
de estufa, em que foi obtida uma umidade na capacidade
de campo de U = 26,32%. As irrigações foram realizadas
diariamente sempre no final da tarde e foi aplicado água
apenas no solo. Por essa razão, objetivou- se, com este trabalho, avaliar
o crescimento e a produção de fitomassa da beterraba, sob
irrigação com água de diferentes concentrações salinas. Beet growth and biomass under supplementary irrigation with water of different saline
concentrations The use of saline water in agriculture is a viable alternative in view of the increased freshwater demand. The objective
was to evaluate the growth and biomass of beet under irrigation with water of different saline concentrations, in an
experiment conducted under field condition in the campus of the Universidade Federal de Alagoas - Arapiraca. The
treatments were five levels of electrical conductivity (S1: 1.0, S2: 2.0, S3: 3.0; S4: 4.0; S5: 5.0 dS m-1). The maximum yield
of beets at 27 days after saline treatments was obtained with salinity of 3.0 dS m-1, for the variables plant height (PH),
stem diameter (SD), root length (RL), shoot dry biomass (SDB), and total dry biomass (TDB). At 42 days after application
of saline treatments, the variables shoot fresh biomass (SFB), root fresh biomass (RFB), total fresh biomass (TFB), shoot
dry biomass (SDB), and total dry biomass (TDB) increased with increasing salinity. Rain may have influenced the results
obtained for the evaluations performed at 42 days after application of saline treatments. Key words: Beta vulgaris L.; electrical conductivity; water quality. Rev. Ceres, Viçosa, v. 63, n.4, p. 509-516, jul/ago, 2016 Rev. Ceres, Viçosa, v. 63, n.4, p. 509-516, jul/ago, 2016 Daniella Pereira dos Santos et al. 510 Alagoano, cujo clima é, de acordo com a classificação de
Köppen, tropical com estação seca de Verão. Rev. Ceres, Viçosa, v. 63, n.4, p. 509-516, jul/ago, 2016 INTRODUÇÃO A irrigação é um instrumento efetivo para o aumento
da produtividade e para a expansão de fronteiras agríco-
las, porém sua utilização inadequada pode causar a
salinização dos solos, principalmente em regiões áridas e
semiáridas, diminuindo os rendimentos das culturas, ou,
até mesmo, resultando no abandono das terras. A
salinização e sodificação é problema com reflexos econô-
micos, sociais e ecológicos (Ferreira et al. 2006). Os dados diários de precipitação pluvial e umidade
relativa do ar do município de Arapiraca, AL, referentes ao
período de março a maio de 2010, época correspondente à
condução do experimento, foram obtidos do Instituto Na-
cional de Meteorologia (INMET) e encontram-se apresen-
tados na Figura 1. As temperaturas do ar máxima, média e
mínima, referentes ao período de condução do experimen-
to, obtidas do Instituto Nacional de Meteorologia
(INMET), estão apresentadas na Figura 2. Segundo Silva et al. (2012), a salinidade induzida pelo
homem é a que traz maiores prejuízos econômicos, pois
ocorre em áreas nas quais se realizaram investimentos ele-
vados, como sistemas de irrigação e fertilizações. A
salinidade induzida está geralmente associada ao manejo
inadequado da irrigação e da fertirrigação, podendo ser
causada tanto pela baixa qualidade da água de irrigação
quanto pela aplicação excessiva de fertilizantes ao solo. O solo utilizado na pesquisa foi classificado como
Argissolo Vermelho distrófico (Embrapa, 2013) e suas ca-
racterísticas químicas, na profundidade de 0-20 cm, estão
apresentadas na Tabela 1. O delineamento estatístico adotado foi o de blocos
casualizados completos, com quatro repetições. Os trata-
mentos foram constituídos da combinação de cinco níveis
de salinidade da água de irrigação (S1: 1; S2: 2; S3: 3; S4: 4
e S5: 5dS m-1), com quatro repetições. Os tratamentos fo-
ram dispostos em 40 vasos, sendo cada parcela constituí-
da por dois vasos de 2 L, espaçados de 0,5 m entre vasos
e 0,9 m entre linhas dos vasos. As águas de diferentes
concentrações salinas foram obtidas a partir de adições de
cloreto de sódio (NaCl) à água de abastecimento, calcula-
das conforme a equação 1, proposta por Richards (1954). O delineamento estatístico adotado foi o de blocos
casualizados completos, com quatro repetições. Os trata-
mentos foram constituídos da combinação de cinco níveis
de salinidade da água de irrigação (S1: 1; S2: 2; S3: 3; S4: 4
e S5: 5dS m-1), com quatro repetições. INTRODUÇÃO Os tratamentos fo-
ram dispostos em 40 vasos, sendo cada parcela constituí-
da por dois vasos de 2 L, espaçados de 0,5 m entre vasos
e 0,9 m entre linhas dos vasos. As águas de diferentes
concentrações salinas foram obtidas a partir de adições de
cloreto de sódio (NaCl) à água de abastecimento, calcula-
das conforme a equação 1, proposta por Richards (1954). É necessário buscar tecnologias alternativas para o
uso condizente dessas águas salinas, com seu maior apro-
veitamento na produção vegetal, diminuindo os impactos
ambientais (Santos et al., 2010). Uma alternativa proposta
é a utilização de culturas tolerantes à salinidade para pro-
dução vegetal intensiva. Utilizada em diversas regiões semiáridas do mundo, a
beterraba (Beta vulgaris L.) é uma opção para produção
em condições de solos salinos (Katerji et al., 1997), visto
que, além de se destacar por sua composição nutricional,
sobretudo em açúcares e pela forma de consumo da raiz
tuberosa, ela se apresenta como uma das hortaliças tole-
rantes a elevados teores de sais (Aquino et al., 2006). C = 640 * CEa (1)
em que: C = 640 * CEa C = 640 * CEa (1) em que: C = concentração de NaCl (mg L-1); C = concentração de NaCl (mg L-1); MATERIAL E MÉTODOS O volume de água consumido foi calculado na manhã
do dia seguinte, pela diferença entre o volume de água
aplicado e o drenado. Estimou-se a evapotranspiração da
cultura (consumo), determinado diariamente. Assim, o vo-
lume de água a ser aplicado foi calculado com base no
consumo do dia anterior. O experimento foi desenvolvido em condições de cam-
po, em área pertencente à Universidade Federal de Alagoas
(UFAL), Campus Arapiraca, nas coordenadas de 9°45´58´´S
e 35°38´58´´O, em altitude de 264 m. Esta região é represen-
tada pela transição entre Zona da Mata e o Sertão Rev. Ceres, Viçosa, v. 63, n.4, p. 509-516, jul/ago, 2016 511 Crescimento e fitomassa da beterraba sob irrigação suplementar com água de diferentes... Crescimento e fitomassa da beterraba sob irrigação suplementar com água de diferentes... A irrigação foi realizada manualmente, com proveta gra-
duada para medir as quantidades a serem aplicadas e dre-
nadas diariamente. De acordo com a equação 2 de Rhoades (1974), e fra-
ção lixiviação 0,15, foram obtidos os tratamentos para esta
fonte de variação. Consequentemente, foi realizado cálcu-
lo idêntico para fração lixiviação 0,20, caracterizando-se,
assim, dez tratamentos com as concentrações salinas. De
acordo com a necessidade hídrica, esse valor variou com
cada etapa do ciclo da cultura. As avaliações foram realizadas aos 27 e 42 dias após
aplicação dos tratamentos salinos. As plantas de cada
unidade experimental foram colhidas separadamente e
acondicionadas em sacos de papel previamente identifica-
dos, transportados para o Laboratório de Física do Solo
da UFAL, onde foram analisadas as variáveis: - altura de
planta (AP), mensurada do colo da planta até a base da
última folha emitida, com régua graduada em cm; - número
de folhas por planta (NF), por meio de contagem direta; -
diâmetro do caule, mensurado com paquímetro graduado;
- área foliar (cm²), por meio de um integrador eletrônico de
área foliar, modelo LI-3100; - comprimento da raiz (CR),
medido com régua graduada, - fitomassas fresca de parte
aérea, de raiz e total; - fitomassas secas de parte aérea, de
raiz e total. VI = VA - VD (2)
1- FL (2) VI = VA - VD
1- FL RESULTADOS E DISCUSSÃO De acordo com o modelo de regressão para compri-
mento de raiz (Figura 4B) o máximo valor, aos 27 dias após
aplicação dos tratamentos salinos, foi atingido com 2,82
dS m-1, o que corresponde a 13,0 cm. Conforme Mohammad
et al.(1998), o incremento da salinidade é acompanhado da
redução do comprimento de raízes, confirmando-se com
isso os resultados obtidos neste trabalho. Houve efeito da salinidade (p < 0,01) sobre as variá-
veis AP, AF, FFPA FSPA, e FST, respectivamente. Houve
efeito quadrático significativo (p < 0,01) sobre AP, DC, AF,
CR, FSPA e FST, em função da salinidade da água de irriga-
ção. De acordo com a equação de regressão para a altura de
planta, Figura 3A, a máxima altura foi obtida com 3,11 dS m-
1, o que corresponde a 12,8 cm aos 27 dias após aplicação
dos tratamentos salinos. O modelo matemático que melhor se ajustou para a
fitomassa seca da parte aérea, aos 27 dias após a aplica-
ção dos tratamentos, foi o quadrático (Figura 5A) sen-
do obtido o máximo com 3,0 dS m-1 e a diferença de 20%
entre o maior (S5) e o menor (S1) nível de salinidade. Chen & Jiang (2010) afirmam que o efeito dos sais pro-
voca redução da parte área de certas espécies em razão
de não apresentarem um ajuste osmótico como meca-
nismo de adaptabilidade ao excesso de sais na solução
do solo. O modelo matemático que melhor se ajustou para diâ-
metro do caule foi o quadrático (Figura 3B). O máximo diâ-
metro do caule, aos 37 dias após aplicação dos tratamen-
tos, ocorreu com 3,0 dSm-1, correspondendo 0,33 mm. Em estudos como o de Silva (2012), atribui-se a redu-
ção dos valores de diâmetro do caule e do consumo hídrico
das plantas ao potencial osmótico da solução do solo,
devido ao excesso de sais presentes, dificultando a absor-
ção de água por parte da planta, fazendo com que ela pre-
cise de maior esforço energético para a absorção de água
e de nutrientes, reduzindo-se, assim, o seu crescimento. Para a variável fitomassa seca total (Figura 5B), aos 27
dias após aplicação dos tratamentos salinos, o máximo ren-
dimento foi obtido com 3,5 dSm-1 e corresponde a 0,25 g. Gondim et al. VI = VA - VD
1- FL De acordo com Tester & Davenport (2003), a
salinidade reduz o potencial osmótico, refletindo na di-
minuição da absorção de água pelas plantas e compro-
metendo os processos fisiológicos; assim, as plantas
podem apresentar modificações morfofisiológicas, a fim
de aumentar sua tolerância à salinidade, com destaque
para a redução da área foliar, em decorrência da diminui-
ção do volume de células. Com redução da AF e aumento
da concentração total de solutos na folha, ocorre ajusta-
mento osmótico das células, garantindo às plantas a ab-
sorção de água. Os resultados foram submetidos à análise de variância
pelo teste F, sendo as médias comparadas pelo teste de
Tukey, a 5% com o Software estatístico SISVAR (Ferreira,
2008); para o fator níveis de salinidade realizou-se teste de
regressão (por polinômios ortogonais), com níveis de
significância de 0,01 ou 0,05 de probabilidade. VI = VA - VD
1- FL em que: VI - Volume de água a ser aplicada na irrigação (mL); VI - Volume de água a ser aplicada na irrigação (mL); VA - Volume de água aplicado na irrigação anterior ou no
período (mL); VD - Volume de água drenada na irrigação anterior ou no
período (mL); FL - Fração de lixiviação (0,15 e 0,20). FL - Fração de lixiviação (0,15 e 0,20). Rev. Ceres, Viçosa, v. 63, n.4, p. 509-516, jul/ago, 2016
Figura 2: Temperaturas do ar máxima, média e mínima durante o período de experimentação. Figura 1: Precipitação pluvial e umidade relativa do ar durante o período de condução do experimento. Figura 1: Precipitação pluvial e umidade relativa do ar durante o período de condução do experimento. Figura 1: Precipitação pluvial e umidade relativa do ar durante o período de condução do experimento. Rev. Ceres, Viçosa, v. 63, n.4, p. 509-516, jul/ago, 2016
Figura 2: Temperaturas do ar máxima, média e mínima durante o período de experimentação. Figura 2: Temperaturas do ar máxima, média e mínima durante o período de experimentação. Rev. Ceres, Viçosa, v. 63, n.4, p. 509-516, jul/ago, 2016 512 Daniella Pereira dos Santos et al. Oliveira et al. (2012) relatam que o maior valor de
área foliar de rabanete irrigado com água salina foi obti-
do com aplicação do menor nível de salinidade (2 dS m-
1), obtendo-se 497,20 cm2, enquanto, no maior nível de
salinidade (10 dS m-1), resultou no menor valor de área
foliar, obtendo-se 220 cm2, correspondentes a uma re-
dução de 55,75%, reafirmando-se os dados obtidos nes-
te estudo, mesmo com maiores concentrações de sais
na água. Para obtenção dessas variáveis, as plantas foram reti-
radas dos vasos, preservando-se suas estruturas, evitan-
do-se danos, tanto para a parte radicular quanto para a
parte aérea. Para determinação das fitomassas fresca e
seca da raiz e de parte aérea, as plantas foram retiradas dos
vasos e lavadas, as raízes e a parte aérea foram pesadas
separadamente. Em seguida, foram acondicionadas em sacos de papel e
colocadas em estufa com circulação forçada de ar, a 65 °C,
por 48 horas, para posterior pesagem em balança analítica
de precisão de 0,01g. As fitomassas frescas e secas totais
foram obtidas com a soma das respectivas massas frescas
e secas. RESULTADOS E DISCUSSÃO (2010), estudando a condutividade elé-
trica na produção e nutrição de alface em sistema de culti-
vo hidropônico NFT, também constataram que a fitomassa
seca total apresentou resposta quadrática ao aumento da
CE da solução, atingindo o máximo de 100,4 g com 2,68 mS
cm-1. A partir dessa CE, houve uma redução de 7,1% na
MST com a CE de 4 mS cm-1. Os resultados da área foliar em função dos tratamen-
tos salinos aplicados encontram-se na Figura 4A. A máxi-
ma produção foi obtida com a salinidade de 2,85 dS m-1,
que corresponde a 21, 4 cm2. A partir desse ponto verifica-
se decréscimo de 24%, quando se compara S1 com S5. Rev. Ceres, Viçosa, v. 63, n.4, p. 509-516, jul/ago, 2016
Tabela 1: Análise química do solo utilizado no experimento
pH
P
M.O
K
Ca
Mg
Al
H+Al
T
V
Fe
Cu
Zn
Mn
(H2O)
(mg dm-3) (g dm-3)
(cmolcdm-3)
(%)
(mg dm-3)
5,7
13
15
0,2
1,4
1,4
0,2
4,0
7,0
42,9
44,5
0,86
2,4
32 Rev. Ceres, Viçosa, v. 63, n.4, p. 509-516, jul/ago, 2016
Tabela 1: Análise química do solo utilizado no experimento
pH
P
M.O
K
Ca
Mg
Al
H+Al
T
V
Fe
Cu
Zn
Mn
(H2O)
(mg dm-3) (g dm-3)
(cmolcdm-3)
(%)
(mg dm-3)
5,7
13
15
0,2
1,4
1,4
0,2
4,0
7,0
42,9
44,5
0,86
2,4
32 Tabela 1: Análise química do solo utilizado no experimento Crescimento e fitomassa da beterraba sob irrigação suplementar com água de diferentes... 513 Hassanli et al. (2010), em estudos sobre a influência de
métodos de irrigação e a qualidade da água na produção
de beterraba açucareira, observaram efeito significativo
da salinidade da água no crescimento. Atribui-se a redu-
ção dos valores ao potencial osmótico acrescido da solu-
ção do solo, em razão do excesso de sais presentes, difi-
cultando a absorção de água por parte da planta, fazendo Houve efeito da salinidade (p < 0,01) sobre FFPA, FFR,
FFT, FSPA, FSR e FST. Houve efeito linear significativo (p
< 0,01) sobre FFPA, FFR, FFT, FSPA, FSR e FST, em fun-
ção da salinidade da água de irrigação, respectivamente. Resultados semelhantes foram observados por Silva et al. (2013), estudando as relações hídricas em cultivares de
beterraba em diferentes níveis de salinidade do solo. RESULTADOS E DISCUSSÃO A B Figura 3: Altura de plantas (A) e Diâmetro do caule (B), aos 27 dias após aplicação dos tratamentos em função da salinidade da água
de irrigação. A
B A B Figura 3: Altura de plantas (A) e Diâmetro do caule (B), aos 27 dias após aplicação dos tratamentos em função da salinidade da água
de irrigação ura de plantas (A) e Diâmetro do caule (B), aos 27 dias após aplicação dos tratamentos em função da salinidade da água Figura 3: Altura de plantas (A) e Diâmetro do caule (B), aos 27 dias após aplicação dos tratamentos em função da salin
de irrigação. B Figura 4: Área foliar (A) e Comprimento da raiz (B), aos 27 dias após aplicação dos tratamentos em função da salinidade da água de
irrigação. A
B A A B Figura 4: Área foliar (A) e Comprimento da raiz (B), aos 27 dias após aplicação dos tratamentos em função da salinidade da água de
irrigação Figura 4: Área foliar (A) e Comprimento da raiz (B), aos 27 dias após aplicação dos tratamentos em função da salinid
irrigação. Área foliar (A) e Comprimento da raiz (B), aos 27 dias após aplicação dos tratamentos em função da salinidade da água B A Rev. Ceres, Viçosa, v. 63, n.4, p. 509-516, jul/ago, 2016
Figura 5: Fitomassa seca da parte aérea (A) e fitomassa seca total (B), aos 27 dias após aplicação dos tratamentos, em função da
salinidade da água de irrigação. A
B A Figura 5: Fitomassa seca da parte aérea (A) e fitomassa seca total (B), aos 27 dias após aplicação dos tratamentos, em função da
salinidade da água de irrigação. Rev. Ceres, Viçosa, v. 63, n.4, p. 509-516, jul/ago, 2016 Daniella Pereira dos Santos et al. 514 retando lixiviação dos sais presentes na zona radicular das
plantas. com que ela precise de maior esforço energético para a
absorção de água e nutrientes, reduzindo-se, assim, seu
crescimento (Silva, 2012). Esses resultados estão em desacordo com os encon-
trados em diversas pesquisas, como as de Putti et al. (2014);
Paulus et al. (2010); Silva et al. (2013); Silva et al. (2014) e
Silva et al. (2014), estudando o efeito dos sais nas diver-
sas culturas. REFERÊNCIAS pécies como o algodão, ou reduzir em 20% o peso da maté-
ria seca da beterraba. Aquino LA de, Puiatti M, Pereira PRG, Pereira FHF, Ladeira IR &
Castro MRS (2006) Produtividade, qualidade e estado nutricional
da beterraba de mesa em função de doses de nitrogênio. Horticultura Brasileira, 24:199-203. De acordo com os estudos de regressão, o modelo
matemático que melhor se ajustou aos dados de fitomassas
fresca total, seca da parte aérea e seca total foi o linear, aos
42 dias após aplicação dos tratamentos salinos (Figuras
7A, B e C). Ayers RS & Westcot DW (1991) Qualidade de água na agricultura. Campina Grande, UFPB. 218p. (Estudos FAO. Irrigação e Dre-
nagem, 29). Verificou-se que, à medida que aumenta a salinidade
da água de irrigação, houve acréscimo das fitomassas
das plantas de beterraba, sendo o maior valor observado
com a salinidade de 5 dS m-1, correspondendo a 3,73; 0,28
e 0,48 g, respectivamente, para as fitomassas fresca total,
seca da parte aérea e seca total. No entanto, esse aumen-
to da massa pode estar relacionado com o fato de as
folhas nos níveis mais altos apresentarem-se mais gros-
sas e menos flexíveis, o que pode estar associado ao fato
de a cultura da beterraba ser considerada tolerante aos
efeitos dos sais, sendo que, para uma salinidade de 4,7
dSm-1, não há redução do rendimento da cultura. Assim,
como os tratamentos estudados estão dentro da
salinidade tolerada pela cultura, não se verificaram per-
das e rendimento (Ayers & Westcott, 2001). No entanto,
deve-se atentar para as chuvas ocorridas no período,
que podem ter lixiviado os sais do solo, comprometendo
o resultado da pesquisa. Chen H & Jiang J (2010) Osmotic adjustment and plant adaptation
to environmental changes related to drought and salinity. Environmental Reviews, 18:309-319. Ferreira DF (2008) Sisvar: um programa para análises e ensino de
estatística. Revista Symposium, 6:36-41. Ferreira PA, Moura RF de, Santos DB dos, Fontes PCR & Melo RF
de (2006) Efeitos da lixiviação e salinidade da água sobre um
solo salinizado cultivado com beterraba. Revista Brasileira de
Engenharia Agrícola e Ambiental, 10:570-578. Filgueira FAR (2008) Novo manual de olericultura: Agrotecnologia
moderna na produção e comercialização de hortaliças. 3a ed. Viçosa, UFV. 421 p. Gondim ARO, Flores MEP, Martinez HEP, Fontes PCR & Pereira
PRG (2010) Condutividade elétrica na produção e nutrição de
alface em sistema de cultivo hidropônico nft. Bioscience Journal,
26:894-904. REFERÊNCIAS Greenway H & Muuns R (1980) Mechanisms of salt tolerance in
nonhalophytes. Annual Review of plant physiology. 31a ed. Nedlans, Web of Science. 41p. Hassanli AM, Ahmadirad S & Beecham S (2010) Evaluation of the
influence of irrigation methods and water quality on sugar beet
yield and water use efficiency. Agricultural Water Management,
97:357-362. No entanto, vale ressaltar, a tolerância depende da ha-
bilidade da planta em controlar o transporte de sais em
cinco pontos específicos: - seletividade no processo de
absorção pelas células das raízes; - carregamento do xilema
preferencialmente com K+, mais do que com Na+; - remo-
ção do sal do xilema na parte superior das raízes, caule,
pecíolo ou bainhas foliares; - retranslocação de Na+ e Cl-
no floema, garantindo a ausência de translocação para te-
cidos da parte aérea em processo de crescimento, e excreção
de sais por glândulas ou pelos vesiculares (Munns et al,
2002). Katerji N, Hoom JW, Hamdy A, Mastrorilli M & Karzel EM
(1997) Osmotic adjustment of sugar beets in response to soil
salinity and its influence on stomatal conductance, growth and
yield. Agricultural Water Management, 34:57-69. Munns R, Husain S, Rivelli AR, Richard AJ, Condon AG, Megan P,
Evans S, Schachtman DP & Hare RA (2002) Avenues for
increasing salt tolerance of crops, and the role of physiologically
based selection traits. Plant and Soil, 247:93-105. Mohammad M, Shibli R & Ajouni M (1998) Tomato root and
shoot responses to salt stress under different levels of
phosphorus nutrition. Journal of Plant Nutrition, 21:1667-
1680. RESULTADOS E DISCUSSÃO Segundo Greenway & Munns (1980), o culti-
vo, com 200 mM de NaCl, por um dado período de tempo,
poderá acarretar a morte de espécies sensíveis, como o
feijão, provocar reduções de até 60% na biomassa de es- Observa-se que, à medida que se aumentou a salinidade
da água de irrigação, houve incremento da fitomassa fres-
ca de parte aérea e de raiz aos 42 dias após aplicação dos
tratamentos salinos (Figura 6A e B). Constata-se, ainda,
que o maior rendimento para ambas as variáveis foi obser-
vado com 5 dSm-1, correspondendo a 3,13 e 0,59 g, respec-
tivamente, o que, provavelmente pode ser justificado pela
elevada precipitação durante o período experimental, acar- B Figura 6: Fitomassa fresca da parte aérea (A) e fitomassa fresca da raiz (B), aos 42 dias após aplicação dos tratamentos, em função
da salinidade da água de irrigação. A
B A B A Figura 6: Fitomassa fresca da parte aérea (A) e fitomassa fresca da raiz (B), aos 42 dias após aplicação dos tratamentos, em função
da salinidade da água de irrigação Figura 6: Fitomassa fresca da parte aérea (A) e fitomassa fresca da raiz (B), aos 42 dias após aplicação dos tratamentos, em função
da salinidade da água de irrigação. Rev Ceres Viçosa v 63 n 4 p 509-516 jul/ago 2016
Figura 7: Fitomassa fresca total (A), fitomassa fresca da parte aérea (B) e fitomassa seca total (C), aos 42 dias após aplicação dos
tratamentos, em função da salinidade da água de irrigação. A
B
C B A B A C C Figura 7: Fitomassa fresca total (A), fitomassa fresca da parte aérea (B) e fitomassa seca total (C), aos 42 dias após aplicação dos
tratamentos, em função da salinidade da água de irrigação. Rev. Ceres, Viçosa, v. 63, n.4, p. 509-516, jul/ago, 2016 Crescimento e fitomassa da beterraba sob irrigação suplementar com água de diferentes... 515 CONCLUSÕES Oliveira AMP, Oliveira AM, Dias NS, Freitas M & Silva KB (2012)
Cultivo de rabanete irrigado com água salina. Revista Verde de
Agroecologia e Desenvolvimento Sustentável, 7:01-05. Os valores máximos na beterraba, aos 27 dias após
aplicação dos tratamentos salinos, foi obtido com uma
salinidade de 3,0 dS m-1, para as variáveis altura de planta,
diâmetro do caule, comprimento da raiz, fitomassa seca da
parte aérea e fitomassa seca total. Paulus D, Dourado Neto D, Frizzone JA & Soares TM (2010)
Produção e indicadores fisiológicos de alface sob hidroponia
com água salina. Horticultura Brasileira, 28:29-35. Putti FF, Silva Junior JF, Ludwig R, Gabriel Filho RA, Cremasco
CP & Klar AE (2014) Avaliação da cultura do rabanete ao longo
do ciclo submetido em diferentes níveis de salinidade. Journal of
Agronomic Sciences, 3:80-90. Aos 42 dias após aplicação dos tratamentos salinos,
as variáveis fitomassa fresca da parte aérea, fitomassa fres-
ca da raiz, fitomassa fresca total, fitomassa seca da parte
aérea e fitomassa seca total aumentaram com o aumento
da salinidade da água. Rhoades JD (1974) Drainage for salinity control. In: Shilfgaarde
J van (Ed.) Drainage for Agriculture. Salt Lake City, American
Society of Agronomy. p.433-462. As chuvas podem ter influenciado o resultado obtido
na avaliação realizada aos 42 dias após aplicação dos tra-
tamentos salinos. Richards LA (1954) Diagnosis and improvement of saline and
alkali soils: united statessalinity laboratory. 60a ed. Washing-
ton, Agriculture Handbook. 160p. Rev. Ceres, Viçosa, v. 63, n.4, p. 509-516, jul/ago, 2016 Rev. Ceres, Viçosa, v. 63, n.4, p. 509-516, jul/ago, 2016 Daniella Pereira dos Santos et al. 516 Silva AO, Soares TM, Silva EFF, Santos AN & Klar AE (2012)
Consumo hídrico da rúcula em cultivo hidropônico nft utilizan-
do rejeitos de dessalinizador em Ibimirim-pe. Irriga, 17:114-
125. Santos AN, Soares TM, Silva EFF, Silva DJR & Montenegro AAA
(2010) Cultivo hidropônico de alface com água salobra subter-
rânea e rejeito da dessalinização em Ibimirim, PE. Revista Bra-
sileira de Engenharia Agrícola e Ambiental, 14:961-969. Silva PF, Cavalcante VS, Santos JCC, Costa ES & Barbosa JTV
(2014) Análise quantitativa da cebolinha irrigada com água sa-
lina. Comunicata Scientiae, 5:241-251. Silva AO (2012) Fertirrigação e controle da salinidade em cultivo
de beterraba em ambiente protegido. Dissertação de Mestrado. Universidade Estadual Paulista, Botucatu. 137p. Tester M & Davenport R (2003) Na+ tolerance and Na+ transport
in higher plants. Annals of Botany, 91:503-527. Silva AO, Soares TM, Silva EFF, Santos AN & Klar AE (2012)
Consumo hídrico da rúcula em cultivo hidropônico nft utilizan-
do rejeitos de dessalinizador em Ibimirim-pe. Irriga, 17:114-
125. Rev. Ceres, Viçosa, v. 63, n.4, p. 509-516, jul/ago, 2016 Tester M & Davenport R (2003) Na+ tolerance and Na+ transport
in higher plants. Annals of Botany, 91:503-527. CONCLUSÕES Silva AO, Klar AE, Silva EFF & Cunha AR (2014)
Evapotranspiração e coeficiente de cultivo para a beterraba sob
estresse salino em ambiente protegido. Irriga, 19:375-389. Silva AO, Klar AE, Silva EFF, Tanaka AA & Silva Junior JF (2013)
Relações hídricas em cultivares de beterraba em diferentes ní-
veis de salinidade do solo. Revista Brasileira de Engenharia Agrí-
cola e Ambiental, 17:1143-1151. Rev. Ceres, Viçosa, v. 63, n.4, p. 509-516, jul/ago, 2016
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https://acp.copernicus.org/articles/11/5505/2011/acp-11-5505-2011.pdf
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English
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Number size distributions and seasonality of submicron particles in Europe 2008–2009
| null | 2,011
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cc-by
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Number size distributions and seasonality of submicron particles in
Europe 2008–2009 Discuss.: 16 March 2011 eceived: 23 February 2011 – Published in Atmos. Chem. Phys. Discuss.: 16 March 2011
evised: 25 May 2011 – Accepted: 26 May 2011 – Published: 15 June 2011 Revised: 25 May 2011 – Accepted: 26 May 2011 – Published: 15 June 2011 and number size distributions between 30 and 500 nm of
dry particle diameter. Spatial and temporal distribution of
aerosols in the particle sizes most important for climate ap-
plications are presented. We also analyse the annual, weekly
and diurnal cycles of the aerosol number concentrations, pro-
vide log-normal fitting parameters for median number size
distributions, and give guidance notes for data users. Em-
phasis is placed on the usability of results within the aerosol
modelling community. and number size distributions between 30 and 500 nm of
dry particle diameter. Spatial and temporal distribution of
aerosols in the particle sizes most important for climate ap-
plications are presented. We also analyse the annual, weekly
and diurnal cycles of the aerosol number concentrations, pro-
vide log-normal fitting parameters for median number size
distributions, and give guidance notes for data users. Em-
phasis is placed on the usability of results within the aerosol
modelling community. Number size distributions and seasonality of submicron particles in
Europe 2008–2009 A. Asmi1, A. Wiedensohler2, P. Laj3, A.-M. Fjaeraa4, K. Sellegri5, W. Birmili2, E. Weingartner6, U. Baltensperger6,
V. Zdimal7, N. Zikova7, J.-P. Putaud8, A. Marinoni9, P. Tunved10, H.-C. Hansson10, M. Fiebig4, N. Kivek¨as11,
H. Lihavainen11, E. Asmi11, V. Ulevicius12, P. P. Aalto1, E. Swietlicki13, A. Kristensson13, N. Mihalopoulos14,
N. Kalivitis14, I. Kalapov15, G. Kiss16, G. de Leeuw11,17, B. Henzing17, R. M. Harrison18, D. Beddows18, C. O’Dowd19,
S. G. Jennings19, H. Flentje20, K. Weinhold2, F. Meinhardt21, L. Ries21, and M. Kulmala1 A. Asmi1, A. Wiedensohler2, P. Laj3, A.-M. Fjaeraa4, K. Sellegri5, W. Birmili2, E. Weingartner6, U. Baltensperger6,
V. Zdimal7, N. Zikova7, J.-P. Putaud8, A. Marinoni9, P. Tunved10, H.-C. Hansson10, M. Fiebig4, N. Kivek¨as11,
H. Lihavainen11, E. Asmi11, V. Ulevicius12, P. P. Aalto1, E. Swietlicki13, A. Kristensson13, N. Mihalopoulos14,
N. Kalivitis14, I. Kalapov15, G. Kiss16, G. de Leeuw11,17, B. Henzing17, R. M. Harrison18, D. Beddows18, C. O’Dowd19,
S. G. Jennings19, H. Flentje20, K. Weinhold2, F. Meinhardt21, L. Ries21, and M. Kulmala1 1Department of Physics, University of Helsinki, P.O. Box 64, Helsinki, Finland
2Leibniz Institute for Tropospheric Research, Permoserstraße 15, 04318 Leipzig, Germany
3Laboratoire de Glaciologie et G´eophysique de l’Environnement Universit´e Joseph Fourier, Grenoble 1/CNRS, 38400 St. Martin d’H`eres, France p
y
,
y
,
,
,
2Leibniz Institute for Tropospheric Research, Permoserstraße 15, 04318 Leipzig, Germany
3Laboratoire de Glaciologie et G´eophysique de l’Environnement Universit´e Joseph Fourier, Grenoble 1/CNRS, 38400 St. Atmos. Chem. Phys., 11, 5505–5538, 2011
www.atmos-chem-phys.net/11/5505/2011/
doi:10.5194/acp-11-5505-2011
© Author(s) 2011. CC Attribution 3.0 License. Atmos. Chem. Phys., 11, 5505–5538, 2011
www.atmos-chem-phys.net/11/5505/2011/
doi:10.5194/acp-11-5505-2011
© Author(s) 2011. CC Attribution 3.0 License. Atmospheric
Chemistry
and Physics Number size distributions and seasonality of submicron particles in
Europe 2008–2009 Martin d’H`eres, France 4Norwegian Institute for Air Research Instituttveien 18, 2027 Kjeller, Norway
5 4Norwegian Institute for Air Research Instituttveien 18, 2027 Kjeller, Norway
5Laboratoire de M´et´eorologie Physique, UMR 6016, CNRS/University of Clermont-Ferrand, Clermont-Ferrand, France Laboratoire de M´et´eorologie Physique, UMR 6016, CNRS/University of Clermont-Ferrand, Clerm 5Laboratoire de Meteorologie Physique, UMR 6016, CNRS/University of Clermont-Ferrand, Clermont-Ferrand, France
6Laboratory of Atmospheric Chemistry, Paul Scherrer Institut, 5232 Villigen PSI, Switzerland Laboratoire de Meteorologie Physique, UMR 6016, CNRS/University of Clermont Ferrand, Clerm
Laboratory of Atmospheric Chemistry, Paul Scherrer Institut, 5232 Villigen PSI, Switzerland 6Laboratory of Atmospheric Chemistry, Paul Scherrer Institut, 5232 Villigen P Laboratory of Aerosol Chemistry and Physics, Institute of Chemical Process Fundamentals of the A
5, 16502 Praha 6, Czech Republic mmission, Joint Research Centre, Institute for Environment and Sustainability, 21027 Ispra (VA), Ita 8European commission, Joint Research Centre, Institute for Environment and Sustainability, 2102
9 9CNR-ISAC, Institute of Atmospheric Sciences and Climate, 40129, Bologna, Italy
10 10Department of Applied Environmental Science (ITM), Stockholm University, Svante Arrhenius v¨ag 8,
10691 Stockholm, Sweden 11Research and Development, Finnish Meteorological Institute, Helsinki, Finland
12 13Department of Physics, Lund University, P.O. Box 118, 221 00 Lund, Sweden
14 p
y
y
vironmental Chemical Processes Laboratory, Department of Chemistry, University of Crete, Greece 14Environmental Chemical Processes Laboratory, Department of Chemistry, Univ
5 15Institute of Nuclear Research and Nuclear Energy, Bulgarian Academy of Sciences, Blvd. Tzarigradsko chaussee, 72,
1784 Sofia, Bulgaria g
emistry Group of the Hungarian Academy of Sciences, University of Pannonia, P.O. Box 158,
szpr´em Hungary 16Air Chemistry Group of the Hungarian Academy of Sciences, University of Pannonia, P.O. Box 158,
8201 Veszpr´em, Hungary 17Netherlands Organisation for Applied Scientific Research TNO, Princetonlaan 6, 3508 TA Utrecht, The Netherlands
18National Centre for Atmospheric Science, School of Geography, Earth and Environmental Sciences, University of
Birmingham, B15 2TT, UK g
pp
18National Centre for Atmospheric Science, School of Geography, Earth and Environmental Sciences, University of
Birmingham, B15 2TT, UK 19National University of Ireland Galway, University Road, Galway, Ireland 20German Meteorological Service, Hohenpeienberg Observatory, Albin-Schwaiger Weg 10,
82383 Hohenpeißenberg, Germany 20German Meteorological Service, Hohenpeienberg Observatory, Albin-Schwaiger Weg 10,
82383 Hohenpeißenberg, Germany 21German Federal Environment Agency (UBA), Messnetzzentrale, Langen, Germany Received: 23 February 2011 – Published in Atmos. Chem. Phys. Discuss.: 16 March 2011
Revised: 25 May 2011 – Accepted: 26 May 2011 – Published: 15 June 2011 Received: 23 February 2011 – Published in Atmos. Chem. Phys. Abstract. Two years of harmonized aerosol number size distribution
data from 24 European field monitoring sites have been anal-
ysed. The results give a comprehensive overview of the Eu-
ropean near surface aerosol particle number concentrations Published by Copernicus Publications on behalf of the European Geosciences Union. A. Asmi et al.: Submicron particles in Europe 2008–2009 A. Asmi et al.: Submicron particles in Europe 2008–2009 5506 Table 1. Nomeclature
Symbol
meaning
unit
dp
electrical mobility particle diameter
nm
ˆdp
nominal (mean) diameter of measured
size interval
nm
n
number concentration (of a size range
or a interval)
cm−3
dn(dp)
dlog10dp
size distribution function
cm−3
N30−50
aerosol number concentration between
30 and 50 nm
cm−3
N50
aerosol number concentration between
50 and 500 nm
cm−3
N100
aerosol number concentration between
100 and 500 nm
cm−3
N250
aerosol number concentration between
250 and 500 nm (Appendix B)
cm−3
x
Arithmetic mean of x
same as x
ˆx
Geometric mean of x
same as x
µp(x)
p-th percentile of x
same as x
σ(x)
(linear) standard deviation of x
same as x
σl(x)
10-logarithmic standard deviation of x
-
A1(x)
Autocorrelation (1-h) of x
- We also show that the aerosol number concentrations of
Aitken and accumulation mode particles (with 100 nm dry
diameter as a cut-off between modes) are related, although
there is significant variation in the ratios of the modal num-
ber concentrations. Different aerosol and station types are
distinguished from this data and this methodology has po-
tential for further categorization of stations aerosol number
size distribution types. The European submicron aerosol was divided into char-
acteristic types: Central European aerosol, characterized by
single mode median size distributions, unimodal number
concentration histograms and low variability in CCN-sized
aerosol number concentrations; Nordic aerosol with low
number concentrations, although showing pronounced sea-
sonal variation of especially Aitken mode particles; Moun-
tain sites (altitude over 1000 m a.s.l.) with a strong seasonal
cycle in aerosol number concentrations, high variability, and
very low median number concentrations. Southern and West-
ern European regions had fewer stations, which decreases the
regional coverage of these results. Aerosol number concen-
trations over the Britain and Ireland had very high variance
and there are indications of mixed air masses from several
source regions; the Mediterranean aerosol exhibit high sea-
sonality, and a strong accumulation mode in the summer. Abstract. The
greatest concentrations were observed at the Ispra station in
Northern Italy with high accumulation mode number concen-
trations in the winter. The aerosol number concentrations at
the Arctic station Zeppelin in Ny- ˚Alesund in Svalbard have
also a strong seasonal cycle, with greater concentrations of
accumulation mode particles in winter, and dominating sum-
mer Aitken mode indicating more recently formed particles. Observed particles did not show any statistically significant
regional work-week or weekday related variation in number
concentrations studied. particle formation in the marine environment (O’Dowd et al.,
2002), BIOFOR for biogenic aerosol production (Kulmala
et al., 2001), or ACE-2 for aerosol-cloud interactions (Raes
et al., 2000). However, this kind of information is sensitive
to the representativeness of the data on temporal and often
also on spatial scales. p
The EUSAAR (European Supersites for Atmospheric
Aerosol Research) project of the Sixth Framework Pro-
gramme of the European Commission is one of the steps to-
wards a reliable and quality-controlled network of measure-
ments (Philippin et al., 2009). The EUSAAR project has im-
proved and homogenized 20 European sites for measuring
aerosol chemical, physical and optical properties following a
standardized protocol of instrument maintenance, measure-
ment procedures and data delivery in common format to a
common data base (Wiedensohler et al., 2010). EUSAAR
also provided intercomparison and calibration workshops as
well as training for the station operators. The improvements
in EUSAAR stations have already proven to be extremely
useful in other European aerosol-related projects, such as
EUCAARI (Kulmala et al., 2009). The German Ultrafine
Aerosol Network (GUAN) is a network of multiple Ger-
man institutes with an interest on submicron aerosol prop-
erties (Birmili et al., 2009a), which has been established in
2008. The methodologies of number size distribution mea-
surements and data handling procedures in both GUAN and
EUSAAR networks are very similar and the size distribu-
tion measurements results are comparable between the two
networks. Analysis products are made for open-access to the research
community, available in a freely accessible internet site. The
results give to the modelling community a reliable, easy-to-
use and freely available comparison dataset of aerosol size
distributions. A. Asmi et al.: Submicron particles in Europe 2008–2009 5507 A. Asmi et al.: Submicron particles in Europe 2008–2009 The representative minimum dry diameter for CCN activa-
tion is often considered to be between 30 to over 100 nm, de-
pending on particle composition, used water supersaturation
and other factors (e.g. Dusek et al., 2006; Kerminen et al.,
2005; Andreae and Rosenfeld, 2008). In modelling stud-
ies, representative CCN numbers are often calculated directly
from dry aerosol size spectrum using a nominal cut-off diam-
eter, such as 70 nm (Spracklen et al., 2005; Makkonen et al.,
2009) or 50 nm (Pringle et al., 2009). The CCNs are not,
however, linearly connected to CDNCs, as the water deple-
tion and other cloud processes can increase the actual cut-off
diameter of cloud activation. In field experiments done at the
top of mountains, a semi-direct measurement of the activated
fraction to CDNC can be sometimes made, with activation
diameters observed from 40 nm to over 200 nm (Henning
et al., 2002; Sellegri et al., 2003; Lihavainen et al., 2008). Nevertheless, fixed activation diameters have been used as
surrogates in several CCN-to-CDNC parameterizations (e.g. Jones et al., 1994; Lin and Leaitch, 1997). Users of field experiment data may not be experts on the
measurement techniques, or be aware of the typical error
sources of experimental atmospheric data. Knowledge on
e.g. how the measurements were made, or which kind of data
are usable for different purposes are questions which are not
always clearly answered in all experimental datasets. The
use of the data requires very close co-operation with the ex-
perimentalist providing the data – a step not always easy to
do and sometimes regrettably overlooked by data users. This article provides an easy-to-use reference on aerosol
number concentrations and size distributions for dry diam-
eters between 30 and 500 nm for the years 2008 and 2009
at the EUSAAR and GUAN stations. We present number
concentrations of different aerosol size ranges and study the
diurnal, weekly and seasonal variability of aerosol number
concentrations at the stations. The analysis focuses on parti-
cle sizes with most potential for climate applications. 1
Introduction Atmospheric aerosols have multiple effects on climate, air
quality, human health and atmospheric visibility (e.g. Charl-
son, 1969; Horvath, 1993; Laden et al., 2006; Lohmann and
Feichter, 2005; Pope and Dockery, 2006; Stevens and Fein-
gold, 2009). To understand the effects of aerosol particles
on climate and health, measurements of their chemical and
physical properties, e.g. size distributions and concentrations
in the atmosphere, are needed. Several European campaign-
type projects have provided important information on the at-
mospheric aerosol properties in Europe, usually by concen-
trating on specific aerosol problems e.g. PARFORCE on new www.atmos-chem-phys.net/11/5505/2011/ Atmos. Chem. Phys., 11, 5505–5538, 2011 1.1
Relevant metrics and properties of sub-micron
size distributions The controlling variable for aerosol indirect effect is usu-
ally considered to be the number of particles activated. Par-
ticle number concentrations are usually dominated by parti-
cles with dry diameters less than 500 nm. We approximate
the N50 and N100 concentrations by integrating the size dis-
tribution to dp = 500 nm: The intent of this article is to produce relevant metrics de-
scribing the aerosol number size distributions observed at 24
EUSAAR and GUAN stations. Almost all stations have com-
parable size distribution measurements in dry particle diam-
eters between 30 and 500 nanometres, and thus we limit our
analysis to this size range only. N50(t) ≈
500nm
X
ˆdp=50nm
ni( ˆdp,t)
(1)
and
N100(t) ≈
500nm
X
ˆdp=100nm
ni( ˆdp,t)
(2) N50(t) ≈
500nm
X
ˆdp=50nm
ni( ˆdp,t)
(1) (1) The aerosol-climate effects are divided into two groups. The direct effect represents the ability of the particle popu-
lation to absorb and scatter short-wave radiation – directly
affecting the radiation balance. These direct effects depend
primarily on the aerosol optical properties and particle size
distribution (Haywood and Boucher, 2000; Charlson et al.,
1992). The indirect effects climate through the effects of
aerosol particles on clouds and is commonly concerned with
cloud albedo (or Twomey) effect and cloud lifetime effect. The cloud albedo effect is the resulting change in cloud ra-
diative properties due to changes in cloud droplet number
number concentration (CDNC); The lifetime effect is con-
nected to the changes in cloud properties and in drizzle and
precipitation (Twomey, 1974; Albrecht, 1989; Lohmann and
Feichter, 2005). 2.1.1
EUSAAR -European Supersites for Atmospheric
Aerosol Research EUSAAR is a EU-funded I3 (Integrated Infrastructures Ini-
tiatives) project carried out in the framework of the specific
research and technological development programme “Struc-
turing the European Research Area – Support for Research
Infrastructures”. It coordinates and harmonizes aerosol mea-
surements at 20 stations across Europe (see Fig. 2), 19 of
which are included in this study. All of the EUSAAR sta-
tions are also EMEP/GAW joint super sites. Another reason to study the sub-50 nm particles is related
to aerosol particle dynamics in atmosphere. By providing
a parameter for the smaller size ranges in the atmosphere,
we can take into account smaller particles generated from
new particle formation and combustion sources. This pro-
vides an additional parameter for model-measurement com-
parisons and a larger part of the particle spectrum and addi-
tional particle processes can be taken into account. We de-
scribe a concentration N30−50 as the number concentration
of particles from 30 nm to 50 nm as The regional background station Aspvreten is located in
S¨ormland, some 70 km south west of Stockholm. The sta-
tion is situated about 2 km from the coast in a rural area cov-
ered by mixed coniferous and deciduous forest with some
meadows. The influence from local anthropogenic activities
is small, and the area around the station is sparsely popu-
lated. The station is operated by the Department of Ap-
plied Environmental Science (ITM), Atmospheric Science
unit. The station is considered to be representative of the
regional background in Mid-Sweden. N30−50(t) =
50nm
X
ˆdp=30nm
ni( ˆdp,t)
(3) (3) where the smaller diameter limit of 30 nm comes mainly
from instrumental and site-related limitations and from try-
ing to limit the effect of actual new particle formation events
to variance. Particles and ions smaller than 30 nm dry diam-
eter were studied in detail by Manninen et al. (2010), using
instrumentation designed and calibrated for extremely small
particle diameters (Asmi et al., 2009). The Birkenes Observatory (BIR) is located in Southern
Norway and is run by NILU – Norwegian Institute for Air
Research. The terrain is undulating and the site is located
in a clearing with relatively free exposure to exchange of air
masses by wind, and with low local sources or pollution. 2.1.1
EUSAAR -European Supersites for Atmospheric
Aerosol Research The
station was moved to a nearby similar location in 2009, and
the data from July–December 2009 were from the new sta-
tion located in 58◦23′ 18′′ N 8◦15′ 7′′ E, 219 m a.s.l. Comparisons between measured and modelled concentra-
tions have usually been done using the arithmetic means of
the relevant quantities. We will show that at many stations
concentration histograms are slightly skewed log-normal dis-
tributions. A typical arithmetic mean comparison is of less
use for these distributions, as the values of linear means are
strongly affected by the outlier values. A way to compare
the results would then be the ability of a model to reproduce
the measured concentration histograms. We consider in this
article mostly the percentile values of the number size distri-
bution to represent the histogram – simultaneously showing
a comparable mean value (median of the distribution) and
some indication of the histogram shape and the concentra-
tion variance (other percentiles). For practical reasons, most
of the percentile values shown are presented in this article
only for the three middle percentile values (16th, 50th and
84th) – although the 5th and 95th percentile values are avail-
able in the comparison database (see Sect. 4.5). The station Pallas (PAL) is run by the Finnish Meteoro-
logical Institute. PAL is located on a top of a treeless hill
in a remote continental area at the northern border of the
boreal forest zone in Europe. The station receives polluted
European air masses, but also clean marine air from the Arc-
tic with very little continental influence. The frequent pres-
ence of clouds allows measurements of cloud microphysical
parameters and, during intensive campaigns, cloud chemical
parameters. The direct aerosol effect results from a combi-
nation of aerosol absorption and scattering. The particle ab-
sorption is primarily controlled by the amount of absorbing
compounds in the particle (e.g. Horvath, 1993). Aerosol scat-
tering is strongly connected to particle surface area and re-
fractive indices and is thus not easy to simulate from size dis-
tribution data. The Preila environmental pollution research
station (PLA) is located in western Lithuania at the coast of
the Baltic Sea, on the Curonian Spit. The station is operated
by Center for Physical Sciences and Technology, Lithuania. This monitoring site was selected according to strict criteria
designed to avoid undue influence from point sources, area
sources and local activities. and and N100(t) ≈
500nm
X
ˆdp=100nm
ni( ˆdp,t)
(2) (2) where ˆdp is the geometric mean diameter of the size inter-
val and ni is the measured aerosol number concentration in
the size interval (cm−3). The N50 and N100 concentrations
present two proxies for CCN-sized aerosol number concen-
trations. Figure 1 shows graphically the size ranges used in
the analyses. Note that the regions overlap for particle di-
ameters larger than 100 nm, and thus N100(t) ≤N50(t),∀t. The cut-off diameters of 50 and 100 nm correspond roughly
to critical supersaturations of 0.8 % and 0.3 % for Finnish
background aerosol (Sihto et al., 2010, κ = 0.18) and 0.4 %
and 0.15 % for pure ammonium nitrate particles (Petters and
Kreidenweis, 2007, κ=0.67). In high-CCN concentration re-
gions there is a need for a proxy for even larger sized CCNs,
and we have also calculated similar factor for particles larger
than 250 nm diameter (N250). The results, potential error
sources and discussion of N250 concentrations are shown in
Appendix B. The aerosol indirect effect is controlled by the ability of
particles to activate to cloud droplets (i.e. Cloud Condensa-
tional Nuclei, CCN) within a cloud (Andreae and Rosenfeld,
2008). This ability is a strong function of particle size, water
supersaturation, and particle hygroscopicity (chemical com-
position) (Lohmann and Feichter, 2005; McFiggans et al.,
2006). An extensive overview of most of these effects are
provided in McFiggans et al. (2006), who specified the par-
ticle size as the most important pre-requisite to get the acti-
vated fraction of particles correct. We consider the aerosol
size as the dominating factor for aerosol particle potential to
act as CCN, and thus the proxies of potential CCN are pro-
duced using only aerosol number size distribution data. From the above discussion, it is obvious that number con-
centrations of particles with dry diameters larger than 50 nm
are critical to the climate effects of particles. There are,
however, reasons to study particles with smaller diameters. Atmos. Chem. Phys., 11, 5505–5538, 2011 www.atmos-chem-phys.net/11/5505/2011/ 5508 A. Asmi et al.: Submicron particles in Europe 2008–2009 joint EUSAAR-GUAN station. joint EUSAAR-GUAN station. Particles with dp < 100 nm have been widely acknowledged
to have potential for adverse health effects (e.g. and Donaldson
et al., 1998; Hoet et al., 2004; Sager and Castranova, 2009),
although the knowledge which particles property is most im-
portant for health effects has not yet determined (Wittmaack,
2007, and related online correspondence). The particle de-
position to alveolar region of lungs is assumed to be espe-
cially efficient for particles of diameters between 10–50 nm
(Oberd¨orster et al., 2005). 2.1
Station descriptions The green area shows the re-
gion used to calculate N30−50 , the orange area N100 and the total
area of orange and teal are the N50 (N100 is part of N50). N250
concentration (Appendix B) is shown in dotted line. BEO Moussala (BEO) is run by the Bulgarian Academy
of Science. Because of the high elevation of the mountain
observatory and because BEO is located far from any local
source of pollution, the air is considered to be representa-
tive of synoptic scale atmospheric composition of the natural
free-tropospheric background (Nojarov et al., 2009). ZEP
ASP
BIR
PAL
PLA
SMR
VHL
CBW
MHD
PDD
JFJ
KPO
MPZ
OBK
BEO
FKL
HWL
CMN
BOE
HPB
SLL
ZSF
WAL
ISP
Fig. 2. Geographic distribution of stations used in this article. EU-
SAAR stations are marked with black and GUAN stations in blue. Station MPZ belongs also to the GUAN network. Circles denote
boundary layer sites and triangles sites of relatively high altitude. See Table 2 for stations’ codes. The Cabauw Experimental Site for Atmos. Res. CESAR
(CBW) is situated in an agricultural area in the western part
of The Netherlands, 44 km from the North Sea, in the vicin-
ity of cities such as Amsterdam, The Hague, Rotterdam and
Utrecht, and near major highways. Hence a variety of air
masses can be encountered from modified clean maritime to
continental polluted. The aerosol measurements used in this
paper are done by TNO. The Finokalia station (FKL) is run by the Environmental
Chemical Processes Laboratory (ECPL) of the University of
Crete. The FKL station is located in the SE Mediterranean
on the island of Crete. It is located far from local sources
of pollution, facing the sea within a sector 270◦to 90◦and
the air is considered to be representative of synoptic scale
atmospheric characteristics. Fig. 2. Geographic distribution of stations used in this article. EU-
SAAR stations are marked with black and GUAN stations in blue. Station MPZ belongs also to the GUAN network. Circles denote
boundary layer sites and triangles sites of relatively high altitude. See Table 2 for stations’ codes. The Harwell Station (HWL) is operated by the Univer-
sity of Birmingham, primarily on behalf of the Department
for Environment, Food and Rural Affairs (DEFRA) as a ru-
ral station representative of large scale air masses affecting
Southern England. 2.1
Station descriptions The SMEAR II station (Station for Measuring Forest
Ecosystem-Atmosphere Relations) in Hyyti¨al¨a (220 km NW
from Helsinki) is run by the University of Helsinki, Finland. Data from 24 stations are used in this study; 18 are EUSAAR
stations, 5 are from German GUAN network and one is a Atmos. Chem. Phys., 11, 5505–5538, 2011 www.atmos-chem-phys.net/11/5505/2011/ A. Asmi et al.: Submicron particles in Europe 2008–2009 5509 10
1
10
2
10
3
Particle diameter d [nm]
10
0
0
500
1000
1500
dn / d(log10dp)
N50
N100
measured in almost all
stations, but not
quantifiably
comparable
-3
[cm ]
N250
N30-50
Fig. 1. Size regions used in the analysis. The red lines show the
size cut-offs from 30 nm to 500 nm. The green area shows the re-
gion used to calculate N30−50 , the orange area N100 and the total
area of orange and teal are the N50 (N100 is part of N50). N250
concentration (Appendix B) is shown in dotted line. 10
1
10
2
10
3
Particle diameter d [nm]
10
0
0
500
1000
1500
dn / d(log10dp)
N50
N100
measured in almost all
stations, but not
quantifiably
comparable
-3
[cm ]
N250
N30-50 pollution, situated in the southernmost part of Sweden. It is
well-suited for studies of the influx of polluted air from con-
tinental Europe to the Nordic countries along a south-north
transect. The K-puszta station (KPO) is run by the Hungarian Me-
teorological Service and University of Vezprem. The sam-
pling site is relatively far from anthropogenic sources, it is
80 km from Budapest in the SE direction and the largest
nearby town is about 15 km SE from the station. Samples
collected at K-puszta are considered to be representative of
the Central-Eastern European regional conditions. 10
3 The Observatory Kosetice (OBK) is a regional background
monitoring station run by the Czech Hydrometeorological
Institute within the national Air Quality monitoring network. The aerosol measurements are carried out by ICPF. The ob-
servatory is located in agricultural countryside outside of ma-
jor population centres in the southern part of the Czech Re-
public far from local source of pollution. Fig. 1. Size regions used in the analysis. The red lines show the
size cut-offs from 30 nm to 500 nm. 2.1
Station descriptions There are however, periods of easterly
winds where it is directly in the plume from London (ap-
prox 80 km distance) and there are very marked differences
between air composition from easterly and westerly trajecto-
ries (Charron et al., 2008). It includes several measurement towers, and a cottage for in-
struments and computers. The air quality at the site is con-
sidered to represent typical regional background conditions
for higher latitudes of Europe. The air masses are influenced
by European pollution but at times very clean Arctic air is
observed. The station is located within a Scots pine stand. It includes several measurement towers, and a cottage for in-
struments and computers. The air quality at the site is con-
sidered to represent typical regional background conditions
for higher latitudes of Europe. The air masses are influenced
by European pollution but at times very clean Arctic air is
observed. The station is located within a Scots pine stand. The high Altitude Research Station Jungfraujoch (JFJ) is
operated by the International Foundation High Altitude Re-
search Stations Jungfraujoch and Gornergrat and is part of
numerous networks. The aerosol measurements are done by
PSI. JFJ is located far from local sources and is well suited to
determine the regional background above a continental area. The station of Vavihill (VHL) is operated since 1984, and
additional laboratory facilities and aerosol equipment were
installed by Lund University starting 1999. VHL is a con-
tinental regional background site with no local sources of Atmos. Chem. Phys., 11, 5505–5538, 2011 www.atmos-chem-phys.net/11/5505/2011/ A. Asmi et al.: Submicron particles in Europe 2008–2009 5510 Table 2. Locations and names of stations used in the data analysis. The site altitudes are given in reference to standard sea level. The areas
indicated are grouped by European sub-divisions using definitions from Central Intelligence Agency (2009). Country codes are given in ISO
3166 standard. Table 2. Locations and names of stations used in the data analysis. The site altitudes are given in reference to standard sea level. The areas
indicated are grouped by European sub-divisions using definitions from Central Intelligence Agency (2009). Country codes are given in ISO
3166 standard. andard. Station name
Station code
Country
Coordinates, altitude
Categorya
Instrument
Reference
Low altitude sites
(less than 1000 m a.s.l.)
Nordic and Baltic
Aspvreten
ASP
SE
58◦48′ N, 17◦23′ E, 30 m
DMPS
Tunved et al. 2.1
Station descriptions (2004)
Birkenesb
BIR
NO
58◦23′ N, 8◦15′ E, 190 m
2
DMPS
Amunsen et al. (1992)
Pallas
PAL
FI
67◦58′ N, 24◦7′ E, 560 m
DMPS
Lihavainen et al. (2008)
Preila
PLA
LT
55◦55′ N, 21◦0′ E, 5 m
6
SMPS
Ulevicius et al. (2010)
SMEAR II
SMR
FI
61◦51′ N, 24◦17′ E, 181 m
DMPS
Hari and Kulmala (2005)
Vavihill
VHL
SE
56◦1′ N, 13◦9′ E, 172 m
DMPS
Kristensson et al. (2008)
Central Europe
B¨osel
BOE
DE
53◦N, 7◦57′ E, 16 m
SMPS
Birmili et al. (2009a)
K-Puszta
KPO
HU
46◦58′ N, 19◦33′ E, 125 m
1
DMPS
Kiss et al. (2002)
Melpitz
MPZ
DE
51◦32′ N, 12◦54′ E, 87 m
DMPS
Engler et al. (2007)
Kosetice
OBK
CZ
49◦35′ N, 15◦5′ E, 534 m
1
SMPS
˘Cervenkov´a and V´a˘na (2010)
Hohenpeissenberg
HPB
DE
47◦48′ N, 11◦E, 988 m
1
SMPS
Birmili et al. (2003)
Waldhof
WAL
DE
52◦31′ N, 10◦46′ E, 70 m
SMPS
Birmili et al. (2009a)
Western Europe
Cabauw
CBW
NL
51◦18′ N, 4◦55′ E, 60 m
3
SMPS
Russchenberg et al. (2005)
Harwell
HWL
UK
51◦34′ N, 1◦19′ W, 60 m
3
SMPS
Charron et al. (2007)
Mace Head
MHD
IE
53◦19′ N, 9◦53′ W, 5 m
5
SMPS
Jennings et al. (1991)
Mediterranean
Finokalia
FKL
GR
35◦20′ N, 25◦40′ E, 250 m
2
SMPS
Mihalopoulos et al. (1997)
JRC-Ispra
ISP
IT
45◦49′ N, 8◦38′ E, 209 m
3
DMPS
Gruening et al. (2009)
Arctic
Zeppelin
ZEP
NO
78◦55′ N, 11◦54′ E, 474 m
DMPS
Str¨om et al. (2003)
High altitude sites
(over 1000 m a.s.l.)
Western Europe
Puy de Dˆome
PDD
FR
45◦46′ N, 2◦57′ E, 1465 m
4
SMPS
Venzac et al. (2009)
Central Europe
Schauinsland
SCH
DE
47◦55′ N, 7◦55′ E, 1210 m
1
SMPS
Birmili et al. (2009a)
Zugspitze
ZSF
DE
47◦25′ N, 10◦59′ E, 2650 m
4
SMPS
Birmili et al. (2009a)
Jungfraujoch
JFJ
CH
46◦32′ N, 7◦59′ E, 3580 m
2
SMPS
Jur´anyi et al. (2011)
Balkans
BEO Moussala
BEO
BG
42◦10′ N, 23◦35′ E, 2971 m
SMPS
Nojarov et al. (2009)
Mediterranean
Monte Cimone
CMN
IT
44◦11′ N, 10◦41′ E, 2165 m
4
DMPS
Marinoni et al. (2008)
n representativeness classification from Henne et al. 2.1
Station descriptions (2010) (if available):
al; (2) Mostly remote; (3) Agglomeration; (4) Weakly influenced; (5) Generally remote; (6) Weakly influenced, generally rem
n was moved in summer 2009 to location 58 ◦23′ N 8◦15′ E, 219 m a.s.l. Since the station is within clouds 40 percent of the time,
aerosol – cloud interactions can be studied as well. and is managed through the Mace Head Management Com-
mittee. MHD is GAW global baseline station a designated
EMEP supersite. MHD has open exposure to the North At-
lantic ocean and is considered to be representative of rela-
tively clean background marine air, although it encounters
some anthropogenic pollution events. The JRC-Ispra atmospheric research station (ISP) is run
by the Institute for Environment and Sustainability of the EC The JRC-Ispra atmospheric research station (ISP) is run
by the Institute for Environment and Sustainability of the EC
– DG Joint Research Centre. ISP is located a tens of kms
from local sources of pollution and is generally represen-
tative of the regional (quite polluted) atmospheric regional
background. – DG Joint Research Centre. ISP is located a tens of kms
from local sources of pollution and is generally represen-
tative of the regional (quite polluted) atmospheric regional
background. Melpitz (MPZ) is an atmospheric research station in East-
ern Germany, 40 km northeast of Leipzig. The station is
surrounded by flat and semi-natural grasslands without any
obstacles, as well as agricultural pastures and forests in the The atmospheric research station Mace Head (MHD) is a
major facility of the National University of Ireland, Galway Atmos. Chem. Phys., 11, 5505–5538, 2011 www.atmos-chem-phys.net/11/5505/2011/ A. Asmi et al.: Submicron particles in Europe 2008–2009 5511 only from five rural regional background stations in GUAN. One of them, MPZ, is a joint station of EUSAAR and GUAN
and was described above. wider environment. Atmospheric observations at MPZ can
be regarded as representative of regional background condi-
tions in Central Europe, as shown by a multiple-site compar-
ison within the GUAN (Birmili et al., 2009a). The air masses
observed at MPZ tend to partition into Atlantic (westerly) air
masses with low particle mass concentrations, and continen-
tal (easterly) air masses with high particle mass concentra-
tions (Engler et al., 2007; Spindler et al., 2010). MPZ is part
of the EUSAAR and GUAN networks. 2.1
Station descriptions B¨osel (BOS) is a regular site in the government air qual-
ity monitoring system of Lower Saxony (L ¨UN, Staatliches
Gewerbeaufsichtsamt Hildesheim). To the south, the sam-
pling site borders agricultural pastures while to the north, it
touches some residential areas of the village of B¨osel. Some
noticeable features of the site include its location in an area
of intense livestock production (including enhanced ammo-
nia emissions), and the limited distance from the North Sea
coast (100 km). The “O. Vittori” Station at Monte Cimone (CMN), hosted
in a military site of the Italian Air Force, is run by ISAC-CNR
and is part of GAW. Monte Cimone is the highest peak of the
Northern Italian Apennines (2165 m a.s.l.), characterized by
a 360◦free horizon. It is located south of the Po Basin and is
a strategic platform to study chemical and physical character-
istics of regional background conditions and air mass trans-
port. The measurements of atmospheric compounds carried
out at CMN can be considered representative of the South
European and North Mediterranean free troposphere (Fischer
et al., 2003). Nevertheless, due to enhanced vertical mixing
and a mountain breeze wind regime, during the warm months
an influence from the lowest layer of the troposphere cannot
be ruled out (Marinoni et al., 2008; Cristofanelli et al., 2007). Waldhof (WAL) is a measurement station of the German
Federal Environment Agency (UBA). The site is located
in the biggest north German forest and heath environment
(L¨uneburger Heide) and is therefore only very little influ-
enced by local anthropogenic sources. Measurements here
are considered to be representative of the remote regional
background in the North German lowlands. The nearest
larger cities are Hanover and Hamburg. WAL is also an
EMEP station and a regional GAW site. Schauinsland (SSL) is another key measurement station of
the German Federal Environment Agency (UBA), with at-
mospheric composition measurements dating back to 1967. The station is situated on a mountain ridge in the Black For-
est, southwest Germany. SSL is located more than 1000 m
above the upper Rhine river valley, where pollution sources
are ubiquitous. As is usual for a mid-level mountain station,
boundary layer dominates during the day, while at night —
especially in winter, the station often resides above the sur-
face inversion. The station is well-suited to characterize air
masses that approach Central Europe from westerly direc-
tions. SSL is also an EMEP station. 2.1
Station descriptions The Puy de Dˆome station (PDD) is run by OPGC/CNRS-
LaMP and is located a few tens of km from Clermont-
Ferrand, France, in the Massif Central mountain chain. Due
to its high altitude (1465 m a.s.l) the air mass composition is
representative of the regional atmospheric background dur-
ing the day and more representative of larger synoptic-scale
air masses during night-time. The Zeppelin Observatory in Ny- ˚Alesund (ZEP) at Sval-
bard is owned and operated by the Norwegian Polar Insti-
tute. NILU is responsible for the scientific programmes at
the station, as part of the largest Arctic research infrastruc-
ture. The ongoing monitoring is performed in cooperation
with Stockholm University (SU). The site is located in an
undisturbed Arctic environment. Zeppelin Mountain is an
excellent site for atmospheric monitoring, with minimal con-
tamination from the local settlement due to its location above
the inversion layer. ZEP is a global GAW site. Hohenpeissenberg (HPB) is a climate observatory of the
German Meteorological Service (DWD) hosting a wide
range of atmospheric in-situ and remote sensing observa-
tions. HPB is also a GAW station. The site is located on
an isolated mountain, about 60 km south of Munich, and
about 40 km north of Zugspitze, Germany’s highest eleva-
tion on the northern edge of the Alps. Separated from the
surrounding countryside by an elevation difference of 300 m,
the HPB site avoids immediate contamination by nearby an-
thropogenic sources. 2.2
Instrumentation Two types of instruments for measuring ultrafine particle size
distributions were used to obtain the results presented in this
article: The Differential Mobility Particle Sizer (or DMPS),
and the Scanning Mobility Particle Sizer (SMPS). Both are
very similar instruments in their operation: they aspirate
dried air, use ionizing radiation to establish an equilibrium
bipolar charge-distribution in the sampled aerosol (Wieden-
sohler, 1988), use a cylindrical differential mobility analyser
to select particles based on their electrical mobility and use a
condensation nuclei counter to measure the resulting concen-
trations in each of the selected size ranges (McMurry, 2000;
Laj et al., 2009; Wiedensohler et al., 2010). The main dif-
ference between these instruments is the mode of operation,
as the DMPS keeps the differential mobility analyser voltage
constant during measurement of a single size interval and the
SMPS scans with continuously differing voltages. The size
range and time resolution of a DMPS or a SMPS system de-
pends on the system architecture (e.g. the physical dimen-
sions of the instrument and the flow rates used) and on user
choice (more size channels vs. faster scanning). in the electrical field is identical to the drag force includ-
ing the Cunningham correction (Hinds, 1999). The choice of
this diameter comes naturally from the experimental set-up,
as the size-resolving instruments in this paper (SMPS and
DMPS) use electrical mobility diameter as the basis of their
size-selection. The most important feature of this diameter
is that for spherical particles the electrical mobility diameter
is identical to the geometric diameter. For DMPS and SMPS
data, the actually reported diameter is usually the interval-
average diameter ˆdp, which is the geometric mean diameter
of the size interval used in the DMPS or SMPS inversion. The number concentration is defined as the number of
aerosol particles in a 1 cubic centimetre (10−6m−3) volume
of air at STP1. The difference from the actual environmental
situation on-site can be significant especially for mountain
sites. The size spectra are reported, both in databases and in
size distribution figures of this article, as a particle number
size distribution function
dn(dp)
dlog10dp . This function provides
comparable size spectra even thought the widths of intervals
are different in different instruments. 1EUSAAR and GUAN follow GAW (Global Atmosphere
Watch) recommendation for STP. The STP air is defined to be
T = 293.15 K, P = 101300 Pa (WMO/GAW, 2003). 2.2.1
Uncertainty of mobility size
spectrometer measurements Two aspects have to be considered determining the uncer-
tainty of particle number size distributions. First, as known
from intercomparison in the past (Khlystov et al., 2001; Dah-
mann et al., 2001; Imhof et al., 2006; Helsper et al., 2008)
and the EUSAAR calibration workshops Wiedensohler et al. (2010), the unit-to unit variability between mobility size
spectrometer can be up to 25 % in the size range 20-500 nm. The instruments used at the EUSAAR stations performed
were within 10 % for the size range 20–200 nm against the
EUSAAR reference system under controlled laboratory con-
ditions, while for sizes up to 500 nm the deviation was up to
25 %. The overall N100 concentrations are usually within 10
% of the reference instrument (Fig. 7 of Wiedensohler et al.,
2010). This data were achieved after averaging ambient mea-
surements of a time period of few hours. Additionally, a short
term variation due to aerosol flow instability might be up to
5 % depending on the internal flow control. These variations
between the instruments and the short term variability (sta-
bility) of the instruments should be taken into account when
drawing conclusions on the variabilities and differences be-
tween aerosol number size distributions. The measured aerosol properties are measured for dry
aerosol samples. The sample air is dried to relative humidity
RH ≤40 % before size selection. The results are categorized by season. We divided the sea-
sons according to the calendar year as indicated in Table 3. This selection is arbitrary, but should show the main meteo-
rological and seasonal variations observed in the datasets and
provides a way to compare the results with short-term sim-
ulations as done with regional Chemical Transport Models
(CTMs). 2.1.2
GUAN – German Ultrafine Aerosol Network The German Ultrafine Aerosol Network (GUAN; Birmili
et al., 2009a) is a cooperation of federal and state-based en-
vironment agencies as well as research institutes. Under
the umbrella of the German Federal Environment Agency
(UBA), GUAN investigates the sources, atmospheric pro-
cesses as well as climatological and health effects related to
ultrafine aerosol particles and soot over Germany. To date,
GUAN conducts particle number size distribution and soot
measurements at 14 measurement stations across Germany. The range of sites in GUAN spans remote regional back-
ground, rural regional background, urban, as well as roadside
observation sites. In this article, we use measurement data Zugspitze-Schneefernerhaus (ZSF) is the highest moun-
tain of the German Alps. It is located in Southern Ger-
many, about 90 km southwest of Munich, at the Austrian
border near the town of Garmisch-Partenkirchen. The GAW
monitoring program is operated jointly by the Federal En-
vironmental Agency (UBA) and the German Meteorological
Service (DWD). Its high altitude causes an annual cycle in
aerosol particle number and mass concentration as a result
of different different boundary layer heights in summer and
winter (Birmili et al., 2009b). The Zugspitze’s elevated posi-
tion allows characterisation of air masses that had only little www.atmos-chem-phys.net/11/5505/2011/ Atmos. Chem. Phys., 11, 5505–5538, 2011 A. Asmi et al.: Submicron particles in Europe 2008–2009
Table 3. Definition of seasons used in analysis given as Day of
Year. Note that 2008 was a leap year. Season
Months
DOY 2008
DOY 2009
Winter
Dec, Jan, Feb
1–60, 336–366
1–59, 335–365
Spring
Mar, Apr, May
61–152
60–151
Summer
Jun, Jul, Aug
153–244
152–243
Autumn
Oct, Sep, Nov
244–335
243–334 A. Asmi et al.: Submicron particles in Europe 2008–2009 5512 contact with the local boundary layer, and may therefore be
representative of a very large continental area. Table 3. Definition of seasons used in analysis given as Day of
Year. Note that 2008 was a leap year. 2.2.3
Data handling The processing of raw data to physical quantities is detailed
in Wiedensohler et al. (2010). The data inversion and pre-
screening were done by the institutes operating the instru-
ments. The data was flagged for instrument failure or main-
tenance breaks. Other important features related to the mea- A. Asmi et al.: Submicron particles in Europe 2008–2009 Table 4. Mean values of the station concentrations. n is the arithmetic mean concentration in the size section in question, ˆn is the geometric
mean concentration and the µx values are the x-th percentage of the concentration distribution. µ50, or median value, is bolded. All
concentrations are in [cm−3] . Refer to Sect. 2.2.1 for details on instrumental uncertainties. A. Asmi et al.: Submicron particles in Europe 2008–2009 N30−50 concentration [cm−3]
N50 concentration [cm−3]
N100 concentration [cm−3]
Station
Code
n
ˆn
µ05
µ16
µ50
µ84
µ95
n
ˆn
µ05
µ16
µ50
µ84
µ95
n
ˆn
µ05
µ16
µ50
µ84
µ95
Aspvreten
ASP
397
279
72
128
284
662
1097
1234
959
246
461
1081
1992
2751
543
396
97
181
421
904
1362
Birkenes
BIR
237
146
27
57
156
393
710
706
443
69
156
511
1288
1947
327
202
38
69
218
583
1007
Pallas
PAL
198
89
12
25
89
345
739
387
187
20
53
205
769
1352
211
80
3
20
111
402
782
Preila
PLA
1042
564
120
236
610
1386
2272
3581
2233
485
1042
2527
4741
7068
2030
1149
256
466
1303
2490
4407
SMEAR II
SMR
345
213
41
85
223
562
1063
1053
779
185
364
878
1730
2487
450
320
76
141
341
752
1156
Vavihill
VHL
550
396
93
180
440
852
1320
1539
1231
360
618
1368
2449
3388
632
490
136
238
527
1041
1471
B¨osel
BOS
1314
1088
411
627
1122
1909
2921
2973
2417
691
1294
2679
4569
6261
1428
1108
275
539
1250
2307
3222
K-Puszta
KPO
979
687
242
372
697
1414
2741
3669
2992
1098
1764
3120
5613
8017
1952
1580
539
901
1660
2976
4366
Kosetice
OBK
958
687
259
386
700
1428
2571
3518
2900
1098
1791
3194
5198
6945
2058
1687
606
1029
1863
3054
4112
Hohenpeißenberg
HPB
502
405
158
229
407
735
1137
1418
1131
295
556
1325
2200
2916
792
594
116
260
739
1270
1657
Melpitz
MPZ
1187
801
222
425
860
1634
3238
2681
2179
723
1222
2327
4078
5999
1487
1193
363
629
1304
2324
3271
Waldhof
WAL
1135
851
252
439
878
1715
2823
2749
2234
694
1157
2434
4270
5770
1341
1081
318
552
1189
2131
2860
Cabauw
CBW
2137
1642
373
846
1914
3401
4599
2910
2883
568
1290
3387
6487
9105
1240
845
159
338
952
2171
3258
Harwell
HWL
770
520
98
211
582
1289
2060
1833
1270
276
524
1332
3282
4991
827
549
131
230
553
1470
2400
Mace Head
MHD
257
107
15
30
105
437
877
632
289
57
95
241
1193
2697
337
167
37
61
142
625
1343
Finokalia
FKL
316
211
55
90
220
498
918
1624
1210
275
542
1345
2707
3861
949
691
150
288
779
1600
2368
JRC-Ispra
ISP
1617
1302
476
734
1341
2386
3714
5571
4165
1040
2032
4448
9334
13918
2888
1943
363
829
2129
4953
8317
Zeppelin
ZEP
82
9
1
2
8
54
147
138
53
3
17
68
213
363
61
27
1
8
37
122
202
Puy de Dˆome
PDD
402
279
75
127
291
624
1112
1155
671
80
203
820
2238
3180
591
283
19
63
381
1203
1821
Schauinsland
SSL
546
407
127
200
418
854
1349
1436
1060
220
469
1242
2407
3362
790
558
98
234
678
1342
1887
Zugspitze
ZSF
269
183
42
79
197
444
724
770
437
66
137
474
1532
2280
451
222
26
60
241
945
1470
Jungfraujoch
JFJ
113
76
21
36
79
166
301
313
184
29
69
195
575
1012
166
81
8
26
87
321
606
BEO Moussala
BEO
272
145
35
56
137
415
936
1072
548
50
144
704
2099
3247
644
293
20
63
399
1338
1933
Monte Cimone
CMN
343
206
48
92
204
486
1104
890
503
48
142
667
1643
2519
504
238
11
51
356
987
1440 3
Results surements were stored in EMEP data flags in the meta data
stored together with the dataset. For the results provided in
this paper, only EUSAAR level II data marked as a “Valid
measurement” were used. The data are provided in the
database as one-hour geometric or arithmetic mean values,
with EUSAAR current recommendation being arithmetic av-
erage. Before analysing the data for this article, the we tested
the averaging methods using high time resolution data from
stations SMR and JFJ. The differences in time-averaging did
not have a substantial effect on any of the concentrations used
in this paper (not shown). The geometric mean value calcu-
lations had a limit for the low value of concentration, which
is set to 10−6 particles per cubic centimetre. This value could
have an effect on the geometric mean values of periods with
extremely low concentrations of particles. 3.1
Data coverage The overall seasonal coverage (Fig. 3) was adequate for de-
termining main features of the concentrations. The data cov-
erages for both years are also tabulated in Table 5. Although
the data coverage was generally good, the coverage was at
some stations fragmentary. 2.2.2
General comments on aerosol properties Particle diameter. In this article particle we use electri-
cal mobility diameter dp, which is defined as the diameter
where the electrostatic force of a (single) charged particle Atmos. Chem. Phys., 11, 5505–5538, 2011 www.atmos-chem-phys.net/11/5505/2011/ 5513 A. Asmi et al.: Submicron particles in Europe 2008–2009 Atmos. Chem. Phys., 11, 5505–5538, 2011 3.2
Seasonality and variability of size distributions Total
2008
2009
σ(n)
σg(n)
A1(n)
σ(n)
σg(n)
A1(n)
σ(n)
σg(n)
A1(n)
ASP
77
61
93
307
2.25
0.85
747
2.09
0.95
383
2.21
0.97
BIR
47
51
43
209
2.62
0.91
575
2.73
0.96
288
2.68
0.97
PAL
80
73
87
229
3.41
0.91
404
3.7
0.97
232
6.02
0.97
PLA
23
0
46
666
2.4
0.91
2202
2.2
0.94
1385
2.31
0.94
SMR
98
99
97
315
2.57
0.89
686
2.28
0.95
324
2.26
0.96
VHL
80
80
79
377
2.21
0.91
888
1.97
0.95
397
2.04
0.96
BOS
53
18
87
724
1.76
0.84
1615
1.93
0.94
865
2.07
0.95
KPO
55
45
64
761
2.67
0.87
2032
2.0
0.95
1101
2.01
0.95
OBK
75
64
87
702
2.67
0.88
1713
2.11
0.93
1018
2.1
0.94
HPB
59
20
98
298
1.79
0.9
781
2.01
0.94
467
2.28
0.95
MPZ
89
92
86
967
2.48
0.82
1517
1.92
0.94
854
1.99
0.96
WAL
50
4
97
774
2.02
0.89
1510
1.9
0.96
752
1.94
0.97
CBW
57
57
58
1238
2.21
0.86
2225
2.31
0.92
931
2.5
0.95
HWL
64
74
54
581
2.44
0.91
1406
2.4
0.96
685
2.44
0.96
MHD
66
66
67
272
3.41
0.93
784
3.14
0.96
396
2.89
0.96
FKL
27
17
37
259
2.31
0.92
1064
2.2
0.95
654
2.28
0.96
ISP
81
92
71
985
1.82
0.83
3868
2.17
0.94
2392
2.51
0.96
ZEP
77
78
77
141
5.33
0.91
133
4.66
0.93
61
5.4
0.98
PDD
57
81
33
309
2.23
0.87
968
3.17
0.95
552
4.18
0.95
SSL
88
93
83
379
2.02
0.9
933
2.29
0.95
535
2.5
0.96
ZSF
61
24
98
206
2.36
0.91
699
3.12
0.97
450
3.68
0.97
JFJ
76
75
78
89
2.25
0.9
293
2.93
0.95
180
3.67
0.95
BEO
43
0
87
306
2.69
0.86
989
3.72
0.95
620
4.43
0.96
CMN
60
51
68
341
2.43
0.88
749
3.39
0.95
450
4.54
0.95 a slightly larger diameter of median diameter of the Aitken
mode. The number size distributions were consistently lower
in winter at all Nordic stations. The influence of nucleation
for the smallest particles is not clearly visible on the 84th
percentile size distributions, although some elevated number
concentrations of sub-20 nm particles are visible at the SMR
and PAL stations in spring and autumn. All of the stations
show a bimodal median number size distributions. 3.2
Seasonality and variability of size distributions As discussed earlier, the use of only median or mean values
are not necessarily representative of the concentrations and
size distributions observed. We present number size distribu-
tions in seasonally calculated percentiles of the size-specific
concentrations. Figures 4, 5, 6 and 7 show the number size
distribution variation of Nordic and Baltic stations, Central
European stations, high altitude (or mountain) stations and
other stations respectively. The Figures show the median,
16th and 84-th percentile of each measured size section. The
shaded areas between 16-th and 84-th percentiles are indica-
tive of each size interval dn/dlog10dp histogram shape. To
guide the eye on the scales, plots have a horizontal dotted
line indicating dn/dlog10dp of 1000 cm−3. Additionally, we
fitted log-normal mode or modes to the median size distribu-
tions. These fitted parameters are discussed in Appendix A. y
p
Before analysis, the files were analysed to ensure that the
time-vectors and the concentration data were of the right for-
mat and comparable with each other. For stations with mul-
tiple size resolutions over the analysis period, the size distri-
bution functions were interpolated to uniform size intervals. This interpolation had no effect on the integrated concentra-
tions. Station specific night-time filters were calculated using
a sunrise/sunset algorithm, defining daytime as time when
any part of the sun was above the visible horizon (Duffett-
Smith, 1988). For the geometric mean and standard devia-
tion calculation, a filter was used to remove zero concentra-
tions from the data and replace them with a concentration
10−6 cm−3. The aerosols at the Nordic stations in Fig. 4 show several
similarities. The number size distributions of particles are
greater in summer than in other seasons, especially for the
Aitken mode particles with dry diameters from 50 to 100 nm. This elevated number size distributions are accompanied by www.atmos-chem-phys.net/11/5505/2011/ Atmos. Chem. Phys., 11, 5505–5538, 2011 A. Asmi et al.: Submicron particles in Europe 2008–2009 5514 Table 5. Data coverage and standard deviations (σ(n), in cm−3), geometric standard deviations (σg(n), unitless) and 1-h autocorrelation
rates (A1, unitless) of concentrations. Data Coverage ( %)
N30−50
N50
N100
Stat. 3.2
Seasonality and variability of size distributions The more
northern PAL station size distributions had similar season-
ality, but number size distribution level was about half that
of the other Nordic stations with more pronounced summer
effect than at ASP, SMR or VHL. The Nordic station size dis-
tributions are well in agreement with earlier studies (Tunved
et al., 2003; Dal Maso et al., 2008). trations in accumulation mode in winter and autumn. The
concentrations of sub-30 nm particles are elevated at HPB
in spring and MPZ and OBK in summer, suggesting con-
tribution of nucleated particles to the size spectrum. All
sites, except HPB – which is close to being a high-altitude
site (988 m) – show remarkably similar median number
size spectra and concentrations suggesting a relatively sta-
ble number size distribution over the central European plain
from Germany to Hungary. At high-altitude sites (defined as height over 1000 m above
mean sea level), aerosol number size distributions are simi-
lar, even though there are large spatial distances between the
stations (Fig. 6). The number concentrations were low com-
pared to nearby lowland sites. The number size distributions
generally show bimodal behaviour, although the modes are
overlapping at some of the stations. The seasonal cycle is
similar at all sites, with greater concentrations during sum-
mer, especially for particles over 70 nm in diameter. The
intra-seasonal variability is considerable especially in sum-
mertime, suggesting a range of different types of airmasses –
most likely boundary layer air during daytime and clear tro-
pospheric air during nighttime (see e.g. Venzac et al., 2009;
Weingartner et al., 1999). The Baltic PLA station has different aerosol distribution
shapes than Nordic stations. The spring and summer number
size distributions shapes were close to those observed at ASP
and VHL, but with with a wider, almost unimodal, distribu-
tion shape. The winter and autumn concentrations had larger
median diameters, but much lower Aitken mode concentra-
tions. The Central European stations in Fig. 5 show generally
greater concentrations than at the Nordic stations. All Cen-
tral European stations have very similar size distributions re-
gardless of the season, although KPO had elevated concen- www.atmos-chem-phys.net/11/5505/2011/ Atmos. Chem. Phys., 11, 5505–5538, 2011 A. Asmi et al.: Submicron particles in Europe 2008–2009 5515 Fig. 3. Data coverage of the stations. Colours indicate the valid
data per season. 3.3
Particle number concentrations in different
size ranges Number concentrations in different size ranges (N30−50 , N50
and N100) of the stations are presented as histograms of the
number concentrations plotted with logarithmically even size
intervals of 20 sections per decade (Fig. 8). All of the his-
tograms are normalized by the total number of valid measure-
ments, and the sum of the seasonally separated histograms
(lines in colour) is equal to the total number concentration
histogram (in black). The annual concentration histogram of
the night-time values (determined as described in Sect. 2.2.3)
is shown as shaded area. The part of the total number concen-
tration histogram above the shaded area is the part of the con-
centrations coming from the day-time measurements. Fig-
ures 9, 10 and 11 show the histograms of the N30−50, N50
and N100 concentrations. Fig. 3. Data coverage of the stations. Colours indicate the valid
data per season. Notable is that all, except one (spring at BIR), of
the stations had at least some coverage in all seasons, and most of
the stations were operating in both years 2008 and 2009. The Western European stations (Fig. 7 top row) show
more inter-station variability. Station CBW size distribu-
tions have similar behaviour as Central European stations,
with high concentrations and almost unimodal size distribu-
tion and small differences between seasons. The HWL and
MHD stations have high seasonal variation and large vari-
ance of intra-seasonal concentrations. The HWL station data
show a prominent spring-summer maximum in all sizes from
30 to 70 nm. Winter and autumn distributions lack this maxi-
mum in the smallest size ranges. During the summer months,
MHD presents relatively large variation with number size
distribution maxima in the 30 to 50 nm diameter – with the
rest of the seasons having much lower concentrations. The
variability was probably due to occurrences of both clean At-
lantic and polluted local airmasses and the maximum value at
MHD during summer months can be attributed to enchanted
marine biota activity which increases the sub-micron parti- The stations in Northern Europe are especially sensitive to
a selection bias of day-night difference, as the length of day
is highly variable between the seasons. Stations PAL and
ZEP have parts of the year completely in night-time and part
completely in day-time, causing the summer and winter to
be overly represented in night-time and day-time histograms. 3.2
Seasonality and variability of size distributions Notable is that all, except one (spring at BIR), of
the stations had at least some coverage in all seasons, and most of
the stations were operating in both years 2008 and 2009. cle concentration of non-seasalt sulphate and organic aerosol
(Yoon et al., 2007). At the Mediterranean station FKL, the aerosol number size
distributions were bimodal for winter with an Aitken mode
around 50 nm and accumulation mode 150 nm. The spring
and summer were dominated by strong accumulation mode at
around 100 nm. At ISP station in Northern Italy (Fig. 7) the
concentrations were much greater. The ISP number concen-
trations were comparable with most polluted Central Euro-
pean station number concentrations in summer and autumn. The winter and autumn median distributions show extremely
high number concentrations of accumulation mode particles
around 100 nm. The only Arctic station, ZEP, has a very distinctive sea-
sonal cycle (Fig. 7). During winter and autumn, the aerosol
concentrations were very low with their maxima at around
200 nm. The springtime distributions are dominated by Arc-
tic haze, strongly increasing the concentrations in accumu-
lation mode. In summertime, the distribution changes to
very clean marine bimodal distribution, with a strong Aitken
mode around 30 nm. This seasonal change is connected
to different meteorological situations, daylight as well as
changes in ocean ice cover. Concentrations at ZEP were very
low compared to European mainland concentrations. 3.3
Particle number concentrations in different
size ranges The percentiles of the concentrations are also tabulated with
respective mean values in Table 4. Atmos. Chem. Phys., 11, 5505–5538, 2011 www.atmos-chem-phys.net/11/5505/2011/ A. Asmi et al.: Submicron particles in Europe 2008–2009 5516 Fig. 4. Median distributions (solid line) and 16th and 84th percentile distributions (shaded areas) divided by seasons at Nordic and Baltic
(PLA) stations. The black dotted line shows the 1000 particles cm−3 dn/dlog10 dp to help the eye on the concentration levels. Blue line and
shading denote winter, green line and shading spring, red line and shading summer and yellow line and shading autumn months datasets. Fig. 4. Median distributions (solid line) and 16th and 84th percentile distributions (shaded areas) divided by seasons at Nordic and Baltic
(PLA) stations. The black dotted line shows the 1000 particles cm−3 dn/dlog10 dp to help the eye on the concentration levels. Blue line and
shading denote winter, green line and shading spring, red line and shading summer and yellow line and shading autumn months datasets. Fig. 5. Median distributions (solid line) and 16th and 84th percentile distributions (shaded areas) divided by seasons at Central European
stations. Notation as in Fig. 5. Fig. 5. Median distributions (solid line) and 16th and 84th percentile distributions (shaded areas) divided by seasons at Central European
stations. Notation as in Fig. 5. Atmos. Chem. Phys., 11, 5505–5538, 2011 www.atmos-chem-phys.net/11/5505/2011/ A. Asmi et al.: Submicron particles in Europe 2008–2009 Overall distribution
Autumn months
Summer months
Winter months
Spring months
=
+
+
+
=
Day time
Night time
+ The FKL station in Greece had greatest N30−50 concen-
trations during spring and winter with no apparent day/night
difference. The concentration levels were relatively low, with
a median concentration of 220 cm−3. ISP histogram has sim-
ilar behaviour with the Central European ones with unimodal
log-normal shape and low seasonality, but with high concen-
trations of 1340 cm−3. Fig. 8. Example of the number concentration histograms used in
Figs. 9–11. The black line shows the histogram (with equal log
distributed bins) of all available data from the station. The seasonal
histograms are normalized to total amount of available data; they
sum up to the total histogram value. The shaded area shows the
similarly normalized histogram of only night-time samples. The
white area above is the fraction of the total histogram which was
from the day-time samples. ZEP station in Svalbard Islands (Fig. 9r), had a unique and
strong N30−50 seasonal cycle, with greater summer N30−50
concentrations of around 100 particles cm−3, compared to
N30−50 concentrations of around 10 cm−3 in other seasons. Some extreme concentrations were also observed in the win-
tertime. The day/night cycle at the ZEP station is very
strongly connected to the seasonal cycle, with almost all
observations larger than 100 cm−3 N30−50 occurring during
summer when the sun was above the horizon. 3.3.1
N30−50 concentrations All of the N30−50 histograms at mountain sites (Fig. 9s–x)
have similarities, with almost log-normal shapes, with clear
concentration tails towards greater concentrations, and sim-
ilar seasonal cycles. The winter conditions, probably more
representative of the free troposphere, were characterized
by lower concentrations. The summertime histograms show
greatest concentrations, probably due the planetary boundary
layer and/or valley winds influence. The greatest concentra-
tions were generally observed during day-time, especially at
stations BEO, PDD and CMN. The median concentrations
varied between 79 cm−3 for JFJ and 418 cm−3 for SSL. The histograms of Nordic and Baltic N30−50 concentrations
are presented in Figs. 9a–f. The Nordic stations ASP, BIR,
SMR and VHL all show similar overall number concentra-
tion histograms in this size class, with a fairly log-normal
shape and somewhat lower wintertime concentrations. The
seasonality (change of position or shape of the coloured
lines) is relatively low at the SMR and VHL stations in this
concentration range. The day-night cycle did not seem to
have a strong influence to number concentrations in these
stations, indicating low sensitivity in respect to diurnal cy-
cles. The more northern PAL station had some similarities
with the rest of the Nordic stations, but the concentrations
seem to be more variable (i.e. histogram is wider) and the ef-
fect of seasonal changes in day-time length is visible in the
differences between the night-time histogram and the total
histogram. The overall median N30−50 of the Baltic PLA
station was also in the same range as most of the Nordic sta-
tions as was the shape of the overall annual histogram. A. Asmi et al.: Submicron particles in Europe 2008–2009 A. Asmi et al.: Submicron particles in Europe 2008–2009 5517 Fig. 6. Median distributions (solid line) and 16th and 84th percentile distributions (shaded areas) divided by seasons at high altitude stations. Notation as in Fig. 4. Fig. 6. Median distributions (solid line) and 16th and 84th percentile distributions (shaded areas) divided by seasons at high altitude stations. Notation as in Fig 4 Fig. 6. Median distributions (solid line) and 16th and 84th percentile distributions (shaded areas) divided by seasons at high altitude stations. Notation as in Fig. 4. Fig. 7. Median distributions (solid line) and 16th and 84th percentile distributions (shaded areas) divided by seasons at West European,
Arctic and Mediterranean stations. Notation as in Fig. 4 . Fig. 7. Median distributions (solid line) and 16th and 84th percentile distributions (shaded areas) divided by seasons at West European,
Arctic and Mediterranean stations. Notation as in Fig. 4 . Atmos. Chem. Phys., 11, 5505–5538, 2011 www.atmos-chem-phys.net/11/5505/2011/ A. Asmi et al.: Submicron particles in Europe 2008–2009 5518 10
100
1000
10000
0
0.01
0.02
0.03
0.04
0.05
CMN
-3
Number concentration of N
(cm )
100
Fraction of all data points having
concentration in a concentration interval
Station 10
100
1000
10000
0
0.01
0.02
0.03
0.04
0.05
CMN
Overall distribution
-3
Number concentration of N
(cm )
100
Fraction of all data points having
concentration in a concentration interval
Autumn months
Summer months
Winter months
Spring months
=
+
+
+
=
Day time
Night time
+
Station
Fig. 8. Example of the number concentration histograms used in
Figs. 9–11. The black line shows the histogram (with equal log
distributed bins) of all available data from the station. The seasonal
histograms are normalized to total amount of available data; they
sum up to the total histogram value. The shaded area shows the
similarly normalized histogram of only night-time samples. The
white area above is the fraction of the total histogram which was
from the day-time samples. of 1914 cm−3. The HWL and MHD stations’ histograms
(Fig. 9n and o) show seasonal variation with greatest con-
centrations during spring time in HWL, and summertime in
MHD. Both stations have a very widely spread histogram
in all seasons suggesting high variability in concentrations. HWL N30−50 concentration median was 580 cm−3. MHD
had in general low N30−50 concentrations, with an annual
N30−50 median at 100 cm−3. 3.3.2
CCN-sized aerosol number concentrations The distributions of N50 and N100 concentrations are shown
in Figs. 10 and 11. The distributions of N50 and N100 concentrations are shown
in Figs. 10 and 11. The CCN-sized aerosol number concentration histograms
of the Nordic stations are similar for both N50 and N100
(Figs. 10a–c, e–f and 11a–c, e–f). The stations in general had
greater concentrations in summertime, although the concen-
trations at ASP and PAL were also elevated during spring. The BIR station had bimodal summer histograms with a
lower N50 concentration mode around 700 particles cm−3. The diurnal cycle was not strongly visible in Nordic sta-
tions histograms, except for the PAL station, where the diur-
nal variation was strongly affected by the seasonal variation. PAL station concentrations have a very wide histogram, es-
pecially in wintertime, suggesting a wide range of sources af-
fecting the concentrations observed at the station. The great-
est N50 and N100 concentrations observed at the PAL station The Central European stations have all a very similar nar-
row log-normal shape of the N30−50 histogram with very low
seasonal variation (Fig. 9g–l). The greatest concentrations
were somewhat increased during day-time at MPZ, OBK and
HPB, but otherwise no clear day/night variation can be seen. The median N30−50 concentrations varied from 410 cm−3 in
HPB to 1120 cm−3 in BOS. Of the western European stations, the CBW station
(Fig. 9m) has an unimodal N30−50 histogram with low
seasonal variation, similar to Central European stations. CBW N30−50
concentrations were high, with a median Atmos. Chem. Phys., 11, 5505–5538, 2011 www.atmos-chem-phys.net/11/5505/2011/ A. 3.3.2
CCN-sized aerosol number concentrations Asmi et al.: Submicron particles in Europe 2008–2009 5519 10
100
1000
10000
0
0.02
0.04
0.06
a) ASP
Relative occurence
10
100
1000
10000
0
0.01
0.02
0.03
0.04
0.05
b) BIR
10
100
1000
10000
0
0.01
0.02
0.03
0.04
c) PAL
10
100
1000
10000
0
0.02
0.04
0.06
d) PLA
Relative occurence
10
100
1000
10000
0
0.02
0.04
0.06
e) SMR
10
100
1000
10000
0
0.02
0.04
0.06
0.08
f) VHL
10
100
1000
10000
0
0.02
0.04
0.06
0.08
0.1
g) BOS
Relative occurence
10
100
1000
10000
0
0.02
0.04
0.06
0.08
h) KPO
10
100
1000
10000
0
0.02
0.04
0.06
0.08
i) OBK
10
100
1000
10000
0
0.02
0.04
0.06
0.08
0.1
j) HPB
Relative occurence
10
100
1000
10000
0
0.02
0.04
0.06
0.08
k) MPZ
10
100
1000
10000
0
0.02
0.04
0.06
0.08
l) WAL
10
100
1000
10000
0
0.02
0.04
0.06
0.08
m) CBW
Relative occurence
10
100
1000
10000
0
0.02
0.04
0.06
n) HWL
10
100
1000
10000
0
0.01
0.02
0.03
0.04
o) MHD
10
100
1000
10000
0
0.02
0.04
0.06
p) FKL
Relative occurence
10
100
1000
10000
0
0.02
0.04
0.06
0.08
0.1
q) ISP
10
100
1000
10000
0
0.01
0.02
0.03
0.04
r) ZEP
10
100
1000
10000
0
0.02
0.04
0.06
0.08
s) PDD
Relative occurence
10
100
1000
10000
0
0.02
0.04
0.06
0.08
t) SSL
10
100
1000
10000
0
0.02
0.04
0.06
u) ZSF
10
100
1000
10000
0
0.02
0.04
0.06
v) JFJ
N30−50 concentration [cm−3]
Relative occurence
10
100
1000
10000
0
0.01
0.02
0.03
0.04
0.05
w) BEO
N30−50 concentration [cm−3]
10
100
1000
10000
0
0.02
0.04
0.06
0.08
x) CMN
N30−50 concentration [cm−3]
Night−time
All data
Winter
Spring
Summer
Autumn
Histograms of N30−50 concentrations at the stations. The concentration bins are evenly distributed in the concentration a
ade) and the y-axes of the figures show the fraction of the values in each bin compared to total number of valid measuremen Fig. 9. Histograms of N30−50 concentrations at the stations. The concentration bins are evenly distributed in the concentration axes (20
bins/decade) and the y-axes of the figures show the fraction of the values in each bin compared to total number of valid measurements. Atmos. Chem. Phys., 11, 5505–5538, 2011 5520 A. 3.3.2
CCN-sized aerosol number concentrations Asmi et al.: Submicron particles in Europe 2008–2009 10
100
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0
0.02
0.04
0.06
0.08
a) ASP
Relative occurence
10
100
1000
10000
0
0.02
0.04
0.06
b) BIR
10
100
1000
10000
0
0.01
0.02
0.03
0.04
c) PAL
10
100
1000
10000
0
0.02
0.04
0.06
0.08
d) PLA
Relative occurence
10
100
1000
10000
0
0.02
0.04
0.06
0.08
e) SMR
10
100
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10000
0
0.02
0.04
0.06
0.08
f) VHL
10
100
1000
10000
0
0.02
0.04
0.06
0.08
g) BOS
Relative occurence
10
100
1000
10000
0
0.02
0.04
0.06
0.08
0.1
h) KPO
10
100
1000
10000
0
0.02
0.04
0.06
0.08
0.1
i) OBK
10
100
1000
10000
0
0.02
0.04
0.06
0.08
0.1
j) HPB
Relative occurence
10
100
1000
10000
0
0.02
0.04
0.06
0.08
k) MPZ
10
100
1000
10000
0
0.02
0.04
0.06
0.08
l) WAL
10
100
1000
10000
0
0.02
0.04
0.06
0.08
m) CBW
Relative occurence
10
100
1000
10000
0
0.02
0.04
0.06
n) HWL
10
100
1000
10000
0
0.01
0.02
0.03
0.04
0.05
o) MHD
10
100
1000
10000
0
0.02
0.04
0.06
p) FKL
Relative occurence
10
100
1000
10000
0
0.02
0.04
0.06
0.08
q) ISP
10
100
1000
10000
0
0.01
0.02
0.03
0.04
r) ZEP
10
100
1000
10000
0
0.01
0.02
0.03
0.04
0.05
s) PDD
Relative occurence
10
100
1000
10000
0
0.02
0.04
0.06
0.08
t) SSL
10
100
1000
10000
0
0.01
0.02
0.03
0.04
0.05
u) ZSF
10
100
1000
10000
0
0.01
0.02
0.03
0.04
0.05
v) JFJ
Relative occurence
N50 concentration [cm−3]
10
100
1000
10000
0
0.01
0.02
0.03
0.04
0.05
w) BEO
N50 concentration [cm−3]
10
100
1000
10000
0
0.01
0.02
0.03
0.04
0.05
x) CMN
N50 concentration [cm−3]
Night−time
All data
Winter
Spring
Summer
Autumn
Histograms of N50 concentrations at the stations. See Fig. 9 for nomenclature. Fig. 10. Histograms of N50 concentrations at the stations. See Fig. 9 for nomenclature. Atmos. Chem. Phys., 11, 5505–5538, 2011 www.atmos-chem-phys.net/11/5505/2011/ A. 3.3.2
CCN-sized aerosol number concentrations These high concentrations happened al-
most exclusively during night-time. The ZEP station in the Arctic has complex histograms with
high seasonality (Figs. 10r and 11r). The day/night varia-
tion was dominated by the seasonal variation of light at the
station, with relatively high concentrations of both N50 and
N100 in spring and summer daytime. The Baltic PLA station (Figs. 10d and 11d) has multi-
modal histograms during springtime, which shows influence
of both cleaner and more polluted airmasses. For other sea-
sons, the PLA station histograms are almost unimodal and
similar to the concentration histograms at Central Europe
(below). The Mountain sites all had wide, almost normal distribu-
tion shaped, histograms (Figs. 10s–x and 11s–x). The sea-
sonal cycle is clearly visible. The wintertime concentrations
are much lower than on other seasons. At most mountain
sites the greatest concentrations were observed during day-
time summer and autumn. This daytime effect could be con-
nected with air masses arriving from lower altitudes bringing
more polluted air from below. The N50 and N100 concentra-
tions are all strongly skewed towards lower concentrations. The lowest concentration tails of the distributions are proba-
bly indicative of concentrations of the free tropospheric air. The concentrations of the stations with highest altitude (JFJ,
BEO and ZSF) had a more pronounced clean mode with N100
concentrations below 100 cm−3. All Central European sites had similar CCN concentration
histograms (Fig. 10g–l and11g–l). The N50 and N100 his-
tograms have very similar shapes, and they only differ by
the magnitude of concentrations. There is very little sea-
sonal change in the histograms. Most of the greatest concen-
trations were observed during daytime. The N50 and N100
concentrations were relatively high at the Central European
stations, with median N50 concentrations of between 1325
and 3120 cm−3 at HPB and KPO, and N100 between 739 and
1863 cm−3 at HPB and OBK. Western European stations do not have as clear a com-
mon behaviour as the Central European stations (Figs. 10m–
o and 11m–o). Station CBW histogram is similar to Cen-
tral European stations with a unimodal shape, high concen-
trations especially for N50 (median 3387 cm−3) and the ab-
sence of seasonal variation. The HWL station histograms
are, as is the case with N30−50 concentrations, widely dis-
persed over the concentration axis with a two-modal his-
togram suggesting influence of cleaner and more polluted
airmasses. 3.3.2
CCN-sized aerosol number concentrations Asmi et al.: Submicron particles in Europe 2008–2009 5521 10
100
1000
10000
0
0.02
0.04
0.06
0.08
a) ASP
Relative occurence
10
100
1000
10000
0
0.01
0.02
0.03
0.04
0.05
b) BIR
10
100
1000
10000
0
0.01
0.02
0.03
0.04
c) PAL
10
100
1000
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0
0.02
0.04
0.06
0.08
d) PLA
Relative occurence
10
100
1000
10000
0
0.02
0.04
0.06
e) SMR
10
100
1000
10000
0
0.02
0.04
0.06
0.08
f) VHL
10
100
1000
10000
0
0.02
0.04
0.06
0.08
g) BOS
Relative occurence
10
100
1000
10000
0
0.02
0.04
0.06
0.08
0.1
h) KPO
10
100
1000
10000
0
0.02
0.04
0.06
0.08
0.1
i) OBK
10
100
1000
10000
0
0.02
0.04
0.06
0.08
0.1
j) HPB
Relative occurence
10
100
1000
10000
0
0.02
0.04
0.06
0.08
k) MPZ
10
100
1000
10000
0
0.02
0.04
0.06
0.08
l) WAL
10
100
1000
10000
0
0.02
0.04
0.06
m) CBW
Relative occurence
10
100
1000
10000
0
0.01
0.02
0.03
0.04
0.05
n) HWL
10
100
1000
10000
0
0.02
0.04
0.06
o) MHD
10
100
1000
10000
0
0.02
0.04
0.06
0.08
p) FKL
Relative occurence
10
100
1000
10000
0
0.02
0.04
0.06
q) ISP
10
100
1000
10000
0
0.01
0.02
0.03
0.04
r) ZEP
10
100
1000
10000
0
0.01
0.02
0.03
0.04
s) PDD
Relative occurence
10
100
1000
10000
0
0.02
0.04
0.06
0.08
t) SSL
10
100
1000
10000
0
0.01
0.02
0.03
0.04
u) ZSF
10
100
1000
10000
0
0.01
0.02
0.03
0.04
v) JFJ
Relative occurence
N100 concentration [cm−3]
10
100
1000
10000
0
0.01
0.02
0.03
0.04
w) BEO
N100 concentration [cm−3]
10
100
1000
10000
0
0.01
0.02
0.03
0.04
0.05
x) CMN
N100 concentration [cm−3]
Night−time
All data
Winter
Spring
Summer
Autumn
Histograms of N100 concentrations at the stations. See Fig. 9 for nomenclature. Fig. 11. Histograms of N100 concentrations at the stations. See Fig. 9 for nomenclature. Atmos. Chem. Phys., 11, 5505–5538, 2011 www.atmos-chem-phys.net/11/5505/2011/ Atmos. Chem. Phys., 11, 5505–5538, 2011 5522 A. Asmi et al.: Submicron particles in Europe 2008–2009 were observed during the summer. The seasonal variations
are large at all Nordic stations, except at VHL. were observed during the summer. The seasonal variations
are large at all Nordic stations, except at VHL. cles cm−3 in N50. 3.3.3
Autocorrelations and variations Standard deviations, geometric standard deviations and 1-
autocorrelations of the main number concentrations were cal-
culated only for valid datapoints without any data padding. The autocorrelation values should be considered only as ap-
proximate values – if the dataset had many instrumental fail-
ures or other unusable data, the relevance of autocorrelation
is degraded. The autocorrelation was calculated as the corre-
lation coefficient between the dataset and dataset shifted with
one hour (i.e. A1(n) = Corr(n(t),n(t +1h))). Large values
of autocorrelation show that the concentrations are typically
changing slowly and smoothly. The results are presented in Table 5. The standard devi-
ations of N30−50 number concentrations were rather large,
with least variation observed at stations BOS, ISP and HPB. In general, stations with high seasonality had greatest mean
variances. The N50 and N100 had generally smaller geomet-
ric standard deviations than N30−50 , with exception of PAL
station and the mountain stations, where the variability of
N100 increased compared to N30−50 aerosols. The hour-to-hour autocorrelation for CCN concentrations
was very high and uniform at all stations. Autocorrelations
of N30−50 concentrations are lower and with some station-to-
station variability. The high correlation coefficients suggest
that the aerosol concentrations rarely changed significantly
over 1-h timescales. Mediterranean FKL station histograms show peaks in N50
and N100 concentrations during spring of over 1000 parti-
cles cm−3 (Figs. 10p and 11p). Another smaller mode of low
concentrations was also visible around 500 particles cm−3
in both N50 and N100 . The ISP station histograms are in
general similar to Central Europe in both N50 and N100 dur-
ing spring and summer (Figs. 10q and 11q). The seasonal
changes of these particles are, however, relatively large and
ISP histogram has a strong signal from autumn and espe-
cially wintertime CCN concentrations at over 10 000 parti- 3.3.2
CCN-sized aerosol number concentrations These results agree with earlier cluster analy-
sis results, where the clean Atlantic and polluted London-
area/continental airmasses were easily distinguishable from
HWL observations (Charron et al., 2008). The MHD sta-
tion histogram shared a two-modal histogram with HWL. Most of the MHD N100 histogram is located at low concen-
trations with approximately 200 particles cm−3, but with a
second mode at about order on magnitude greater concen-
trations. This is well in line with previous studies from the
MHD station showing the importance of the difference be-
tween airmasses arriving over the relatively clean Atlantic
ocean contrasted by polluted airmasses arriving from Britain
and mainland Europe (McGovern et al., 1996). The CCN
concentrations in the mode with greater concentrations were
of same magnitude as the more polluted mode of HWL. None
of the Western European stations have strong seasonal sig-
nals in N50 or N100 concentrations. 3.3.4
Weekday variations The weekday variations were studied by grouping the daily
averages of the N30−50 , N50 and N100 concentrations of
each station on for each day of the week, and analysing the Atmos. Chem. Phys., 11, 5505–5538, 2011 www.atmos-chem-phys.net/11/5505/2011/ A. Asmi et al.: Submicron particles in Europe 2008–2009 5523 resulting concentration histograms. The daily averaging was
to remove the high autocorrelation of the concentrations and
thus providing independent samples for testing. We tested
the statistical significance of the difference between these
day of the week concentrations to other weekdays distri-
butions utilising a non-parametric Wilkinson rank-sum test
using a ranksum function in Matlab (Higgins, 2004; Math-
Works, Inc., 2010). The null-hypotheses was that each week-
day’s concentration distribution (i.e. histogram) was of sim-
ilar shape and with a similar median as the combined his-
togram of all other days of the week. Even at the relatively
highly polluted, and potentially anthropogenic-influenced
Central European stations, the histograms differences could
not be considered statistically significant with p = 0.05. The
only exception is station BOS N100 concentrations, which
showed a statistically significant increase in Wednesday con-
centrations compared to the rest of the weekdays. an extremely seasonal station such as ZEP, comparisons be-
tween modelled and measured concentrations should take
the seasonality differences into account. Year-to-year vari-
abilities were generally low for the stations with both years
of data. The weekday analysis shows that, for the concentrations
studied, only a few stations per test had any statistically
significant variation of concentrations between weekdays or
work-week and weekend. As the analysis was done for the
whole dataset, periods of significant weekly cycles can oc-
cur at the stations intermittently and the signal can be over-
shadowed by the other changes in concentrations. The par-
ticle concentrations nearer to sources are known to show a
weekly cycle in number concentrations (van Dingenen et al.,
2004; Rodriguez et al., 2007), but the relative remoteness
of most of the stations and annual averaging could have re-
moved an anthropogenic weekly signal from our analysis. A
more comprehensive time-series analysis of these signals us-
ing e.g. wavelet analysis could provide more information on
these cycles. As the rank-sum test above only compares the each week-
day median to the median of other weekdays separately,
we repeated the test using similar non-parametric Kruskal-
Wallis test (function kruskalwallis in Matlab) to compare the
medians of all weekday samples. 4.2
Station representativeness and categorisation The aerosol modelling community often works with mod-
els with relatively low spatial resolution, often on a scale
of parts of degrees. This low resolution has caused concern
for model-measurement comparisons, especially for polluted
or otherwise potentially heterogeneous regions. The differ-
ences in absolute concentrations shown in this paper can vary
within a region, but in general the size distribution shape,
histograms of concentrations and seasonal behaviours of the
aerosol populations are relatively homogeneous over large
geographical areas – even in areas with known anthropogenic
aerosol sources. The high autocorrelations support this, indi-
cating low temporal (and thus most probably – spatial) vari-
ability near the station for the size ranges in question. The
variability is larger for N30−50 concentrations, than for other
concentrations, possibly explained through the lower lifetime
of N30−50 particles and thus larger sensitivity to sources and
sinks (Tunved et al., 2004). 3.3.4
Weekday variations The p value gained from
this test is the probability for the null hypothesis that all
samples are drawn from the same population (Kruskal and
Wallis, 1957; MathWorks, Inc., 2010). None of the stations
did showed any statistically significant differences between
weekdays with p = 0.05 in any concentration tested. To fur-
ther extend the analysis the test was also repeated by group-
ing the data only to two groups: one with weekend days (Sat-
urday and Sunday) and one with work week days (Monday,
Tuesday, Wednesday, Thursday and Friday). Here, only HPB
N30−50 concentrations and ZSF N100 concentrations were
different between groups (p < 0.05). 4
Discussion The results shown are relevant to climate and air qual-
ity studies in several ways. Even though the earlier com-
parisons brought important aspects, such as mean concen-
trations, of European submicron particle distributions (e.g. van Dingenen et al., 2004; Tunved et al., 2003), this study
shows the concentrations from comparable regional back-
ground stations, using the same general period of time and
using comparable instruments and data analysis methods. The results of regional background aerosol number concen-
trations and variability should be usable in e.g. model-to-
measurement comparisons of regional and potentially global
aerosol models. The question of representativeness is dependent on which
kind of representativeness is actually expected by the data
user. Different definitions of representativeness were dis-
cussed in Henne et al. (2010), who studied the representative-
ness of O3 measurements at ground-based stations in Europe
for comparisons with medium-to-large scale models. They
categorized the European stations into 6 groups based on es-
timated O3 source and deposition patterns around the catch-
ment areas of the stations. Of the 24 stations in this study, 15
stations were also categorised by Henne et al. and the result-
ing representability classes are shown in Table 2. The groups
they determined most useful for large-scale model compar-
isons were classes 5 (generally remote), 2 (mostly remote). Class 3 (agglomeration) was considered to be least represen-
tative of the area around it. 4.1
Temporal variation of concentrations The aerosol seasonal variation depends on the site of study. For central European sites, seasonal changes do not have
a major effect in CCN concentrations. In comparison, for Atmos. Chem. Phys., 11, 5505–5538, 2011 www.atmos-chem-phys.net/11/5505/2011/ A. Asmi et al.: Submicron particles in Europe 2008–2009 5524 0
0.5
1
1.5
2
2.5
x 10
10
1
1.5
2
2.5
3
3.5
4
4.5
5
x 10
7
BIR
PLA
KPO
OBK
HPB
CBW
HWL
MHD
FKL
PDD
SSL
ZSF
JFJ
CMN
NOR
CEN
WST
MED
ITA
ARC
MNT
N100
ASP
BIR
PAL
PLA
SMR
VHL
BOS
OBK
H B
P
MPZ
WAL
CBW
HWL
MHD
FKL
ZEP
PDD
SSL
ZSF
JFJ
BEO
Geographical categorisation
CMN
KPO
UNK
RUR
MRE
AGG
WIF
GRE
WGR
c)
NOR
CEN
WST
MED
ITA
ARC
MNT
N30-50
ASP
BIR
PAL
SMR
VHL
BOS
KPO
OBK
H B
P
MPZ
WAL
CBW
HWL
MHD
FKL
ZEP
PDD
SSL
ZSF
JFJ
BEO
Geographical categorisation
CMN
UNK
RUR
MRE
AGG
WIF
GRE
WGR
Henne et al. (2010) representability
a)
PLA
Geometric standard deviation of concentrations
-3
Annual median concentration (cm )
1.8
2.0
2.2
2.4
2.6
2.8
3.0
3.5
4+
1000
1000
1500
1500
500
500
100
100
2000
2000
d)
Population times residence time [s] (Henne et al, 2010)
Henne et al. (2010) representability
Total dry deposition times residence time
[cm] (Henne et al, 2010)
N100
0
0.5
1
1.5
2
2.5
x 10
10
1
1.5
2
2.5
3
3.5
4
4.5
5
x 10
7
BIR
KPO
OBK
CBW
HWL
MHD
FKL
ISP
PDD
SSL
ZSF
JFJ
CMN
PLA
b)
Population times residence time [s] (Henne et al, 2010)
Total dry deposition times residence time
[cm] (Henne et al, 2010)
N30-50
HPB
ISP
ISP
ISP
g. 12. Station categorisation according to geographical location and Henne et al. (2010) (see text) (a) and (c). The category abbreviations NOR
CEN
WST
MED
ITA
ARC
MNT
N30-50
ASP
BIR
PAL
SMR
VHL
BOS
KPO
OBK
H B
P
MPZ
WAL
CBW
HWL
MHD
FKL
ZEP
PDD
SSL
ZSF
JFJ
BEO
Geographical categorisation
CMN
UNK
RUR
MRE
AGG
WIF
GRE
WGR
Henne et al. 4.1
Temporal variation of concentrations (2010) representability
a)
PLA
ISP 0
0.5
1
1.5
2
2.5
x 10
10
1
1.5
2
2.5
3
3.5
4
4.5
5
x 10
7
BIR
KPO
OBK
CBW
HWL
MHD
FKL
ISP
PDD
SSL
ZSF
JFJ
CMN
PLA
b)
Population times residence time [s] (Henne et al, 2010)
Total dry deposition times residence time
[cm] (Henne et al, 2010)
N30-50
HPB NOR
CEN
WST
MED
ITA
ARC
M
Geographical categorisation OR
CEN
WST
MED
ITA
A
Geographical categorisation NOR
CEN
WST
MED
ITA
ARC
MNT
N100
ASP
BIR
PAL
PLA
SMR
VHL
BOS
OBK
H B
P
MPZ
WAL
CBW
HWL
MHD
FKL
ZEP
PDD
SSL
ZSF
JFJ
BEO
Geographical categorisation
CMN
KPO
UNK
RUR
MRE
AGG
WIF
GRE
WGR
c)
Henne et al. (2010) representability
ISP 0
0.5
1
1.5
2
2.5
x 10
10
1
1.5
2
2.5
3
3.5
4
4.5
5
x 10
7
BIR
PLA
KPO
OBK
HPB
CBW
HWL
MHD
FKL
PDD
SSL
ZSF
JFJ
CMN
NOR
CEN
WST
MED
ITA
ARC
MNT
N100
ASP
BIR
PAL
PLA
SMR
VHL
BOS
OBK
H B
P
MPZ
WAL
CBW
HWL
MHD
FKL
ZEP
PDD
SSL
ZSF
JFJ
BEO
Geographical categorisation
CMN
KPO
UNK
RUR
MRE
AGG
WIF
GRE
WGR
c)
d)
Population times residence time [s] (Henne et al, 2010)
Henne et al. (2010) representability
Total dry deposition times residence time
[cm] (Henne et al, 2010)
N100
ISP
ISP 0
0.5
1
1.5
2
2.5
x 10
10
1
1.5
2
2.5
3
3.5
4
4.5
5
x 10
7
BIR
PLA
KPO
OBK
HPB
CBW
HWL
MHD
FKL
PDD
SSL
ZSF
JFJ
CMN
d)
Population times residence time [s] (Henne et al, 2010)
Total dry deposition times residence time
[cm] (Henne et al, 2010)
N100
ISP NOR
CEN
WST
MED
ITA
AR
Geographical categorisation Geographical categorisation etric standard deviation of concentrations
-3
Annual median concentration (cm )
2.2
2.4
2.6
2.8
3.0
3.5
4+
1000
1000
1500
1500
500
500
100
100
2000
2000 Fig. 12. Station categorisation according to geographical location and Henne et al. (2010) (see text) (a) and (c). The category abbreviations
are: WGR – “Weakly influenced, generally remote”, GRE – “generally remote”, WIF – “weakly influenced”, AGG – “agglomeration”,
MRE – “mostly remote”, RUR – “Rural”, UNK – unknown, not categorized. 4.3
Modal concentrations The size distribution functions or size range concentrations
histograms in Sect. 3 give statistical information on concen-
trations observed at each station. However, they do not di-
rectly inform about the correlation between different parti-
cle sizes. We investigate the modal correlations by group-
ing the data according to geographical regions (see Table 2)
and showing the concentrations of particles between 30 and
100 nm of dry particle diameter (representing Aitken mode)
as a function of N100 concentrations (representing accumu-
lation mode). The 100 nm diameter is chosen to present
the approximate location of the Hoppel gap (Hoppel and
Frick, 1990) between the modes and to be consistent with
the 100 nm cut-off of the N100 concentrations. For data visu-
alization purposes, we separated areas with greatest density
of observations of each geographical category with contours
containing 83 percent of the observations (Fig. 13, see Ap-
pendix C for details on the separation). The shape of the
scatter density contour can be interpreted to show the typi-
cal relationship between concentrations of the two modes for
specific types of stations. If the modal concentrations often
change in the same way, the shape of the contour is narrow
and symmetrical along diagonal lines of the figure. A narrow,
but not a diagonal shape of the contour suggests that the two
modes are correlated, but one of the modes has much greater
variability in the size distributions observed at the station. If
the shape of the contour is circular, the two mode concentra-
tions are not strongly correlated. From the Henne et al. (2010) point of view, the most com-
parable ”Generally remote” classification only had one sta-
tion, MHD, which based on particle data and multi-peaked
histograms does instead show high heterogeneity in particle
concentrations. This large variability does not agree with
high representability in this sense. The Central European
sites are categorized in the “Rural” category which from the
ozone point of view was not one of the most representative
groupings in Henne et al. (2010) (i.e. one could expect high
variability), but from a particle point-of-view show very sim-
ilar behaviour over a large area with low variability, i.e. po-
tentially high representativeness for the region. Groupings
where the Henne et al. (2010) produced relatively similar
concentrations and variations were “Weakly influenced” and
“mostly remote” which both were considered to be usable for
model comparison. As Henne et al. A. Asmi et al.: Submicron particles in Europe 2008–2009 A. Asmi et al.: Submicron particles in Europe 2008–2009 either variability or median concentrations on either concen-
tration studied. If this categorization would be usable directly for num-
ber concentrations of aerosol particles, we would assume
that the variability of the aerosol concentrations would be
lower in stations categorized as more representative. Es-
pecially the concentration histograms should have relatively
unimodal shape, as several peaks in histograms are signs of
non-homogenous airmasses. Aerosol number concentrations
should also follow the main source and sink parameters used
in the categorization. Overall, the Henne et al. (2010) categorization does not
seem to agree well with all aerosol measurements. Based on
the main parameters used in their categorization, the source
term used in their approach do have some similarities with
observed aerosol concentrations (i.e. the concentrations were
generally greater with bigger source term), but the use of dry
deposition parameter as the main sink term does not have
good agreement with our results. We recommend caution
on using site categorizations derived from non-aerosol pollu-
tants to describe the expected representability of a station in
aerosol number concentration point-of-view. g
We explore the effectiveness of different categorisations
in Fig. 12a and c, which show the stations categorized by ge-
ographical and Henne et al. (2010) categories, also showing
the annual median concentrations and geometric standard de-
viations of N30−50 and N100 concentrations. The geographic
categorization seems to capture most of the overall concen-
tration levels quite successfully (i.e. the markers are gener-
ally of same size in vertical columns), and the standard de-
viations of the stations seem to be also reasonably similar. HPB, with relatively high altitude, starts to have lower me-
dian concentrations and greatest variability for Central Eu-
ropean stations, a common feature with the SSL mountain
station. The largest discrepancies are within the Western Eu-
ropean group, where CBW is much closer to polluted Central
European sites and the differences between HWL and MHD
are relatively large. 4.1
Temporal variation of concentrations The geographical areas are NOR – Nordic and Baltic, CEN
– Central Europe, WST – Western Europe, MED – Mediterranean, ITA – North Italy, ARC – Arctic, MNT – Mountains. (a) for N30−50
concentrations and (c) for N100. On subfigures (b) and (d) are shown the station concentrations and standard deviations as function of Henne
et al. (2010) main categorizing parameters, the “catchment area population times residence time” and the “catchment area total O3 deposition
times residence time”. The values are from 24 h catchment areas. The colour of the station indicators describe the annual geometric standard
deviations of the concentrations in question and the sizes of the indicators show the annual median number concentrations. Atmos. Chem. Phys., 11, 5505–5538, 2011 www.atmos-chem-phys.net/11/5505/2011/ 5525 4.3
Modal concentrations Scatter plot of particle concentration between 30 and 100 nm as a function of particle concentrations above 100 nm (grey points
The station groupings are shown with different colours, with the contours showing the areas with greatest density of each station groupin
The contours are drawn to include 83 % of the each group data points. The colour coded numbers in the plot indicate the coordinates of th
geometric means of both concentrations for each station. The insert shows an interpretation of the approximate aerosol types in the sam
axes. See text for details. Atlantic
Arctic Haze
Clean
Mountain
Clean continental
Polluted
continental
V. Polluted
continental
Mediterranean
1
10
100
1000
10000
100000
1
10
100
1000
10000
100000
2:1
10:1
1:1
1:2
1:10
Nordic and Baltic (6 st.)
Central Europe (6 st.)
Mountains (6 st.)
Western Europe (3 st.)
Mediterranean (FKL)
N. Italy (ISP)
Arctic (ZEP)
30nm
100nm
1
2
3
4
5
6
7
8 9
10
11
12
13
14
15
16
17
18
19
20
22
23
24
1
ASP
2
BIR
3
PAL
4
PLA
5
SMR
6
VHL
7
BOS
8
KPO
9
OBK
10 HPB
11 MPZ
12 WAL
13 CBW
14 HWL
15 MHD
16 FKL
17 ISP
18 ZEP
19 PDD
20 SSL
21 ZSF
22 JFJ
23 BEO
24 CMN
21
-3
N
number concentration [cm ]
100
-3
N
number concentration [cm ]
30-100 10
100
-3
N
number concentration [cm ]
30-100 10 -3
N
number concentration [cm ]
100 Fig. 13. Scatter plot of particle concentration between 30 and 100 nm as a function of particle concentrations above 100 nm (grey points). The station groupings are shown with different colours, with the contours showing the areas with greatest density of each station grouping. The contours are drawn to include 83 % of the each group data points. The colour coded numbers in the plot indicate the coordinates of the
geometric means of both concentrations for each station. The insert shows an interpretation of the approximate aerosol types in the same
axes. See text for details. station has a unique, very low-concentration contour, where
the two modes are widely varying in their concentrations. 500 cm−3 N100 concentrations and station HWL contribut-
ing to the concentrations between these extremes. 4.3
Modal concentrations (2010) classification did not have overall
a good agreement with our analysis, we also used the param-
eters of the classification separately, to find out the reasons
for the discrepancies. Fig. 12b and d show the stations as
a function of two Henne et al. (2010) classification param-
eters, the 24-h catchment area “population times residence
time” (representing the potential immediate sources of pollu-
tants) and the 24-h catchment area “O3 total deposition times
the residence time” (representing potential sink terms). The
N30−50 concentrations increase as the population parameter
increases, with some indication of decreasing variability. For
N100 concentrations, the population parameter has less influ-
ence on the concentration levels or on variation. The deposi-
tion parameter however does not have such a clear trend on The Central European stations concentrations are concen-
trated in a grouping between 500 and 5000 N100 cm−3, with
a relationship between the bimodal concentrations (Fig. 13). A similar behaviour can be seen in the low-concentration
end of station ISP contour, but with high concentrations of
accumulation mode starting to dominate the size distribu-
tions at over 2000 cm−3 N100 concentrations. The Western
European stations are heterogeneous (Fig. 13 yellow con-
tour); Station CBW contributing to the high-concentration
end, with similar or even greater, concentrations compared
to Central European stations; Station MHD contributing to
the narrow low concentration part of the contour below Atmos. Chem. Phys., 11, 5505–5538, 2011 www.atmos-chem-phys.net/11/5505/2011/ A. Asmi et al.: Submicron particles in Europe 2008–2009 A. Asmi et al.: Submicron particles in Europe 2008–2009 5526 Atlantic
Arctic Haze
Clean
Mountain
Clean continental
Polluted
continental
V. Polluted
continental
Mediterranean
1
10
100
1000
10000
100000
1
10
100
1000
10000
100000
2:1
10:1
1:1
1:2
1:10
Nordic and Baltic (6 st.)
Central Europe (6 st.)
Mountains (6 st.)
Western Europe (3 st.)
Mediterranean (FKL)
N. Italy (ISP)
Arctic (ZEP)
30nm
100nm
100nm
1
2
3
4
5
6
7
8 9
10
11
12
13
14
15
16
17
18
19
20
22
23
24
1
ASP
2
BIR
3
PAL
4
PLA
5
SMR
6
VHL
7
BOS
8
KPO
9
OBK
10 HPB
11 MPZ
12 WAL
13 CBW
14 HWL
15 MHD
16 FKL
17 ISP
18 ZEP
19 PDD
20 SSL
21 ZSF
22 JFJ
23 BEO
24 CMN
21
-3
N
number concentration [cm ]
100
-3
N
number concentration [cm ]
30-100
Fig. 13. 4.5
Data usage and access for end-user communities We have created usable and relevant statistics from the data. The actual choice of what should be used for the models
to compare with, depends on application and complexity
needed. The most straightforward way is just to compare
one or more mean parameters, such as median concentra-
tions. This approach is simple to do, but can easily lose many
features of the data, and, in cases of strongly bimodal his-
tograms, can even be misleading. Comparing modelled his-
tograms to results should pay attention to the histogram mode
location (mean or median concentration), width and relative
abundance (height) of each mode in the histograms. One
method would be to calculate parametric or non-parametric
statistical error parameters to compare with modelled and
measured distributions. 0
Particle diameter [nm] Fig. 14. Cumulative number concentration of the median size distri-
butions at EUSAAR and GUAN stations. The summation was done
starting from the largest available size. The plot shows how much
particles were above each diameter in the median size distribution
interval. Although this paper produces a good comparison dataset
to compare modelled aerosol concentrations, the ability of a
model to correctly produce the measured size distribution or
CCN-sized aerosol number distributions in some or all of the
stations provided is not enough to ensure the correct distri-
bution of actual CCNs let alone CDNCs. Getting the correct
size distribution is a critical first step in this process, but only
the first step. There are several complications before a model
can be considered to have a realistic representation of CCNs,
such as limited horizontal spatial coverage of the measure-
ments, surface measurements instead of cloud-base aerosol
concentrations, implicit assumption that aerosol concentra-
tion histograms are independent of the cloud formation prob-
ability, particle composition, size and surface effects and ac-
tual cloud processes dominating the relative humidities lead-
ing to droplet activation. These kinds of complex processes
need to be taken into account before realistic cloud activation
and thus realistic aerosol climate effects in the models can be
achieved. relatively low concentrations and low correlation between the
modes. For the other aerosol types, we use the interpretations from
the N100 concentration histograms to separate portions of the
contours most likely representing specific aerosol types. 4.4
High activation diameters The size ranges used in this article are not necessarily the
best choices for all environments. Figure 14 shows the cu-
mulative annual median distributions at the stations, calcu-
lated from the largest particle size available towards smaller
sizes, showing the median concentration of particles above a
certain diameter. In Central European stations, station PLA
in Baltic and station ISP in Northern Italy, the median con-
centration of N100 particles are over 1000 cm−3. These are a
high, but not unrealistic, amount of particles to be activated
as cloud droplets (Martinsson et al., 2000). In these relatively
polluted stations, a more useful metric could be derived from 4.3
Modal concentrations The moun-
tain stations have a distribution of observations from polluted
Central-Europe-type aerosol concentrations to very low ac-
cumulation mode and Aitken mode tail. This cleaner end of
the mountain station concentrations, with dominant Aitken
mode, is likely descriptive of the free tropospheric air ob-
served at the highest mountain stations. Nordic stations form
more round shape in middle concentrations, showing greater
inter-modal variability and possibly more varied sources of
the two modes. Mediterranean FKL station has lower con-
centrations and slightly more accumulation-mode dominated
concentrations than Central European stations. Arctic ZEP Using the interpretations of N100
histograms from
Sect. 3.3.1, we distinguish phenomenological aerosol types
from these Aitken/accumulation mode concentrations (see
Fig. 13 insert). The Central European and ISP concentra-
tions show the mode concentration range of the polluted and
very polluted European background air. The Mediterranean
FKL station contour is considered to be representative of typ-
ical Mediterranean regional background, with lower concen-
trations than Central Europe, but strong correlation between
the modes. We interpret the Nordic station contour as repre-
sentative of clean continental regional background air, with Atmos. Chem. Phys., 11, 5505–5538, 2011 www.atmos-chem-phys.net/11/5505/2011/ 5527 A. Asmi et al.: Submicron particles in Europe 2008–2009 larger particle sizes. For this reason, histograms of an addi-
tional CCN number concentration N250 are shown and dis-
cussed in Appendix B. 10
2
10
310
1
10
2
10
3
10
4
Particle diameter [nm]
Cumulative concentration [cm-3]
ASP
BIR
PAL
PLA
SMR
VHL
BOS
KPOOBK
HBP
MPZWAL
CBW
HWL
MHD
FKL
ISP
ZEP
PDD
SSL
ZSF
JFJ
BEO
CMN
Nordic and Baltic (6 st.)
Central Europe (6 st.)
Mountains (6 st.)
Western Europe (3 st.)
Mediterranean (FKL)
N. Italy (ISP)
Arctic (ZEP)
Fig. 14. Cumulative number concentration of the median size distri-
butions at EUSAAR and GUAN stations. The summation was done
starting from the largest available size. The plot shows how much
particles were above each diameter in the median size distribution
interval. 10
2
10
310
1
10
2
10
3
10
4
Particle diameter [nm]
Cumulative concentration [cm-3]
ASP
BIR
PAL
PLA
SMR
VHL
BOS
KPOOBK
HBP
MPZWAL
CBW
HWL
MHD
FKL
ISP
ZEP
PDD
SSL
ZSF
JFJ
BEO
CMN
Nordic and Baltic (6 st.)
Central Europe (6 st.)
Mountains (6 st.)
Western Europe (3 st.)
Mediterranean (FKL)
N. Italy (ISP)
Arctic (ZEP) Cumulative concentration [cm-3] 4.5
Data usage and access for end-user communities The
low N100 concentration end of the mountain station con-
centrations (below 100 N100 cm−3) is identified as possi-
ble free tropospheric or clean mountain air aerosol, show-
ing dominant Aitken mode concentrations. This hypothesis
is supported by removing the relatively low altitude moun-
tain stations (PDD and SSL, both below 2000 m a.s.l.) from
the analysis (not shown), which partially removes the area
of the mountain contour which overlaps the Central Euro-
pean contour. The low concentration end of station MHD
N100 concentrations is interpreted as clean Atlantic marine
aerosol. This marine air has low concentrations of accumu-
lation mode particles, but high variability in Aitken mode. The (for the station) high concentrations of N100 particles in
ZEP station in relation to N30−50 and N50 particles are in-
terpreted as instances of Arctic haze, identified as the bottom
part of the Arctic contour. The datasets used in this paper are available directly as
time-series from NILU EBAS databank at address http://
ebas.nilu.no/. The data presented in this article (histograms,
seasonal profiles, etc) are also stored on a publicly available
server at http://www.atm.helsinki.fi/eusaar/. More informa-
tion on data formats and suggestions on data usage is in Ap-
pendix C. 5
Conclusions This study shows the importance of standardized long-term
measurements to provide reliable information on statistical
behaviour of atmospheric aerosols. Although study encom-
passes a period of only two years, the data already provides
a previously unavailable variety of information on the sub- Atmos. Chem. Phys., 11, 5505–5538, 2011 www.atmos-chem-phys.net/11/5505/2011/ A. Asmi et al.: Submicron particles in Europe 2008–2009 5528 CENTRAL EUROPE
ARCTIC
N. ITALY
MOUNTAINS
WESTERN EUROPE
NORDIC AND
BALTIC
MEDITERRANEAN
Summer
Winter
Autumn
Spring
1000
500
250
2000
84th percentile
Median
16th percentile
Seasonal concentrations of 100-500 nm
particles in EUSAAR and GUAN stations
2008-2009
High concentrations
Low seasonal cycle
Unimodal size distributions
Uniform airmasses
Low variability
Low concentrations
High seasonal cycle
Maximum levels in
summer
Bi-modal size distributions
Moderate variability
Heterogenous airmasses
Varied concentration levels
High seasonal cycle
High variability
Heterogenous airmasses
Very low concentrations
Dominated by seasonal cycle
Arctic haze in wintertime
High
concentrations
Winter maximum
Usually low
concentrations
High variability
Seasonal cycle with
summer maximum
Heterogenous
airmasses
Medium
concentrations
High seasonality
Spring-summer
maximum
GUAN
Crete
Svalbard Is. cm-3
Fig. 15. Overview of EUSAAR and GUAN station measurements – spatial distribution of particle number concentrations on particle (in cm−3
STP). The coloured areas are relative to the median concentration observed in each season, and the lower and higher arcs show 16th and 84th
percentile concentrations. The colours and location of the segments show different seasons. The locations of the stations are approximate. Stations ZEP (Arctic) and FKL (Mediterranean) are located in inserts. Seasonal concentrations of 100-500 nm
particles in EUSAAR and GUAN stations
2008-2009 Fig. 15. Overview of EUSAAR and GUAN station measurements – spatial distribution of particle number concentrations on particle (in cm−3
STP). The coloured areas are relative to the median concentration observed in each season, and the lower and higher arcs show 16th and 84th
percentile concentrations. The colours and location of the segments show different seasons. The locations of the stations are approximate. Stations ZEP (Arctic) and FKL (Mediterranean) are located in inserts. WAL, MPZ, OBK and KPO, although some seasonal
signal was observed in OBK. Stations HPB and SSL
had many similarities with these stations, but due to
their relatively high altitude they also showed similar-
ities with mountain stations, including greater variabil-
ity and skewed N100 concentrations histograms. 5
Conclusions The
station CBW in Netherlands has many features in com-
mon with the Central European stations and can be cat-
egorised in this group. micron aerosol physical properties and variability in Europe. Such information would be hard to achieve based on infor-
mation collected from separately managed stations, espe-
cially if the instrumentation and data handling are not har-
monized. The analysis in this paper shows that the aerosol
concentrations in Europe are highly variable spatially. We
consider both the spatial and temporal variations of the
aerosol number concentrations in Europe to be generally
much greater than instrumental uncertainties. The particle
concentrations, derived statistics, size distribution functions
and modal fitting parameters at the stations show groupings
based on geographic location (see Fig. 15): 2. The “Northern European Aerosol”, has clearly lower
concentrations,
with
decreasing
concentrations
at
higher latitudes. Although the similarities are not as ob-
vious as in Central Europe, the stations are still similar
enough in regards of seasonal and size distribution be-
haviour that these stations can be described by one type. The seasonal cycle has a strong effect on the particles in
this region, and the overall variation is relatively large. The summer concentrations are usually greatest, espe-
cially for smaller particle sizes, which also affects the 2. The “Northern European Aerosol”, has clearly lower
concentrations,
with
decreasing
concentrations
at
higher latitudes. Although the similarities are not as ob-
vious as in Central Europe, the stations are still similar
enough in regards of seasonal and size distribution be-
haviour that these stations can be described by one type. The seasonal cycle has a strong effect on the particles in
this region, and the overall variation is relatively large. The summer concentrations are usually greatest, espe-
cially for smaller particle sizes, which also affects the 1. The “Central European Aerosol”, observed at low-land
stations from the Netherlands to Hungary showed low
seasonal changes, high particles concentrations, almost
unimodal median distributions and relatively low vari-
ability. The concentration histograms are practically
log-normal and not strongly affected by diurnal varia-
tion. This aerosol type was observed the stations BOE, 1. The “Central European Aerosol”, observed at low-land
stations from the Netherlands to Hungary showed low
seasonal changes, high particles concentrations, almost
unimodal median distributions and relatively low vari-
ability. The concentration histograms are practically
log-normal and not strongly affected by diurnal varia-
tion. 5
Conclusions This aerosol type was observed the stations BOE, Atmos. Chem. Phys., 11, 5505–5538, 2011 www.atmos-chem-phys.net/11/5505/2011/ 5529 A. Asmi et al.: Submicron particles in Europe 2008–2009 observed differences of day and night-time values due
to differing length of day in the northern latitudes. The
concentration distributions often show multiple modes,
suggesting a combination of more polluted airmasses
and cleaner air from the Arctic or Atlantic oceans. The
stations included in this group were BIR, VHL, ASP
and SMR. PAL has many similarities with other Nordic
stations, but had even more extreme seasonal variation
and thus had some parameters in common with Arctic
aerosol concentrations and variability. The Baltic PLA
station was only partially similar to the Nordic stations,
with both concentration histograms and size distribu-
tions showing influence from multiple source areas of
particles and some similarities in concentration levels
with Central European aerosol. termined using a combination of methods: comparing sta-
tion concentrations in a representable area and by observ-
ing the histogram widths, seasonality and number of modes
visible. A more detailed generalization of the representabil-
ity for aerosol number concentrations would require simi-
lar underlying methodology as Henne et al. (2010), proba-
bly with longer back-trajectories, including wet deposition as
the main deposition proxy and by including secondary parti-
cle formation as a source of particle number concentrations. However, considering the difficulties of modelling wet depo-
sition and secondary particle formation this could be a diffi-
cult task. We have also shown that even though the aerosol number
concentrations of Aitken and accumulation mode particles
are generally related, there is significant variation in the ratio
of concentrations of these two modes. Different aerosol and
station types can be distinguished from these concentrations
and this methodology has potential for further categorization
of stations. 3. “Mountain aerosol” which is characterized by episodes
of extremely clean air, most probably from the free tro-
posphere, mixed by episodes of relatively polluted air-
masses, especially during daytime in summer. These
changes of airmass produce complex, strongly skewed
and very wide concentration histograms, with in many
cases strong diurnal cycles. The Central European SSL
and HPB stations were borderline mountain stations
with some similarities with boundary layer Central Eu-
ropean stations. The stations with this kind of aerosol
were JFJ, BEO, ZSF and CMN. The measured aerosol number concentrations did not have
strong weekly variance in the annual concentration his-
tograms. 5
Conclusions The statistical tests done did not support statis-
tically significant differences in CCN-sized aerosol num-
ber concentrations between individual weekdays or between
work-week and week-end. This result is different previous
studies using particle mass or AOT as the aerosol tracer,
where weekly variation was detected even in multi-year
weekday means (B¨aumer et al., 2008; Barmet et al., 2009). For the other areas, the number of stations is too low to
clearly distinguish the aerosol type in this way, as the spa-
tial coverages of the stations are unknown. We can however
state, that with greater uncertainty, the aerosol types are The produced datasets are designed from the model-
measurement comparison point-of-view. The datasets are
available for the modellers in easy-to-use format for parti-
cle sizes which have potential for climate relevance. This
dataset is a valuable comparison toolbox for the use of the
global and regional modelling communities. 4. “Arctic aerosol” with high seasonality, very low over-
all number concentrations and evidence of Arctic haze
events during dark winter periods; The main future lines of improvement of such European
long-term studies is to make the coverage of the stations a
more uniform. The lack of stations in Eastern Europe, and
partially in the Mediterranean basin are clear open areas in
this field of study. The only EUSAAR station on the Iberian
Peninsula (MSY, Montseny) was not included in this study
due lack of validated data for this period, but in future this
will somewhat increase our knowledge of the Mediterranean
size distributions in long-term basis. 5. “North Italian aerosol” with very high number concen-
trations, especially during winter in accumulation mode
and with a unimodal median particle number size distri-
bution; 6. “Mediterranean aerosol” with medium number concen-
trations and maximum number concentrations during
summer and spring; and The EUSAAR and GUAN networks are globally unique
both in data quality and relatively dense network. Build-
ing a similar global network is a major undertaking, but
would enable the community to efficiently characterize the
aerosol number distribution, and thus improve the potential
of characterizing the climate impacts of the aerosols in global
boundary layer. 7. “Western European” aerosol with clear influence of
multiple sources of aerosol (clean and polluted), and
with a high seasonal cycle for smaller particle sizes. This grouping does not have high similarity between the
stations. The site categorisation developed by Henne et al. 5
Conclusions (2010)
does not have extremely good agreement with aerosol prop-
erties studied in this paper, the underlying methodology
does have potential also for aerosol studies. The site repre-
sentability regarding some particle size ranges could be de- A. Asmi et al.: Submicron particles in Europe 2008–2009 AW
was partly in charge of MPZ and GUAN datasets, was the
manager of the size distribution measurement planning, stan-
dards and intercalibration. He also contributed to writing of
the manuscript. PL was the project manager of the EUSAAR
network, took a part on the representability analysis and con-
tributed to PDD station data. He also contributed to writing
of the manuscript. AMF was responsible for the database,
data format definitions and other characteristics of stations. KS contributed to writing of the manuscript and was respon-
sible for PDD dataset. WB was in charge of GUAN mea-
surements and the data from MPZ station. EW and UB were
in charge of JFJ measurements and contributed to writing of
the manuscript. VZ was responsible for calibration of sta-
tion OBK, SMPS and its CPC during workshops, installa-
tion of SMPS, its proper long-term operation, maintenance
and enabling the data transfer to the tropos.de and EBAS
databases. NZ did the statistical analysis of the SMPS data
from Kosetice station, checked carefully data from the OBK
station given in Figures and Tables and produced corrected versions of them. J-PP was responsible for the data from ISP
station and he contributed significantly on the early version
of the manuscript. AM was responsible for aerosol measure-
ments at Monte Cimone station, she did the day-to-day up-
keep and data collection and submission to EBAS; She also
commented extensively on the manuscript. PT and HCH
were responsible of the data from ZEP and ASP stations. MF was the instrument operator and took care of the daily
maintenance of station BIR. NK, HL and EA were in charge
of instrumentation and data handling from PAL station; EA
also did the modal fitting. VU contributed for the measure-
ments from station PRL. PPA contributed to day-to-day op-
eration and data quality from SMR and KPO stations. ES
is responsible for the aerosol measurements at the VHL sta-
tion and designed the DMPS system together with AK. NM
is the FKL station head investigator and NK was in charge
of system maintaining, data collecting and analysis. IK was
responsible of PLA station data. GK was responsible for the
KPO station. GdL and BH were responsible for the CBW
dataset and GdL commented extensively on the document. RMH manages the contract under which the HWL station
operates and supervises the processing of the data. DCSB Atmos. Chem. A. Asmi et al.: Submicron particles in Europe 2008–2009 A. Asmi et al.: Submicron particles in Europe 2008–2009 Table 6. Modal fit parameters for median size distributions in the stations. σg is the geometric standard deviation of the mode, Nm is
the mode number concentration, ˆdp,m is the geometric mean dry diameter of the mode and R2 is the coefficient of determination between
observed and fitted size distribution. 1-Mode fit
2-Mode fit
Station
σg
Nm
ˆdp,m
R2
σg,m1
Nm1
ˆdp,m1
σg,m2
Nm2
ˆdp,m2
R2
(cm−3)
(nm)
(cm−3)
(nm)
(cm−3)
(nm)
ASP
2.12
1405
69
0.95
1.87
1233
64
1.36
144
222
0.99
BIR
2.03
700
60
0.91
1.63
539
53
1.41
142
170
1.00
PAL
2.57
337
58
0.84
1.71
221
48
1.45
81
176
0.99
PLA
2.09
3281
80
1.00
2.04
3175
78
1.27
84
207
1.00
SMR
2.32
1202
63
0.97
1.96
1008
57
1.36
144
201
1.00
VHL
2.17
2069
61
0.97
1.86
1756
56
1.41
253
195
1.00
BOS
2.77
5500
48
0.99
1.36
432
21
2.44
4710
57
1.00
KPO
2.32
4353
81
0.98
2.20
2788
54
1.73
1590
134
1.00
OBK
2.31
4183
87
0.97
2.26
2479
51
1.78
2019
131
1.00
HPB
2.73
2173
66
0.96
2.39
1609
45
1.66
554
148
1.00
MPZ
2.74
3941
65
0.99
2.49
3516
58
1.60
338
203
1.00
WAL
2.51
4078
61
0.99
2.37
3837
58
1.38
175
194
1.00
CBW
1.82
6352
48
0.98
1.40
1721
35
1.76
4450
60
0.99
HWL
2.43
2568
49
0.98
2.06
2196
46
1.55
256
196
1.00
MHD
5.00
717
30
0.82
1.79
303
35
1.70
152
174
0.99
FKL
2.06
1613
99
0.99
1.94
1359
86
1.41
228
189
1.00
ISP
2.24
6849
69
0.98
2.19
2828
31
1.87
4344
91
1.00
ZEP
2.20
157
117
0.81
1.65
50
39
1.61
109
150
1.00
PDD
2.57
1482
57
0.98
2.25
1223
48
1.54
207
147
1.00
SSL
2.71
2109
62
0.96
2.32
1709
51
1.48
322
166
1.00
ZSF
2.76
908
54
0.97
2.29
729
45
1.49
132
157
1.00
JFJ
2.20
324
60
0.96
1.70
221
47
1.46
84
136
1.00
BEO
2.16
871
90
0.97
1.76
514
60
1.53
330
156
1.00
CMN
2.47
1005
69
0.95
1.98
716
52
1.50
237
157
1.00 ready-made datasets in databank, programmed the analysis,
wrote most of the article, decided on the chosen metrics,
methods of analysis and made the figures and tables. Author contributions In author order (only initials used): AA had the original idea
of the paper jointly with COD, did the data collection from www.atmos-chem-phys.net/11/5505/2011/ Atmos. Chem. Phys., 11, 5505–5538, 2011 5530 N250 concentrations The instrumentation used in this article have generally
greater discrepancies with the reference instrument on par-
ticles with diameters larger than 200 nm, due to usually low
number concentrations in that range. There however are sta-
tions where the concentration of these particles is enough for
a metric of this range to be relevant. dn
dlog10dp
=
Nm
√
2π log10(σg,m)
exp
−
log10dp −log10 ˆdp,m
2
2
log10(σg,m)
2
(A1) We calculated N250 concentrations for particles between
250 and 500 nm diameter similarly as other integrated con-
centrations in this paper. The resulting histograms are shown
in Fig. 16. The overall picture is that the N250 concentrations
are of greater variability than N100. The Nordic station sea-
sonality is similar as N100, although the summer maximum
is of much less prominence compared to N100 histograms. The Central European stations had a similar histogram shape
seasonality as in N100 cases, although with approximately
one order of magnitude less concentration, suggesting that
the particles between 100 and 250 nm have similar sources
and sinks as particles larger than 250 nm in Central Europe. The Western Europe, North Italy, Mediterranean and Arc-
tic concentration histogram shapes were also very similar to
N100, although the greater concentration maxima of MHD
was much less visible in the N250 distribution. This indicates
that the polluted mode of the MHD station is mainly from
particles between 100 and 250 nm of diameter. The moun-
tain stations had significantly higher variability in N250, es-
pecially for the lowest concentrations. where Nm is the modal concentration (cm−3), σg,m is the ge-
ometric standard deviation of the mode and ˆdp,m is the mode
peak location (nm) (Heintzenberg, 1994). The goodness of fit was determined by the coefficient of
determination R2 = 1−
PN
i (yi −fi)2
PN
i (yi −y)2
(A2) (A2) where N is the number of size bins used in the fit, yi are the
measured size distribution function values for each bin, y is
the mean measured size distribution function value and the
fi are the size distribution function values derived from the
fit for each bin. Results of the mode fitting are in Table 6. The single-
mode fits have a relatively large R2 parameter (>0.95) for
Central European stations, PLA, HWL, CBW, FKL, ISP and
mountain stations. Appendix B We fitted log-normal modes to the median size distributions
of the stations. These help to demonstrate the modality of the
aerosol population at each station. The fit was done only for
particle sizes larger than 20 nm dry particle diameter to avoid
the need of a third mode and to keep the size distribution
measurements comparable. The was done at least-squares
sense using either one or two log-normal modes of form A. Asmi et al.: Submicron particles in Europe 2008–2009 Phys., 11, 5505–5538, 2011 www.atmos-chem-phys.net/11/5505/2011/ A. Asmi et al.: Submicron particles in Europe 2008–2009 5531 mode was more representative of traditional Aitken mode. At CBW and ISP, the two modes were strongly overlapping. The geometric standard deviations of 1-mode fit were gen-
erally large, with an exception in the case of station CBW,
where the geometric standard deviation was almost identical
in 1-mode and 2-mode fits. collates the data from the HWL station and conducts data
analyses and interpretation. COD and SGJ were responsible
for the MHD dataset and COD took a part in the early plan-
ning of this study. HF, KW, FM and LR were responsible of
the GUAN aerosol measurements. MK was responsible of
the design of SMR and KPO measurements and took a part
in the early planning of the work. Not surprisingly, two mode fits improve the goodness of
fit. The median size distribution of most of the stations can
however be rather well captured by a unimodal fit. At the
Nordic stations, Arctic ZEP station and marine-influenced
MHD station the bimodal fit is most likely needed to capture
the overall shape of the distribution. All authors contributed to the final version of the document
with their comments. Atmos. Chem. Phys., 11, 5505–5538, 2011 N250 concentrations The single mode fit did less well on the
more bimodal aerosol populations of the Nordic stations,
MHD and ZEP, resulting in extremely wide geometric stan-
dard deviations. The bimodal fitting parameters have very
large R2 values suggesting that two modes give a very good
approximation of annual median size distribution function. The bimodality of the station ZEP distribution is however
somewhat misleading, as the two modes rarely appear at the
same time (see Section 3.2 for details). The peak diameter
of the smaller (Aitken) mode was for most stations around
50–60 nm, with the exception of BOS, where the smaller
mode was fitted on observed nucleation mode, and the larger The results generally show similar patterns compared to
N100 concentrations, even though the comparability between
instruments is not so good as with other measurements. The
N250 histograms can still be useful, as they represent a sig-
nificant fraction of potential CCN sized aerosol number espe-
cially on the relatively high concentration stations in Central
Europe and Northern Italy. The usability of this metric in the
lowest end of the concentration scale is limited, as the instru-
mental noise starts to affect the concentrations significantly,
probably explaining part of the greater variability of the N250
concentrations compared to other properties in this article. Atmos. Chem. Phys., 11, 5505–5538, 2011 www.atmos-chem-phys.net/11/5505/2011/ 5532 A. Data availability and suggestions on data usage The datasets used in this paper are available directly as time-
series from the graphical interface at NILU EMEP database
(EBAS) at address http://ebas.nilu.no/. The data presented in
this article (histograms, seasonal profiles, etc) are also stored
on publicly available server at http://www.atm.helsinki.fi/
eusaar/. 5. Compare all the available size ranges provided. Getting
a single range, e.g. N50 correct can easily be interpreted
as getting the CCN-sized aerosol “right” in statistical
sense, but can be “correct” from wrong reasons. Inclu-
sion of the three main provided size ranges gives at least
a more comparable set of different processes related to
aerosols which the model has to get correct. The his-
tograms of N250 (Appendix B) have higher possibility
of error, but can be useful as tracers of the above 100 nm
sub-micron aerosol. The data format of data presented in this article is ASCII
text files, with a README file to explain the data structuring
used. The datasets are provided for the following aerosol and
supporting data groups: – Standard size intervals where the size-dependent data
are interpolated – Standard time-axis for using the data (hourly) 6. In comparing with mountain sites, consider the effect of
local topography, which is not usually well described in
the large-scale (and often a large-grid-size) models. The
local wind effects will move polluted boundary layer air
to mountain sites, which can make comparison difficult. In these cases, compare the histograms carefully, with
special attention to the lower concentration ”tails” of the
mountain site concentration profiles. – Station-specific “bad data” vectors, to possibly remove
those same periods from modelled datasets – Aerosol size distribution medians and percentiles per-
size-bin basis. The data is interpolated to standard
size intervals (above) and presented with total, sea-
sonal, daytime, night-time and ECHAM5-HAM sam-
pling. This data is also available separately for 2008
and 2009. 7. In comparing with Arctic site, the effect of Arctic haze
can dominate the aerosol distribution during wintertime. – N30−50, N50 and N100 concentration histograms for
identical logarithmically evenly distributed concentra-
tion bins. The data is separated for total, seasonal, day-
time, night-time and ECHAM5-HAM sampling. 8. A scatterplot of “Aitken” and “accumulation” mode
concentrations can also be useful in trying to see if
the modelled surface concentrations are similar to the
measured ones (see Fig. 13). N250 concentrations Asmi et al.: Submicron particles in Europe 2008–2009 5533 A. Asmi et al.: Submicron particles in Europe 2008–2009 4. If the histogram has multiple peaks, it often suggests
multiple sources of aerosols, e.g. airmasses from dif-
ferent source areas. If your modelled data lacks one
of the peaks detected, the reason might also be on the
model advection scheme instead of aerosol processes or
sources. N250 concentrations Asmi et al.: Submicron particles in Europe 2008–2009 .1
1
10
100
1000
0
0.01
0.02
0.03
0.04
0.05
a) ASP
Relative occurence
.1
1
10
100
1000
0
0.01
0.02
0.03
0.04
b) BIR
.1
1
10
100
1000
0
0.01
0.02
0.03
0.04
c) PAL
.1
1
10
100
1000
0
0.01
0.02
0.03
0.04
0.05
d) PLA
Relative occurence
.1
1
10
100
1000
0
0.02
0.04
0.06
e) SMR
.1
1
10
100
1000
0
0.01
0.02
0.03
0.04
0.05
f) VHL
.1
1
10
100
1000
0
0.02
0.04
0.06
0.08
g) BOS
Relative occurence
.1
1
10
100
1000
0
0.02
0.04
0.06
0.08
h) KPO
.1
1
10
100
1000
0
0.02
0.04
0.06
0.08
i) OBK
.1
1
10
100
1000
0
0.02
0.04
0.06
j) HPB
Relative occurence
.1
1
10
100
1000
0
0.02
0.04
0.06
0.08
k) MPZ
.1
1
10
100
1000
0
0.02
0.04
0.06
0.08
l) WAL
.1
1
10
100
1000
0
0.01
0.02
0.03
0.04
0.05
m) CBW
Relative occurence
.1
1
10
100
1000
0
0.01
0.02
0.03
0.04
0.05
n) HWL
.1
1
10
100
1000
0
0.01
0.02
0.03
0.04
0.05
o) MHD
.1
1
10
100
1000
0
0.01
0.02
0.03
0.04
0.05
p) FKL
Relative occurence
.1
1
10
100
1000
0
0.02
0.04
0.06
q) ISP
.1
1
10
100
1000
0
0.01
0.02
0.03
0.04
r) ZEP
.1
1
10
100
1000
0
0.01
0.02
0.03
0.04
s) PDD
Relative occurence
.1
1
10
100
1000
0
0.01
0.02
0.03
0.04
0.05
t) SSL
.1
1
10
100
1000
0
0.01
0.02
0.03
u) ZSF
.1
1
10
100
1000
0
0.01
0.02
0.03
v) JFJ
Relative occurence
N250 concentration [cm−3]
.1
1
10
100
1000
0
0.01
0.02
0.03
0.04
w) BEO
N250 concentration [cm−3]
.1
1
10
100
1000
0
0.01
0.02
0.03
0.04
x) CMN
N250 concentration [cm−3]
Night−time
All data
Winter
Spring
Summer
Autumn
Histograms of N250 concentrations in the stations. See Fig. 9 for nomenclature. Note the different scale on x-axis co
. 10
100
N250 concentration [cm−3] Fig. 16. Histograms of N250 concentrations in the stations. See Fig. 9 for nomenclature. Note the different scale on x-axis compared to
Figs. 9–11. www.atmos-chem-phys.net/11/5505/2011/ Atmos. Chem. Phys., 11, 5505–5538, 2011 A. Asmi et al.: Submicron particles in Europe 2008–2009 A. Data availability and suggestions on data usage Asmi et al.: Submicron particles in Europe 2008–2009 (c) Map C(i,j) 2D array into a single-dimension array
Cs1(l)
C(i,j) = Cs1(l), ∀i,j
(C3)
where the l = i +(j −1)ns = 1...n2
s. Then sort the
densities in numerical order (from largest density to
lowest)
Cs = sort(Cs1) ⇒Cs(l) ≥Cs(l +1),∀l
(C4) (c) Map C(i,j) 2D array into a single-dimension array
Cs1(l) the person responsible for the data quality in the meta-data
headers of the data files. This contact is very important not
only for the data user, who gets information on the dataset
and possible caveats of use of the data, but also for the data
provider who will then know how and how much the data
is used and can thus partly justify the resources used for the
upkeep of the station. C(i,j) = Cs1(l), ∀i,j
(C3) (C3) where the l = i +(j −1)ns = 1...n2
s. Then sort the
densities in numerical order (from largest density to
lowest) Cs = sort(Cs1) ⇒Cs(l) ≥Cs(l +1),∀l
(C4) (C4) Acknowledgements. This work was performed in the framework
of the Research Infrastructure Action under the FP6 “Structuring
the European Research Area” Programme, EUSAAR Contract
no. RII3-CT-2006-026140. The work has been partly funded
by FP6 Integrated project EUCAARI, Contract no. 36833. The
financial support by the Academy of Finland Centre of Excellence
program (project no. 1118615) is gratefully acknowledged. The
authors thank the Italian Air Force for the fruitful collaboration at
Mt. Cimone and SHARE Project (Ev-K2-CNR) for the financial
support. Authors wish to thank Mila Vana, head of the station KOS
for his support of EUSAAR measurements. The Harwell station
is operated with financial support from the UK Department for
Environment, Food and Rural Affairs. The UK National Centre
for Atmospheric Science is funded by the Natural Environment
Research Council. The authors acknowledge the Swedish Envi-
ronmental Monitoring Program. Thanks to the Norwegian Polar
Institute and the technicians that help maintaining the equipment
at Zeppelin station. Contributions from OPGC, French National
Agency (ANR) and ADEME to support measurements at PDD is
greatly acknowledged. Data availability and suggestions on data usage (d) Find value of lm ∈l where the cumulative sum of
the Cs is closest to 83 percent of the number of ob-
servations (d) Find value of lm ∈l where the cumulative sum of
the Cs is closest to 83 percent of the number of ob-
servations |0.83n−
lm
X
o=1
Cs(o)| = min
|0.83n−
lX
o=1
Cs(o)|
! (C5) (C5) (e) Use this value to draw a contour around regions
of C(i,j) > Cs(lm). We used value of 30 for ns,
but testing showed that the main properties of the
contours were not sensitive to doubling of ns. The
method could also be described in writing as i. Create equal log separated grid of concentra-
tions ii. See how many observations go to each grid box iii. Sum from the greatest observation density grid
box towards lower concentration grid boxes un-
til you end up with 83 percent of the observa-
tions. Draw a contour around these grid-boxes. Edited by: I. Riipinen As a note on the sampling frequency, several large-scale
models do not automatically output information on hourly
basis for performance reasons. As an example ECHAM5-
HAM global climate model outputs data on every six hours
UTC (00:00 h, 06:00 h, 12:00 h and 18:00 h) as instantaneous
concentrations. As we used hourly means in our study, we
could only approximate this imperfect sampling by using the
hourly means of the hours starting with the same UTC hours. Only few of the stations seemed to be sensitive to this change
of sampling, the maximum error of annual median being in
all size ranges less than 5 %, with most stations having er-
ror less that 2 %. The error from this sampling can raise
as high as 14 % for 5th percentile of N100 on mountain sta-
tions, showing that the mountain stations can be sensitive to
non-ideal sampling. Overall the effect on middle percentiles
shown in this article are minimal. The results are well in
line with the extremely high autocorrelation rates of the con-
centrations, suggesting that small variations in sampling or
short-period gaps in the data do not significantly affect the
annual or seasonal mean concentrations. Data availability and suggestions on data usage A way to quickly calcu-
late approximate modal concentrations from concentra-
tions, approximate the Aitken mode concentration by
N30−100 = N50 −N100 + N30−50 and an accumulation
mode with N100. The actual method to draw the “83
percent of observations lines” is as follows: For a successful data-measurement comparison, we sug-
gest that the modeller will at least consider the following
steps 1. Make sure you are comparing similar concentrations. Change the modelled concentrations to particle num-
ber in cm−3 under STP condition air and model the
aerosol to low relative humidity conditions to be com-
parable with measurement techniques and conditions of
this paper (a) Assume n co-incident observations of N30−100 and
N100. Divide concentrations axes into ns logarith-
mically even intervals between concentrations of 1
and 100 000 cm−3. Denote the interval borders as
Ki and Lj (i = j = 1...ns + 1) for N30−100 and
N100, respectively. (a) Assume n co-incident observations of N30−100 and
N100. Divide concentrations axes into ns logarith-
mically even intervals between concentrations of 1
and 100 000 cm−3. Denote the interval borders as
Ki and Lj (i = j = 1...ns + 1) for N30−100 and
N100, respectively. 2. Use long enough datasets to be comparable with the sea-
sonal data provided in this paper. Although e.g. individ-
ual days might be outside of the 16th–84th percentile
range provided in most figures here, they still might be
perfectly reasonable for the most polluted episodes of
the station. (b) Calculate the concentration density function for
each size interval C(i,j): C(i,j) =
n
X
k=1
3(N30−100(k),N100(k),Ki,Ki+1,Lj,Lj+1) (C1) C(i,j) =
n
X
k=1
3(N30−100(k),N100(k),Ki,Ki+1,Lj,Lj+1) (C1) for all i = 1...ns and j = 1...ns, and the summation
function 3 is defined as 3. Do not only compare median concentrations or size
ranges, but instead use histogram information to get
the statistical distributions of the particles correct. The
cloud generation in the model will then at least sample
from similar size distributions of potential CCNs. 3(C1,C2,M11,M12,M21,M22) 3(C1,C2,M11,M12,M21,M22) =
1, if M11 < C1 ≤M12 and M21 < C2 ≤M22
0, otherwise
(C2) =
1, if M11 < C1 ≤M12 and M21 < C2 ≤M22
0, otherwise
(C2) =
1, if M11 < C1 ≤M12 and M21 < C2 ≤M22
0, otherwise
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A total blood volume or more transfused during pregnancy or after childbirth: Individual patient data from six international population-based observational studies
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Citation McCall, S. J., Henriquez, D., Edwards, H. M., Akker, T. van den, Bloemenkamp, K. W. M., Bom,
J. van der, … Knight, M. (2021). A total blood volume or more transfused during pregnancy or
after childbirth: individual patient data from six international population-based observational
studies. Plos One, 16(1). doi:10.1371/journal.pone.0244933 Version:
Publisher's Version
License:
Creative Commons CC BY 4.0 license
Downloaded from:
https://hdl.handle.net/1887/3276466 License: Downloaded from: Note: To cite this publication please use the final published version (if applicable). PLOS ONE RESEARCH ARTICLE Abstract Copyright: © 2021 McCall et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited. Background This study aimed to compare incidence, management and outcomes of women transfused
their blood volume or more within 24 hours during pregnancy or following childbirth. Data Availability Statement: Data cannot be
shared publicly because of confidentiality issues. Request for access to the data should be directed
to the steering committee of the relevant country. Access to the Australian (AMOSS) data requires
specific ethics committee approval and an
application to the AMOSS Steering committee, who
can be contacted using amoss@uts.edu.au;
requests for access to the French data (EPIMOMS)
should be directed to epimoms@inserm.fr; A total blood volume or more transfused
during pregnancy or after childbirth:
Individual patient data from six international
population-based observational studies Stephen J. McCallID1,2*, Dacia Henriquez3,4☯, Hellen McKinnon Edwards5☯, Thomas van
den Akker1,3, Kitty W. M. Bloemenkamp6, Johanna van der Bom4, Marie-Pierre BonnetID7,8
Catherine Deneux-Tharaux8, Serena Donati9, Ada Gillissen3,4, Jennifer J. KurinczukID1,
Zhuoyang Li10, Alice Maraschini9, Aure´lien Seco7,11, Elizabeth Sullivan10,
Simon Stanworth12,13,14, Marian Knight1 a1111111111
a1111111111
a1111111111
a1111111111
a1111111111 a1111111111
a1111111111
a1111111111
a1111111111
a1111111111 1 National Perinatal Epidemiology Unit, Nuffield Department of Population Health, University of Oxford,
Headington, Oxford, United Kingdom, 2 Center for Research on Population and Health, Faculty of Health
Sciences, American University of Beirut, Riad El Solh, Beirut, Lebanon, 3 Department of Obstetrics and
Gynaecology, Leiden University Medical Centre, Leiden, Netherlands, 4 Jon J van Rood Center for Clinical
Transfusion Research, Sanquin Research & Department of Clinical Epidemiology, Leiden University Medical
Center, Leiden, Netherlands, 5 Department of Obstetrics and Gynaecology, Copenhagen University Hospital
Herlev, Herlev, Denmark, 6 Department of Obstetrics, Birth Centre Wilhelmina’s Children Hospital, Division
Woman and Baby, University Medical Centre Utrecht, Utrecht, Netherlands, 7 Department of
Anaesthesiology and Critical Care, Armand Trousseau Hospital, Assistance Publique des Hoˆpitaux de Paris,
Paris, France, 8 INSERM U1153, Obstetrical, Perinatal and Pediatric Epidemiology Research Team
(EPOPe´), Research Center for Epidemiology and Biostatistics (CRESS), Paris University, Paris, France,
9 National Centre for Disease Prevention and Health Promotion, Istituto Superiore di Sanità - Italian National
Institute of Health, Rome, Italy, 10 Faculty of Health and Medicine, The University of Newcastle, Newcastle,
Australia, 11 Clinical Research Unit of Paris Descartes Necker Cochin, APHP, Paris, France, 12 Oxford
University Hospitals NHS Trust, Department of Haematology, Oxford, United Kingdom, 13 NIHR BRC Blood
Theme, University of Oxford, Oxford, United Kingdom, 14 NHS Blood and Transplant, John Radcliffe
Hospital, Oxford, United Kingdom OPEN ACCESS Citation: McCall SJ, Henriquez D, Edwards
HMKinnon, van den Akker T, Bloemenkamp KWM,
van der Bom J, et al. (2021) A total blood volume
or more transfused during pregnancy or after
childbirth: Individual patient data from six
international population-based observational
studies. PLoS ONE 16(1): e0244933. https://doi. org/10.1371/journal.pone.0244933 ☯These authors contributed equally to this work. * sm227@aub.edu.lb ☯These authors contributed equally to this work. * sm227@aub.edu.lb Editor: Frank T. Spradley, University of Mississippi
Medical Center, UNITED STATES
Received: November 6, 2020
Accepted: December 20, 2020
Published: January 22, 2021 Interpretation There was considerable variation in the incidence of massive transfusion associated with
major obstetric haemorrhage across six high-income countries. There were also large dis-
parities in both transfusion and obstetric management between these countries. There is a
requirement for detailed evaluation of evidence underlying current guidance. Furthermore,
cross-country comparison may empower countries to reference their clinical care against
that of other countries. Competing interests: The authors have declared
that no competing interests exist. Findings The incidence of massive transfusion was approximately 21 women per 100,000 maternities
for the United Kingdom, Australia and Italy; by contrast Denmark, the Netherlands and
France had incidences of 82, 66 and 69 per 100,000 maternities, respectively. There was
large variation in obstetric and haematological management across countries. Fibrinogen
products were used in 86% of women in Australia, while the Netherlands and Italy reported
lower use at 35–37% of women. Tranexamic acid was used in 75% of women in the Nether-
lands, but in less than half of women in the UK, Australia and Italy. In all countries, women
received large quantities of colloid/crystalloid fluids during resuscitation (>35 litres). There
was large variation in the use of compression sutures, embolisation and hysterectomy
across countries. There was no difference in maternal mortality; however, variable propor-
tions of women had cardiac arrests, renal failure and thrombotic events from 0–16%. Funding: SM was funded by the Medical Research
Council (UK) and the Nuffield Department of
Population Health, University of Oxford. EPIMOMS
was funded by a grant from the French National
Research Agency (ANR) and the Ile-de-France
Regional Health Agency. The Italian study was
funded by the Italian Ministry of Health. The
AMOSS study “Life-threatening massive obstetric
haemorrhage requiring rapid, high-volume blood
transfusion” was funded by the Australian Red
Cross Blood Service and the Royal Hospital for
Women Foundation. The TeMpOH-1 study in the
Netherlands was funded by a grant from Sanquin
Research, grant No. PPOC-11-032. Methods Combined analysis of individual patient data, prospectively collected in six international pop-
ulation-based studies (France, United Kingdom, Italy, Australia, the Netherlands and Den-
mark). Massive transfusion in major obstetric haemorrhage was defined as transfusion of
eight or more units of red blood cells within 24 hours in a pregnant or postpartum woman. Causes, management and outcomes of women with massive transfusion were compared
across countries using descriptive statistics. 1 / 15 PLOS ONE | https://doi.org/10.1371/journal.pone.0244933
January 22, 2021 PLOS ONE Massive blood transfusion during pregnancy or after childbirth requests for access to the Italian data (ItOSS)
should be made to direzione.cnapps@iss.it; access
to the data from the Netherlands can be requested
from info@studies-obsgyn.nl. Request to access
the Danish data should be directed to the Danish
Health Data Authority, who can be contacted at
dataanmeldelser.herlev-og-gentofte-
hospital@regionh.dk. Requests for access to the
UK dataset will be considered by the National
Perinatal Epidemiology Unit Data Sharing
committee. Access to the UK data can be
requested from general@npeu.ox.ac.uk. PLOS ONE | https://doi.org/10.1371/journal.pone.0244933
January 22, 2021 Case definition Massive transfusion in major obstetric haemorrhage was defined as a receipt of eight or more
units of red blood cells within 24 hours in a pregnant or postpartum woman of at least 20
weeks of gestation. Overall description Member countries of the International Network of Obstetric Survey Systems were invited to
provide national/sub national data on obstetric haemorrhage, and this included published and
non-published data. In each country, data were collected either nationally or from a number
of regions on a population basis using tailored data collection systems. A detailed explanation
of the methodology for each country is summarised in Table 1. In short, these population-
based studies collected data, using enhanced systems, from the medical records of the women
who met the case definition for each of the respective studies. Danish data were solely based
on information entered into the Danish Medical Birth Registry and the Danish Transfusion
Database, which included data from all hospitals in Denmark. Three of the included datasets
were national except for the Netherlands, France, and Italy. The TeMpOH-1 study in the Neth-
erlands collected data from 75% of national births. Italy collected data from six regions and
these represented 49% of births in Italy. The EPIMOMS study in France collected data from
six regions and these regions represented 18% of national births. Study description This international population-based study of massive transfusion in MOH, used secondary
analyses of data from six population-based studies from Australia, Denmark, France, Italy, the
Netherlands and the UK. Introduction The most common form of major obstetric haemorrhage (MOH), postpartum haemorrhage
(PPH) remains a major cause of maternal mortality and morbidity. PPH occurs in 3–7% of
deliveries in high-income settings [1]. In France, Italy and the United States, haemorrhage is
the leading cause of maternal mortality responsible for 11%, 15% and 14% of maternal deaths,
respectively [2–4] and haemorrhage related mortality in the United Kingdom (UK) nearly
doubled during the period 2010–12 to 2013–15 [5]. In addition, major obstetric haemorrhage
(MOH) is a common reason for admission to intensive care [6–9]. Management of MOH, often defined pragmatically as transfusion of a total body blood vol-
ume (8–10 units of red blood cells) or more within 24 hours of delivery [10–12] and also
referred to as massive transfusion, focusses on transfusion and fluid resuscitation, alongside
planning for definitive obstetric and surgical interventions [13]. The literature in major haem-
orrhage caused by trauma has emphasised the importance of damage control resuscitation,
including timely transfusion support, early use of coagulation factors and minimising use of
crystalloids, which have all contributed to improved clinical outcomes. However, it is unclear
how far these protocols should be applied in an obstetric setting [14]. Multiple guidelines to direct transfusion practice in MOH exist and these vary in terms of
when and what quantity of blood components should be given. The reasons for variation in
practice are unclear but are likely in part to reflect lack of high-quality data [15, 16]. A number
of national studies have reported the transfusion management of PPH [13, 17, 18], but there
has been little comparison reported at a national level to explore regional variations in practice. As part of an initiative to inform the international research agenda, this exploratory study was PLOS ONE | https://doi.org/10.1371/journal.pone.0244933
January 22, 2021 2 / 15 PLOS ONE Massive blood transfusion during pregnancy or after childbirth planned to describe and compare the incidence, characteristics, aetiology, management and
outcomes of pregnant or postpartum women who have been transfused their total blood vol-
ume or more within 24 hours, based on regional or national data collection systems across six
countries. Case notification and data collection United Kingdom and Australia. Both countries used a national surveillance system
identify cases of massive transfusion from consultant led obstetric units nationally in the identify cases of massive transfusion from consultant led obstetric units nationally in the
Table 1. Summary of data collection systems and data collected. Country
Name of system or dataset
Type of system
Date collected
Australia
Australasian Maternity Outcomes Surveillance System
(AMOSS). [35]
National obstetric surveillance system. Collected data from
hospitals with more than 50 births per year. One year (July 2014-June
2015)
Denmark
Danish Medical Birth Registry and the Danish
Transfusion Database
Routinely collected national data on both birth and
transfusions. Five years (2010–2015)
France
E´pide´miologic de la Morbidite´ Maternelle Se´vère study
(EPIMOMS) [36]
Prospective Population-based study from all maternity
units in 6 regions. One year (2012–2013)
Italy
Italian Obstetric Surveillance System (ItOSS)
Population-based study from all maternity units in 6
regions in Italy. Two years (September
2014-August 2016)
The
Netherlands
Transfusion strategies in women during Major Obstetric
Haemorrhage study (TeMpOH-1) [37]
Collected data from 75% of national births
Two years (January 2011 and
January 2013)
United
Kingdom
United Kingdom Obstetric Surveillance System (UKOSS)
[38]
Obstetric surveillance system which collected data
nationally from all consultant-led hospitals. One year (June 2013- July
2014)
https://doi.org/10.1371/journal.pone.0244933.t001
PLOS ONE | https://doi org/10 1371/journal pone 0244933
January 22 2021
3 / 15 Table 1. Summary of data collection systems and data collected. 3 / 15 PLOS ONE Massive blood transfusion during pregnancy or after childbirth United Kingdom and from hospitals with over 50 births per year in Australia. Reporters were
asked to complete a data collection form using the medical records of the women. Data were
collected anonymously. The findings from the United Kingdom study have been published
previously [11, 12]. During the period 2014–2015, AMOSS identified women who had 5 or more units of RBCs
transfused within 24 hours at 20 weeks’ gestation or more. Between June 2013- July 2014,
women who had 8 or more units of RBCs transfused within 24 hours at 20 weeks’ gestation or
more were identified using the United Kingdom Obstetric Surveillance System. France. This study included data from the EPIMOMS study that prospectively identified
cases of severe maternal morbidity from 6 regions in France: Alsace Champagne-Ardenne,
Lorraine, Auvergne, Rhone-Alpes, Ile-de-France and Normandy. Cases were identified for a
year between 2012–2013 from 113 intensive care units and 118 maternity units. Case notification and data collection Selected
maternal morbidities were identified by a local reporter in each hospital. Data about demo-
graphics, previous medical history, current pregnancy, causes, management and maternal and
perinatal outcomes were collected from the case notes of each women. Major obstetric haemorrhage was one of the severe maternal morbidities included in the
EPIMOMS study. To be included as a MOH case, women had to be at least 22 weeks’ of gesta-
tion or more and had to meet one of the following criteria: 1500mls blood loss, blood
transfusion 4 RBC, uterine artery embolisation, ligation or compressive uterine sutures or
have an emergency peripartum hysterectomy [19]. Italy. The Italian Obstetric Surveillance (ItOSS) system in Italy identified women who
had 4 or more units of RBCs transfused at 22 weeks’ gestation or greater. This prospective pop-
ulation-based study identified women during 2014–2016 from six regions in Italy, namely
Piedmont and Emilia-Romagna, Tuscany, Lazio, Campania and Sicily. The clinical reporter
from each facility identified and collected data on women with PPH, uterine rupture, abnor-
mally invasive placenta and peripartum hysterectomy. Clinical and demographic data were
collected from the woman’s medical records and entered into an online data collection form. The completeness of the reported cases was verified through the Hospital Discharge Database. The Netherlands. TeMpOH-1 was a national retrospective cohort study conducted
between January 2011 and January 2013 in the Netherlands. Women were identified using rec-
ords from blood transfusion services and birth registries of participating hospitals. Women
were included if they had received four units or more of RBCs or a multicomponent blood
transfusion as a result of obstetric haemorrhage. Data were collected from medical records and
included information about previous medical history, current pregnancy, causes of haemor-
rhage, haematological parameters, blood components, fluid resuscitation and surgical and
medical management. Denmark. The Danish data were identified and collected using the Danish National Birth
Registry (DNBR) and the Danish Transfusion Database (DTD). During the period 2010–2015,
women who had a birth identified in the DNBR and had transfused 8 or more units of RBC
within 24 hours identified from DTD were included in study. The DNBR collects information
on all births in Denmark and this includes clinical and demographic data from each birth. The
DTD is a national database and contains information of the quantity and type of blood prod-
ucts transfused and the time of each transfusion. Statistical analysis Women’s characteristics, medical history, obstetric and transfusion management and maternal
outcomes were compared across countries. Normality was assessed using histograms. Nor-
mally distributed continuous variables were presented as means with standard deviations and
skewed continuous variables were presented as medians with interquartile ranges. The exact
binomial distribution was used to estimate confidence intervals for proportions. Statistical
hypothesis testing was carried out to test for statistical differences between countries and
respective characteristics, management and outcomes of women undergoing massive transfu-
sion. Descriptive analyses used the following tests where appropriate: Analysis of Variance
(ANOVA), Wilcoxon rank-sum test, Kruskal-Wallis test and chi square tests or the Fisher-
Freeman-Halton’s test. In order to reduce the risk of a type I error a p-value <001 was used to
indicate statistical significance. Missing data were included as a ‘missing’ category for categori-
cal variables. If a country did not collect the specified variable this was indicated by a dash in
tables. Complete case analysis was used for continuous variables. Categorisation of aetiology of MOH. France had multiple causes of MOH coded, so a
primary cause was devised using a hierarchical approach. The primary cause was defined in
the following order: first those with abnormal placentation, second women with abruption,
third women with trauma and lastly those with atony. Comparability of datasets With reference to the literature, the clinical and demographic variables required to answer the
objectives were requested from the participating countries. Available desired variables were
provided by each country. Using data dictionaries from each country, each variable was 4 / 15 PLOS ONE | https://doi.org/10.1371/journal.pone.0244933
January 22, 2021 PLOS ONE Massive blood transfusion during pregnancy or after childbirth mapped to assess the availability and comparability between datasets. If the components of a
particular variable were not comparable between the datasets, then a common definition for
that variable was created. If this was not possible the variable was excluded from the analysis. A mapping exercise was completed to assess whether the coding matched; where coding dif-
fered, a harmonised coding was devised and applied. The included variables and issues with
comparability can be found in S1 Table. PLOS ONE | https://doi.org/10.1371/journal.pone.0244933
January 22, 2021 Results Denmark reported the highest incidence of massive transfusion at 82 (95% CI: 73–92) women
per 100,000 deliveries; followed by France and the Netherlands with an incidence of 69 per
100,000 deliveries (95% CI: 58–82) and 66 per 100,000 deliveries (95% CI: 57–77), respectively. The UK, Australia and Italy had a similar incidence of massive transfusion at approximately 21
women per 100,000 deliveries (Table 2). These variable rates are seen despite many similarities
in baseline characteristics of women presenting with PPH, including age and BMI. Mean age of women with massive transfusion was 32 years (SD ±57), and the majority
were multiparous (57%). Amongst women with a previous pregnancy, there were differences
between countries in the proportion of women with previous caesarean section and previous
postpartum haemorrhage (previous caesarean section: highest in the UK (66%) and lowest in
Australia (39%)). Amongst cases, there were differences in the proportions of women with multiple pregnancies
between countries (Denmark 24%, France 14% and UK 4%, respectively). In addition, the major-
ity of women were delivered by caesarean section in each country (range excluding the Nether-
lands: 59%-68%), while the Netherlands had the lowest proportion of caesarean deliveries at 42%. The most common cause of MOH in all countries was atony, with a prevalence ranging
from 40% in the UK to 63% in the Netherlands (S2 Table). The second leading cause was
abnormal placentation with a prevalence ranging from 22% in Italy and the Netherlands to
32% in Australia. The least common cause was placental abruption with a proportion ranging
from 3% in the Netherlands and Italy to 9% in the UK. Use of blood components varied across countries, Italy had a smaller median use of fresh
frozen plasma (FFP) and platelets compared to other countries (Table 3). France had the high-
est use of FFP with a median of 7 units (IQR: 5–9) compared with the other countries. The
inter country difference in use of FFP was statistically significant (P<001). Different concentrated sources of fibrinogen were used, the UK and Australia mainly
administered cryoprecipitate while France, Italy and the Netherlands used fibrinogen concen-
trate. The highest proportion of women receiving a fibrinogen product was in Australia (86%),
while the Netherlands and Italy had the lowest use (35–37%) compared to the other countries
(64%-86%). Ethics approval and data sharing agreements This study was a secondary data analysis of data collected from primary studies. All countries
had the required ethics approval for their primary studies and performed research following
the World Medical Association’s Declaration of Helsinki [20]. The UK, Italy, TeMpOH-1 (the
Netherlands), France and Australia had obtained ethics approval for the collection of anon-
ymised information without seeking the consent of the women from the London Multi-centre
Research Ethics Committee (MREC reference 04/MRE02/45), Italian National Institute of
Health (Prot. PRE-839/13, Rome 19/12/2013), Leiden University Medical Center, National
Data Protection Authority in France (CNIL, authorisation no. 912210, 14/3/2012), and from
the Human Research Ethics Committee, New South Wales, Australia and from Human
Research Ethics Committee for individual sites across Australia, respectively. Australia
obtained ethics approval for this secondary data analysis; this study was approved by the
Human Research Ethics Committee, New South Wales, Australia. The TeMpOH-1 study was
approved by the ethics committee of the Leiden University Medical Center and by the institu-
tional review board of each participating hospital; the TeMpOH-1 and ItOSS steering commit-
tees approved the study protocol and the data transfer agreement. The ethics committee at the
Leiden University Medical Center approved the study protocol. The French data collaborators
gained approval for this analysis from the EPIMOMS steering committee at INSERM, Paris. Ethics committee approval for the secondary analysis of UK data was not required. The Danish
collaborators acquired permission from the Danish Data Protection Agency (J.nr: HGH-2016-
066), no permission was required from the Danish Ethics Committee according to Danish law. 5 / 15 PLOS ONE | https://doi.org/10.1371/journal.pone.0244933
January 22, 2021 PLOS ONE Massive blood transfusion during pregnancy or after childbirth PLOS ONE | https://doi.org/10.1371/journal.pone.0244933
January 22, 2021 PLOS ONE PLOS ONE Table 2. Sociodemographic, previous medical history and current pregnancy information in women undergoing massive transfusion for major obstetric
haemorrhage. PLOS ONE UK n = 162
Australia
n = 61
Italy n = 99
the
Netherlands
n = 179
France
n = 126
Denmark
n = 288
P-value
National denominator
787,105
302,666
458,995
270,101
182,309
349,998
Incidence per 100,000 maternities
206 (175–
240)
202 (154–
259)
216(175–
263)
663 (569–
767)
691 (579–
823)
823 (731–
924)
Age
Mean (Std)
33
(59)
33
(53)
35
(61)
32
(53)
33
(59)
32
(54)
<0001
BMI
Median (IQR)
25
(22–
29)
25
(21–
28)
23
(21–
25)
23
(21–
26)
24
(21–
27)
23
(21–
27)
<0001
Previous medical history
Parity
Nulliparous
60
(373)
20
(328)
48
(485)
84
(469)
56
(448)
128
(448)
0185
Multiparous
101
(627)
41
(672)
51
(515)
95
(531)
69
(552)
158
(552)
Missing
1
0
0
0
1
2
Previous caesarean section
None
35
(343)
25
(61)
23
(469)
51
(537)
38
(559)
77
(481)
0020
Yes
67
(657)
16
(39)
26
(531)
44
(463)
30
(441)
83
(519)
Nulliparous
60
20
48
84
56
128
Missing
0
0
2
0
2
0
Number of previous caesarean section
0
35
(343)
25
(61)
23
(469)
-
-
38
(559)
-
-
0.036
1
38
(373)
10
(244)
16
(327)
-
-
21
(309)
-
-
2+
29
(284)
6
(146)
10
(204)
-
-
9
(132)
-
-
Nulliparous
60
20
48
-
-
56
-
-
Missing
0
0
2
2
Previous postpartum haemorrhage
None
78
(765)
29
(853)
41
(911)
86
(915)
64
(928)
-
-
0007
Yes
24
(235)
5
(147)
4
(89)
8
(85)
5
(72)
-
-
Nulliparous
60
20
48
84
56
-
-
Missing
0
7
6
1
1
-
-
Current pregnancy
Hypertension prior to pregnancy or current
problem
None
161
(994)
59
(967)
94
(949)
153
(855)
124
(992)
-
-
<0001
Yes
1
(06)
2
(33)
5
(51)
26
(145)
1
(08)
-
-
Missing
0
0
0
0
1
-
-
Infection in current pregnancy
No
161
(994)
56
(949)
99
(100)
179
(100)
117
(975)
-
-
0006
Yes
1
(06)
3
(51)
0
(0)
0
(0)
3
(25)
-
-
Missing
0
2
0
0
6
-
-
Inherited bleeding disorders
No
160
(988)
55
(982)
-
-
-
-
121
(968)
-
-
0602
Yes
2
(12)
1
(18)
-
-
-
-
4
(32)
-
-
Missing
0
5
-
-
-
-
1
-
-
Multiple pregnancy
No
155
(957)
58
(951)
92
(929)
167
(938)
108
(857)
219
(76)
<0001
Yes
7
(43)
3
(49)
7
(71)
11
(62)
18
(143)
69
(24)
Missing
0
0
0
1
0
0
Induced
None
103
(652)
20
(541)
56
(737)
117
(654)
52
(416)
-
-
<0001
Yes
55
(348)
17
(459)
20
(263)
62
(346)
73
(584)
-
-
Missing
4
24
23
0
1
-
-
Delivery type
Vaginal delivery
50
(316)
25
(41)
37
(374)
104
(584)
47
(376)
92
(319)
<0001
Caesarean
delivery
108
(684)
36
(59)
62
(626)
74
(416)
78
(624)
196
(681)
N/A Pregnancy
loss
4
0
0
1
1
0
Used Fisher’s exact test. Used Fisher s exact test.
Missing data were not included in the percentage. A dash indicated that the country did not collect data on that specific variable. a were not included in the percentage. A dash indicated that the country did not collect data on that specific variable. Results Between 13–16% of women received factor VIIa in Italy, France and the Netherlands while
the United Kingdom reported the lowest use at 8%, although this difference was not statisti-
cally significant. Use of tranexamic acid (TXA) also varied between countries, the Netherlands
used TXA in 75% of women with massive transfusion while it was used in less than a half of
women in the UK, Australia and Italy, and these differences were statistically significant. Large
amounts of colloid and crystalloid fluids were transfused; the median fluid transfusion was
between 3.5 and 4.5 litres in Italy, the Netherlands and the UK. The use of surgical techniques and interventional radiology also varied between countries
(Table 4). An intrauterine balloon was used in at least half of all women with massive transfusion
except in France where a balloon was only used in a third of women. Use of arterial embolisation
and major vessel ligation was more common in France and the Netherlands (71% and 52% of
women, respectively) compared to the UK and Italy (16% and 4% of women, respectively). Uterine compression sutures were more commonly used in Australia, France and the UK
(36%-24%) compared to the remaining countries (13–14%). In Italy, 74% of women with a
massive transfusion had a hysterectomy, compared with less than half of women in the United
Kingdom, France, and a third of women in the Netherlands. Overall there were fourteen maternal deaths which gave a case fatality of 1.5% (95% CI: 0.8–
2.6) (Table 5). Nevertheless, maternal mortality was rare across all countries. Fifteen women had 6 / 15 PLOS ONE | https://doi.org/10.1371/journal.pone.0244933
January 22, 2021 Massive blood transfusion during pregnancy or after childbirth PLOS ONE Massive blood transfusion during pregnancy or after childbirth PLOS ONE Table 3. Haematological management by country. only recorded use of 5 and 25% albumin administration. y
a were not included in the percentage. A dash indicated that the country did not collect data on that specific variable. PLOS ONE Missing data were not included in the percentage. A dash indicated that the country did not collect data on that specific variable. edical history and current pregnancy information in women undergoing massive transfusion for major obstetric PLOS ONE | https://doi.org/10.1371/journal.pone.0244933
January 22, 2021 7 / 15 https://doi.org/10.1371/journal.pone.0244933.t003 Australia only recorded use of 5 and 25% albumin administration. Australia only recorded use of 5 and 25% albumin administration.
Missing data were not included in the percentage A dash indicated that the country did not collect data on that specific variable lbumin administration.
tage. A dash indicated that the country did not collect data on that specific variable. PLOS ONE Blood products
UK n = 162
Australia
n = 61
Italy n = 99
the Netherlands
n = 179
France
n = 126
Denmark
n = 288
P-value
Number of RBC units
Median
(IQR)
10
(8–14)
11
(9–14)
9
(8–11)
11
(9–15)
10
(9–
12)
11
(8–
15)
<0001
Received Fresh frozen plasma
n (%)
162
(100)
59
(967)
84
(848)
179
(100)
126
(100)
247
(858)
<0001
Fresh frozen plasma
Median
(IQR)
6
(4–8)
6
(4–8)
4
(2–8)
6
(4–8)
7
(5–9)
6
(4–8)
<0001
Received Platelets
n (%)
126
(778)
51
(836)
40
(404)
179
(100)
126
(100)
205
(712)
<0001
Platelets
Median
(IQR)
1
(1–2)
1
(1–2)
0
(0–2)
2
(1–3)
1
(1–3)
2
(0–3)
<0001
Source of concentrated fibrinogen
administered
No
58
(358)
8
(136)
62
(626)
116
(648)
27
(223)
-
-
<0001
Yes
104
(642)
51
(864)
37
(374)
63
(352)
94
(777)
-
-
Missing
0
2
0
0
5
-
-
Fibrinogen concentrate used
No
152
(938)
54
(100)
62
(626)
116
(648)
27
(223)
-
-
<0001
Yes
10
(62)
0
(0)
37
(374)
63
(352)
94
(777)
-
-
Missing
0
7
0
0
5
-
-
Cryoprecipitate used
No
55
(355)
8
(136)
-
-
-
-
-
-
-
-
0002
Yes
100
(645)
51
(864)
-
-
-
-
-
-
-
-
Missing
7
2
-
-
-
-
-
-
-
-
Number of units of cryoprecipitate
Median
(IQR)
2
(0–2)
1
(1–2)
-
-
-
-
-
-
-
-
0.07
Use of cell salvage
No
115
(742)
46
(852)
-
-
-
-
-
-
-
-
0098
Yes
40
(258)
8
(148)
-
-
-
-
-
-
-
-
Missing
7
7
-
-
-
-
-
-
-
-
If Yes, amount transfused (ml)
Median
(IQR)
1150
(500–
1900)
884
(565–
1377)
-
-
-
-
-
-
-
-
0.95
Recombinant Factor VIIa
No
149
(92)
48
(906)
86
(869)
151
(844)
103
(851)
-
-
0224
Yes
13
(8)
5
(94)
13
(131)
28
(156)
18
(149)
-
-
Missing
0
8
0
0
5
-
-
Tranexamic acid
No
87
(537)
41
(745)
57
(576)
45
(251)
47
(392)
-
-
<0001
Yes
75
(463)
14
(255)
42
(424)
134
(749)
73
(608)
-
-
Missing
0
6
0
0
6
-
-
Any fluid used during resuscitation
No
4
(25)
42
(778)
0
(0)
0
(0)
0
(0)
-
-
<0001
Yes
157
(975)
12
(222)
55
(100)
167
(100)
108
(100)
-
-
Missing
1
7
44
12
18
-
-
Total amount (ml)
4000
(3000–
6000)
3500
(3000–
5000)
4500
(3000–
6000)
0205
Colloid
Yes
139
(885)
-
-
49
(98)
160
(964)
87
(829)
-
-
<0001
Missing
5
-
-
49
13
21
-
-
If Yes, amount transfused (ml)
Median
(IQR)
1000
(650–
2000)
-
-
1000
(500–
1500)
1500
(1000–
2000)
-
-
-
-
<0001
Crystalloid
Yes
152
(95)
-
-
49
(100)
137
(100)
103
(954)
-
-
0013
Missing
2
-
-
50
42
18
-
-
If Yes, amount transfused (ml)
Median
(IQR)
3000
(1500–
4000)
-
-
2500
(1700–
3500)
2500
(1600–
4300)
-
-
-
-
0442
Used Fisher’s exact test. PLOS ONE Italian and Danish data include intrauterine packing. Italian and Danish data include intrauterine packing. The Netherlands: intra-abdominal packing was after hysterectomy and was not included. Missing data were not included in the percentage. A dash indicated that the
country did not collect data on that specific variable. e packing. as after hysterectomy and was not included. Missing data were not included in the percentage. A dash indicated that the
ariable. https://doi.org/10.1371/journal.pone.0244933.t004 a thrombotic event; women in France had about double the proportion of these events compared
to the UK and Australia (6% vs. ~3%, respectively). In addition, France had a higher proportion
of renal failure compared to other countries (16% vs. ~1–2%, respectively). Furthermore, France
had a slightly higher proportion of cardiac arrests compared to other countries (8% vs. ~1–5%). PLOS ONE Massive blood transfusion during pregnancy or after childbirth PLOS ONE Table 4. Obstetric management by country. UK n = 162
Australia
n = 61
Italy n = 99
the Netherlands
n = 179
France n = 126
Denmark
n = 288
P-value
Medical management
Oxytocin
No
6
(37)
7
(117)
26
(263)
65
(363)
47
(395)
-
-
<0001
Yes
156
(963)
53
(883)
73
(737)
114
(637)
72
(605)
-
-
Missing
1
0
7
-
-
Ergometrine
No
69
(426)
32
(561)
-
-
160
(894)
-
-
-
-
<0001
Yes
93
(574)
25
(439)
-
-
19
(106)
-
-
-
-
Missing
4
-
-
Prostaglandin
No
64
(395)
30
(526)
49
(495)
30
(168)
16
(136)
-
-
<0001
Yes
98
(605)
27
(474)
50
(505)
149
(832)
102
(864)
-
-
Missing
4
0
8
-
-
Misoprostol
No
73
(451)
29
(518)
-
-
117
(654)
118
(992)
-
-
<0001
Yes
89
(549)
27
(482)
-
-
62
(346)
1
(08)
-
-
Missing
5
7
-
-
Any uterotonic treatment
No
5
(31)
3
(5)
21
(212)
13
(73)
9
(75)
-
-
<0001
Yes
157
(969)
57
(95)
78
(788)
166
(927)
111
(925)
-
-
1
6
-
-
Surgical management
Intrauterine balloons
No
68
(42)
26
(448)
45
(455)
77
(43)
88
(721)
246
(854)
<0001
Yes
94
(58)
32
(552)
54
(545)
102
(57)
34
(279)
42
(146)
Missing
0
3
0
0
4
Intra-abdominal packing
No
147
(907)
47
(839)
-
-
-
-
109
(901)
-
-
0344
Yes
15
(93)
9
(161)
-
-
-
-
12
(99)
-
-
Missing
5
-
-
-
-
5
-
-
Embolisaton or ligation
No
136
(84)
41
(672)
95
(96)
86
(48)
36
(295)
286
(993)
<0001
Yes
26
(16)
20
(328)
4
(4)
93
(52)
86
(705)
2
(07)
Missing
4
Compressive sutures
No
123
(759)
38
(644)
86
(869)
154
(86)
90
(738)
247
(858)
<0001
Yes
39
(241)
21
(356)
13
(131)
25
(14)
32
(262)
41
(142)
Missing
0
2
0
0
4
Hysterectomy
No
87
(537)
29
(475)
26
(263)
126
(704)
66
(545)
223
(774)
<0001
Yes
75
(463)
32
(525)
73
(737)
53
(296)
55
(455)
65
(226)
Missing
0
0
0
0
5
0 Table 4. Obstetric management by country. https://doi.org/10.1371/journal.pone.0244933.t004 PLOS ONE Australia only recorded use of 5 and 25% albumin administration. PLOS ONE | https://doi.org/10.1371/journal.pone.0244933
January 22, 2021 8 / 15 Discussion Maternal outcomes: Cardiac arrest: UK included—cardiac arrest, cardiac infection and cardioversion and inotropic support; Italy, Australia and France: cardiac arrest. Infection: UK: chest infection, septicaemia, septic shock, sepsis, enterococcus infection, necrotising fasciitis, C. difficile, abscess, suspected tuberculosis or meningitis,
wound infection and wound dehiscence. Australia: sepsis. Italy and France: septicaemia. Thrombotic: pulmonary embolism or deep venous thrombosis. Missing data were not included in the percentage. A dash indicated that the country did not collect data on that specific variable. ITU: Intensive therapy unit. Maternal outcomes: Cardiac arrest: UK included—cardiac arrest, cardiac infection and cardioversion and inotropic support; Italy, Australia and France: cardiac arrest. Infection: UK: chest infection, septicaemia, septic shock, sepsis, enterococcus infection, necrotising fasciitis, C. difficile, abscess, suspected tuberculosis or meningitis,
wound infection and wound dehiscence. Australia: sepsis. Italy and France: septicaemia. Thrombotic: pulmonary embolism or deep venous thrombosis. Missing data were not included in the percentage. A dash indicated that the country did not collect data on that specific variable. ITU: Intensive therapy unit. Maternal outcomes: Cardiac arrest: UK included—cardiac arrest, cardiac infection and cardioversion and inotropic support; Italy, Australia and France: cardiac arrest. Infection: UK: chest infection, septicaemia, septic shock, sepsis, enterococcus infection, necrotising fasciitis, C. difficile, abscess, suspected tuberculosis or meningitis,
wound infection and wound dehiscence. Australia: sepsis. Italy and France: septicaemia. Thrombotic: pulmonary embolism or deep venous thrombosis. Missing data were not included in the percentage. A dash indicated that the country did not collect data on that specific variable. ITU: Intensive therapy unit. https://doi.org/10.1371/journal.pone.0244933.t005 guideline writers, in each country different obstetric and haematological management is pro-
vided for the management of women undergoing transfusion in major obstetric haemorrhage. Although there were no differences in maternal mortality between countries, these numbers
are low, and there was evidence of highly variable rates of cardiac arrest, thrombotic events
and renal failure. The greatest variation in resuscitation was seen for use of fibrinogen prod-
ucts, tranexamic acid and recombinant factor VIIa. Again, there was striking variation in
obstetric management including the use of intrauterine balloons, embolisation and hysterec-
tomy between countries. The variation in incidence between countries is likely to be multifactorial. Variations in
obstetric management may be a relevant factor, for example, differential thresholds for peri-
partum hysterectomy may be a potential determinant of the inter country differences. Discussion This population-based study has shown a five-fold variation in the incidence of massive trans-
fusion across six high-income countries with similarly well-developed healthcare systems and
medical resources. Despite the same primary evidence being available to clinicians and PLOS ONE | https://doi.org/10.1371/journal.pone.0244933
January 22, 2021 9 / 15 Massive blood transfusion during pregnancy or after childbirth PLOS ONE Table 5. Maternal outcomes by country. UK n = 162
Australia n = 61
Italy n = 99
the Netherlands
n = 179
France n = 126
Denmark n = 288
P-value
Maternal outcome
Maternal death
No
160
(988)
61
(100)
96
(97)
175
(978)
123
(976)
286
(993)
037
Yes
2
(12)
0
(0)
3
(3)
4
(22)
3
(24)
2
(07)
Cardiac arrest
No
154
(951)
57
(966)
94
(949)
-
-
116
(921)
285
(99)
0005
Yes
8
(49)
2
(34)
5
(51)
-
-
10
(79)
3
(1)
Missing
0
2
0
0
0
Renal failure
No
160
(988)
58
(983)
97
(98)
-
-
106
(841)
284
(986)
<0001
Yes
2
(12)
1
(17)
2
(2)
-
-
20
(159)
4
(14)
Missing
2
0
0
Infection
No
157
(969)
56
(966)
97
(98)
-
-
118
(937)
-
-
0399
Yes
5
(31)
2
(34)
2
(2)
-
-
8
(63)
-
-
Missing
0
3
0
0
Thrombotic event
No
158
(975)
57
(966)
99
(100)
-
-
118
(937)
287
(997)
0001
Yes
4
(25)
2
(34)
0
(0)
-
-
8
(63)
1
(03)
Missing
0
2
0
-
-
0
0
ITU admission
No
30
(185)
2
(33)
15
(153)
32
(179)
44
(349)
-
-
<0001
Yes
132
(815)
59
(967)
83
(847)
147
(821)
82
(651)
-
-
Used Fisher’s exact test. Maternal outcomes: Cardiac arrest: UK included—cardiac arrest, cardiac infection and cardioversion and inotropic support; Italy, Australia and France: cardiac arrest. Infection: UK: chest infection, septicaemia, septic shock, sepsis, enterococcus infection, necrotising fasciitis, C. difficile, abscess, suspected tuberculosis or meningitis,
wound infection and wound dehiscence. Australia: sepsis. Italy and France: septicaemia. Thrombotic: pulmonary embolism or deep venous thrombosis. Missing data were not included in the percentage. A dash indicated that the country did not collect data on that specific variable. ITU: Intensive therapy unit. Table 5. Maternal outcomes by country. Used Fisher’s exact test. PLOS ONE | https://doi.org/10.1371/journal.pone.0244933
January 22, 2021 https://doi.org/10.1371/journal.pone.0244933.t005 Discussion More
timely recognition, and control of bleeding may prevent worsening to more severe bleeds (8
units), and therefore factors that delay presentation and definitive management need to be
explored between countries. National support for robust MOH protocols through appropriate
fluid and haematological management may also control the bleeding at earlier stages. Other
factors include case-mix and variation in risk factors such as the caesarean section rate, and
rate of multiple pregnancies. However, the data collected in each country did not include
information about the general obstetric population, so there may be other differences in back-
ground risks for PPH between countries that have not been captured. Alternatively, the varia-
tion in incidence of massive transfusion may be a reflection of differential transfusion policies. 10 / 15 PLOS ONE | https://doi.org/10.1371/journal.pone.0244933
January 22, 2021 PLOS ONE Massive blood transfusion during pregnancy or after childbirth Guidelines for the haematological and fluid resuscitation management of haemorrhage
vary by country and medical specialty [16]. Consequently, it is not surprising that clinical prac-
tice and use of blood components differed by country. Clinicians in the UK and Australia mainly used cryoprecipitate as a source of concentrated
fibrinogen while in Italy, France and the Netherlands clinicians only used fibrinogen concen-
trate. There is a paucity of evidence regarding the efficacy of, optimal source and dose of
fibrinogen. The FIB-PPH trial compared the use of fibrinogen concentrate to placebo in the
initial treatment of PPH, but the intervention did not reduce the primary outcome of postpar-
tum bleeding [21]; the study explored the efficacy of fibrinogen concentrate among a wide
range of cases including non-severe PPH. The FIBRES trial conducted in cardiac surgery
showed that fibrinogen was noninferior to cryoprecipitate [22]. While this study focused on
sources of concentrated fibrinogen, FFP is also a source of fibrinogen; however, FFP has a
much lower concentration of fibrinogen than fibrinogen concentrate or cryoprecipitate. Con-
sequently, FFP is not recommended as a primary agent for fibrinogen concentration [23, 24]. At the time of the data collection for this study there was a lack of high-quality evidence
about the use of TXA in the treatment of obstetric haemorrhage, and there was variation in
use. Trials were either too small to detect a statistically significant difference in the primary
outcome [25] or did not have the power to examine safety issues of the drug [26]. Discussion The findings
of the WOMAN trial have now been published and it would be of interest to understand how
this trial has changed TXA use [27]. In the majority of countries, current guidelines and expert reviews recommend that recom-
binant factor VIIa should not be given in obstetric haemorrhage [16, 28, 29]. A Cochrane
review reported no evidence to support the efficacy of recombinant factor VIIa across a range
of clinical settings as prophylaxis or therapeutically; but with evidence to indicate an increased
risk of thromboembolic events [30]. French guidelines allow for the use of recombinant factor
VIIa as a “compassionate treatment” to avoid a hysterectomy in nulliparous women or for life-
threatening situations [31]. Overall, despite lack of evidence of benefit alongside risks and high
costs, approximately 12% of women across all 6 countries received the pro-haemostatic agent
in this international study. Previous research has shown that obstetric management of PPH varies internationally [32–
34]. The findings from this study are consistent with this, as embolisation use was higher in
France and the Netherlands in comparison to the UK. This most likely reflects the availability
of the infrastructure required to provide an embolisation service as well as perceived efficacy. Intrauterine balloon tamponade is the most common second stage management of MOH and
its lower use in France most likely reflects higher use of embolisation. This study also showed a significant disparity in the use of hysterectomy where three-quar-
ters of women had a hysterectomy in Italy while only a third of women did so in the Nether-
lands and less than a fifth in Denmark. Consistent with previous research, a similar proportion
of women in the UK and France had a hysterectomy [32]. With no difference in maternal mor-
tality between countries, this suggests that the future fertility of some of these women could
have been saved. There was no difference in maternal mortality between countries. However, this study may
not have been adequately powered to test for differences in this rare event. A higher number of
women with massive transfusion in France had renal failure, cardiac arrests and thrombotic
events. The reasons are unclear but higher rates of pro-haemostatic agents were reported. However, the EPIMOMS study included specific questions on these outcomes within their
data collection form, which may have resulted in better ascertainment than the UK, Italy and
Australia. PLOS ONE | https://doi.org/10.1371/journal.pone.0244933
January 22, 2021 Discussion 11 / 15 PLOS ONE | https://doi.org/10.1371/journal.pone.0244933
January 22, 2021 PLOS ONE Massive blood transfusion during pregnancy or after childbirth This study was unique in its ability to compare all women between populations, based on
national or regional studies with women who had been transfused at least eight of more units
of red blood cells. This study used similar methods to an individual patient data meta-analysis. This methodology allowed the harmonisation of definitions and creation of comparable data-
sets, which in turn allowed robust comparisons between nations. All countries except for Den-
mark had tailored data collection forms, which collected detailed information about each
woman, and enabled this unique comparison. This analysis should be interpreted in the context of its limitations. Each national study col-
lected slightly different items. To reduce errors due to misclassification all countries had their
cases cross-checked against hospital records, other than Denmark, which was solely based on
ICD-10-CM codes. This analysis was also susceptible to survival bias, as those women who died or had a hyster-
ectomy before they received 8 units of RBC would not have been included in the study popula-
tion. To overcome this limitation, future epidemiological studies should use a composite of
inclusion criteria to prevent survival bias. This composite should include women who died
from a haemorrhage, women who have a hysterectomy or surgical management due to uncon-
trolled bleeding, women who meet a certain number of RBC units transfused or where a major
obstetric haemorrhage protocol has been activated. Furthermore, women with haemorrhage
before 20 weeks of gestation were not included in the case definition, thus most women with a
major haemorrhage as a result of an ectopic pregnancy would have been excluded. In addition,
the surveillance systems in UK, Australia and Italy may not have identified women with sec-
ondary PPH who were not readmitted to an obstetric unit, whilst hospital databases and blood
banks in Denmark, the Netherlands and France were interrogated to ensure obstetric related
haemorrhages after pregnancy were included. It is unlikely, however, that differences in regis-
tration are responsible for the marked variation seen in incidence. Conclusion There was a considerable variation in the incidence of massive transfusion associated with
major obstetric haemorrhage between countries. This may be the result of disparities in the
timely management of less severe bleeds between countries or differences in transfusion poli-
cies; however, more research is required to ascertain reasons for these differences. Obstetric
management and the use of blood products and haemostatic agents varied substantially. How-
ever, despite these variations in management, the rate of maternal mortality was similar. Therefore, there is need for a detailed evaluation of the evidence underlying current guidance,
including the use of fibrinogen concentrate and other pro-thrombotic agents. g
y
Therefore, there is need for a detailed evaluation of the evidence underlying current guidance,
including the use of fibrinogen concentrate and other pro-thrombotic agents. Acknowledgments This formed part of Stephen McCall’s DPhil in Population Health, University of Oxford. This formed part of Stephen McCall’s DPhil in Population Health, University of Oxford. S1 Table. Availability and comparability of variables from each respective dataset.
(DOCX) S2 Table. Aetiology of the massive obstetric haemorrhage by country. Legend: France had a
hierarchy of primary cause of PPH and were coded using this system: 1st Abnormal placenta-
tion, 2nd Abruption, 3rd Trauma and 4th was Atony. Denmark did not have data on cause of
haemorrhage. (DOCX) Project administration: Stephen J. McCall. Project administration: Stephen J. McCall. Supervision: Thomas van den Akker, Jennifer J. Kurinczuk, Simon Stanworth, Marian
Knight. Writing – original draft: Stephen J. McCall, Alice Maraschini, Aure´lien Seco. Writing – review & editing: Dacia Henriquez, Hellen McKinnon Edwards, Thomas van den
Akker, Kitty W. M. Bloemenkamp, Johanna van der Bom, Marie-Pierre Bonnet, Catherine
Deneux-Tharaux, Serena Donati, Ada Gillissen, Jennifer J. Kurinczuk, Zhuoyang Li, Eliza-
beth Sullivan, Simon Stanworth, Marian Knight. Summary In the absence of high-level evidence, each country has a preferred treatment regimen, which
may lead to the use of unnecessary costly haematological products, exposure of women to
potential adverse effects and radical management such as hysterectomy. With wide variation
in obstetric practice, including hysterectomy and embolisation, significant improvement in
the evidence base is required particularly if a woman’s future fertility could be otherwise pre-
served. In extreme scenarios there is a delicate balance between saving a woman’s life and her
fertility, however, with a 40% difference in use of hysterectomy between Italy and the Nether-
lands and no difference in maternal mortality, there must be an examination and comparison
of the clinical pathway to ascertain if hysterectomy was necessary. PLOS ONE | https://doi.org/10.1371/journal.pone.0244933
January 22, 2021 12 / 15 PLOS ONE Massive blood transfusion during pregnancy or after childbirth Supporting information
S1 Table. Availability and comparability of variables from each respective dataset. (DOCX)
S2 Table. Aetiology of the massive obstetric haemorrhage by country. Legend: France had a
hierarchy of primary cause of PPH and were coded using this system: 1st Abnormal placenta-
tion, 2nd Abruption, 3rd Trauma and 4th was Atony. Denmark did not have data on cause of
haemorrhage. (DOCX) Formal analysis: Stephen J. McCall. Funding acquisition: Johanna van der Bom, Catherine Deneux-Tharaux, Serena Donati, Eliz-
abeth Sullivan, Marian Knight. Investigation: Stephen J. McCall, Dacia Henriquez, Hellen McKinnon Edwards, Thomas van
den Akker, Kitty W. M. Bloemenkamp, Marie-Pierre Bonnet, Catherine Deneux-Tharaux,
Serena Donati, Ada Gillissen, Jennifer J. Kurinczuk, Zhuoyang Li, Alice Maraschini, Aure´-
lien Seco, Elizabeth Sullivan, Simon Stanworth, Marian Knight. Methodology: Stephen J. McCall, Thomas van den Akker, Ada Gillissen, Simon Stanworth,
Marian Knight. Author Contributions Conceptualization: Stephen J. McCall, Thomas van den Akker, Jennifer J. Kurinczuk, Simon
Stanworth, Marian Knight. Data curation: Stephen J. McCall, Dacia Henriquez, Hellen McKinnon Edwards, Thomas van
den Akker, Kitty W. M. Bloemenkamp, Johanna van der Bom, Catherine Deneux-Tharaux,
Serena Donati, Ada Gillissen, Zhuoyang Li, Alice Maraschini, Aure´lien Seco, Elizabeth Sul-
livan, Marian Knight. Formal analysis: Stephen J. McCall. 1.
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January 22, 2021
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The effect of lay responders’ experience on response time to medical emergencies in a rural area: An observational study
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Simon Michael Starck
(
simon.m.starck@gmail.com
) Simon Michael Starck
(
simon.m.starck@gmail.com
) The effect of lay responders’ experience on
response time to medical emergencies in a rural
area: An observational study
Simon Michael Starck
(
simon.m.starck@gmail.com
)
Odense University Hospital: Odense Universitetshospital
https://orcid.org/0000-0003-1393-004X
Jonas Junghans Jensen
Odense Universitetshospital, Department of Cardiology
Laura Sarkisian
Odense Universitetshospital, Department of Cardiology
Henrik Schakow
, Emergency Medical Service, Region of Southern Denmark
Claus Andersen
Odense Universitetshospital, Department of Anaesthesiology
Finn Lund Henriksen
Odense Universitetshospital, Department of Cardiology
Original research
Keywords: Response time, community first responder, automated external defibrillator, AED, bystander
defibrillation
Posted Date: June 14th, 2022
DOI: https://doi.org/10.21203/rs.3.rs-1743036/v1
License:
This work is licensed under a Creative Commons Attribution 4.0 International
License.
Read Full License The effect of lay responders’ experience on
response time to medical emergencies in a rural
area: An observational study
Simon Michael Starck
(
simon.m.starck@gmail.com
)
Odense University Hospital: Odense Universitetshospital
https://orcid.org/0000-0003-1393-004X
Jonas Junghans Jensen
Odense Universitetshospital, Department of Cardiology
Laura Sarkisian
Odense Universitetshospital, Department of Cardiology
Henrik Schakow
, Emergency Medical Service, Region of Southern Denmark
Claus Andersen
Odense Universitetshospital, Department of Anaesthesiology
Finn Lund Henriksen
Odense Universitetshospital, Department of Cardiology
Original research
Keywords: Response time, community first responder, automated external defibrillator, AED, bystander
defibrillation
Posted Date: June 14th, 2022
DOI: https://doi.org/10.21203/rs.3.rs-1743036/v1
License:
This work is licensed under a Creative Commons Attribution 4.0 International
License. Read Full License The effect of lay responders’ experience on
response time to medical emergencies in a rural
area: An observational study The effect of lay responders’ experience on
response time to medical emergencies in a rural
area: An observational study
Simon Michael Starck
(
simon.m.starck@gmail.com
)
Odense University Hospital: Odense Universitetshospital
https://orcid.org/0000-0003-1393-004X
Jonas Junghans Jensen
Odense Universitetshospital, Department of Cardiology
Laura Sarkisian
Odense Universitetshospital, Department of Cardiology
Henrik Schakow
, Emergency Medical Service, Region of Southern Denmark
Claus Andersen
Odense Universitetshospital, Department of Anaesthesiology
Finn Lund Henriksen
Odense Universitetshospital, Department of Cardiology
Original research
Keywords: Response time, community first responder, automated external defibrillator, AED, bystander
defibrillation
Posted Date: June 14th, 2022
DOI: https://doi.org/10.21203/rs.3.rs-1743036/v1
License:
This work is licensed under a Creative Commons Attribution 4.0 International
License. Read Full License Simon Michael Starck
(
simon.m.starck@gmail.com
)
Odense University Hospital: Odense Universitetshospital
https://orcid.org/0000-0003-1393-004X
Jonas Junghans Jensen
Odense Universitetshospital, Department of Cardiology
Laura Sarkisian
Odense Universitetshospital, Department of Cardiology
Henrik Schakow
, Emergency Medical Service, Region of Southern Denmark
Claus Andersen
Odense Universitetshospital, Department of Anaesthesiology
Finn Lund Henriksen
Odense Universitetshospital, Department of Cardiology Results A total of 7273 CFR activations were included. Median response time for the CFR arriving first on-site (n =
3004) was 4:05 min (IQR 2:42 − 6:01) and median response time for the arrival of the CFR with an
automated external defibrillator (n = 2594) was 5:46 min (IQR 3:59 − 8:05). Median response times were
5:53 min (3:43 − 8:29) for ≤ 10 calls (n = 1657), 5:39 min (3:49 − 8:01) for 11–24 calls (n = 1396), 5:45
min (3:49 − 8:00) for 25–49 calls (n = 1586), 5:07 min (3:38 − 7:26) for 50–99 calls (n = 1548) and 4:46
min (3:14 − 7:32) for ≥ 100 calls (n = 1086) (p < 0.001). There was a significant negative correlation
between experience and response times (p < 0.001, Spearman’s rho=-0.0914). Abstract The aim of this retrospective observational study was to determine how response times correlated to the
experience of the community first responders (CFRs) using data collected from the Danish Island of
Langeland via a global positioning system (GPS)-based system. Conclusion This study found that response times are significantly reduced as CFRs become more experienced which
could lead to increased survival after a time-critical incident. Methods All medical emergency calls involving CFRs in the time period from 21st of April 2012 to 31st of
December 2017 were included. Each emergency call activated 3 CFRs. Response times were calculated
using the time from when the system alerted the CFRs to CFR time of arrival at the emergency site
measured by GPS. CFRs response times were grouped depending on their level of experience according to
≤ 10, 11–24, 25–49, 50–99, ≥ 100 calls accepted and arrived on-site. Original research Posted Date: June 14th, 2022 License:
This work is licensed under a Creative Commons Attribution 4.0 International
License. License:
This work is licensed under a Creative Commons Attribution 4.0 International
License. Read Full License Page 1/18 Page 1/18 Page 1/18 Page 1/18 Introduction Critically illness should be treated as fast as possible in case of life-threatening events such as sudden
cardiac arrest to increase their odds for better mental outcome and survival. To shorten response times in
areas with prolonged arrival times for emergency medical services (EMS), it has been proposed that
citizens trained as community first responder (CFRs) can be activated to perform cardiopulmonary
resuscitation (CPR) before EMS arrival. A randomized study carried out in Sweden showed an absolute
increase of 14% in bystander-initiated CPR with the use of a global positioning system (GPS)-based Page 2/18 Page 2/18 message system1. Other lay responder systems have been successfully used in the Netherlands2,3,
Switzerland4, France5 and are under development in the United Kingdom6 and North America7. The global resuscitation alliance postulated a frame of survival as an extension of the chain of survival. The frame of survival states that leadership and training are needed to improve the quality of
resuscitation and thereby lead to a culture of excellence8. One way to improve quality is faster initiation
of CPR and we theorize that more experienced and trained CFRs arrive faster at the emergency site. We
assume that more experienced CFRs would have increased knowledge of the location of the local AED-
cabinets and that they would have established routines to arrive quicker at the site. On the Danish island
of Langeland, a GPS-based community first responder system was initiated in 2012 to locate and
dispatch CFRs alongside standard emergency medical services response9. In this retrospective observational study, our main purpose was to determine a possible correlation
between response times and experience of the community first responders by using data collected from
the island of Langeland. Study design and approval Study design and approval This retrospective observational study presents data collected from a GPS-based system on the Danish
Island of Langeland. Approval for this study was given by The Danish Data Protection Agency (Journal
no. 17/32047) and Danish Patient Safety Authority under the administration of Danish Health Authority
(no. 3-3013-2848/1, ref.LOSC) and the volunteers accepted during their registration in the project, that
data was collected from their smartphones, stored on the server and used for research purposes. In
Denmark, ethical approval is not needed for this kind of study. Setting The island of Langeland has a population of approximately 12,000 inhabitants, but during summertime
approximately 260,000 tourists visit the holiday island. It is 52 km long and 11 km at its widest and is
bridge connected to the mainland9. There are 5 larger villages, and the population is a typical island
community, where most of the island’s inhabitants have lived most of their life on the island. Langeland
has one ambulance and one paramedic vehicle and in case the ambulance is reserved for another duty,
response times increase drastically as another ambulance needs to arrive from the city of Svendborg
placed on the mainland 20–50 km away9. The island has 96 AEDs placed strategically with no more than
2 km between each of them. Recruitment of lay responders Recruitment of lay responders CFRs were recruited to the project through local advertisement and were mostly laypersons but included
some off-duty healthcare providers. To become CFR certified, the CFRs needed to be at least 18 years of
age and complete a 12-hour basic life support training course provided by Langeland AED Association. The CFRs were trained by ERC certified staff. Each CFR is manually approved by an administrator before
being able to accept assignments in the system. To remain active as a CFR an annual 3-hour retraining
course is required. Data collection All medical emergency calls involving CFRs on Langeland in the time period from 21st of April 2012 to
31st of December 2017 were included. CFRs were excluded from analysis if they did not arrive on-site or
if their data did not appear to be appropriate (arrived on-site > 2 hours after accepting the call). The time for arrival at the emergency site was automatically logged by the GPS-based system. Time and
date for when the system alerts the CFRs, the emergency site’s address and CFRs arrival time at the
emergency site were collected from the project-server. Emergency service records were acquired and were
used to correct any missing data as far as possible in the CFR information. GPS system GPS system A GPS-based system to activate CFRs was initiated in 2012 on Langeland. Each time an ambulance was
dispatched to an emergency, the system identified the nine closest CFRs in a five km radius from the Page 3/18 Page 3/18 emergency site via GPS and notified them by an alarm sent to their smartphone. The CFRs could then
choose to accept or decline the call. Based on the choices made, the system within 20 seconds identified
the three closest CFRs and sent a map to the location of the emergency site and tasks. Two CFRs were
dispatched directly to the emergency site, while the third was sent to pick up an automated external
defibrillator (AED) before proceeding to the emergency site. These tasks are selected by the system based
on the CFRs GPS location and nearest AED. After each activation, there was a possibility for a debriefing
for the CFRs9,10. Statistics The data were analysed using Stata 16 and statistical significance was set to p = 0.05. To validate if data
were normally distributed a Q-Q plot was used. Non-normally distributed outcomes were reported as
median with interquartile ranges (IQR). Categorical data were described as absolute numbers and
percentages. A Spearman’s rank-order correlation was used to assess if there was a monotonic
relationship between experience and response times. To evaluate non-normally distributed outcomes, a
Kruskal Wallis test was used if there were three or more unmatched groups and a Mann-Whitney U-test
was used to test for statistically significant differences between two groups. A multiple regression
analysis was used to analyse the relationship between CFR response times and the independent
variables (experience, AED, year, time of day, season, Rudkoebing vs. the rest of Langeland). Definition of variables CFR experience was measured as the number of times a CFR was activated and arrived to the emergency
site, and was divided into the following groups based on milestones from the Langeland AED Association
as the association gives rewards based on the categories: ≤10, 11–24, 25–49, 50–99, ≥ 100 calls
accepted. We presumed these milestones to be applicable and good indicators for CFR experience. These
groups were used to investigate the link between CFRs experience and response time; e.g. the response
times of a CFR who was called out 34 times was placed in the category ≤ 10 calls the first 10 times
he/she was called out, then placed in the category 11–24 calls for the next 14 calls, and finally placed in
the category 25–49 for the last 10 calls. Response times were calculated using the time from alerting the
CFRs to CFRs time of arrival at the emergency site. Variables of interest Page 4/18 The median response time could be influenced by multiple variables and were therefore analysed by the
effect of a CFR acquiring an AED on the way to the emergency site, the time of day and season. Time of
day was categorised according to hours at work (7:00 to 15:00), spare time (15:00 to 23:00) and
nighttime (23:00 to 7:00). The season was defined meteorologically as winter (December, January,
February), spring (March, April, May), summer (June, July, August) and fall (September, October,
November). As Rudkoebing is the biggest City on Langeland, we wanted to see if this had an impact
compared to the rest of Langeland. Results Our study included 3063 emergency calls with activated CFRs resulting in 9189 possible CFR activations,
of which 7273 activations were included and 1916 were excluded. We excluded 712 activations with no
response times generated, 177 activations had no response times available because no willing CFRs were
available within a five kilometre search radius. The remaining 535 possible activations had no response
times due to only one or two of the three potential CFRs being available within a five kilometre radius. The
CFRs did not arrive on-site in 1192 activations and were thus excluded. We also excluded 12 activations
with a response time over 2 hours (Fig. 1). At least one CFR arrived at the emergency site in 3004 emergency calls (98.1%). An AED arrived at the
emergency site in 2594 emergency calls (84.7%). The annual mean number of alerts for each CFR in the
study period was approximately 6.25 alerts divided between 204 CFRs. The median response time for the CFRs who arrived first on-site (n = 3004) was 4:05 min (IQR 2:42 −
6:01). Median response time for the arrival of the CFRs with an AED (n = 2594) was 5:46 min (IQR 3:59 −
8:05). The median response times for all CFRs (n = 7273) was 5:29 min (IQR 3:41 − 7:54). CFRs response
times generally decreased with increasing experience when divided into the five experience groups (p <
0.001) (Table 1; Fig. 2). Likewise, the median response times generally decreased with experience when
analysed according to subgroups: picking up an AED and time of day. Median response times for all
CFRs were lowest if the CFR did not run for an AED (5:18 vs 5:46), during spare time (5:03 vs 5:13 at work Page 5/18 Page 5/18 hours and 7:07 at nighttime) (Table 2). CFR response times according to season were 5:26 min in spring,
5:40 min during summer, 5:28 min in fall and 5:23 min winter. Results Table 1
– Summary of outcomes
Outcome
n (%)
Median time in minutes:seconds (IQR)
First CFR on-site
3005 calls (98.1)
4:05 (2:42 − 6:01)
CFR with AED on-site
2594 calls (84.7)
5:46 (3:59 − 8:05)
Median CFR response time
7273 activations (100)
5:29 (3:41 − 7:54)
Experience groups*
· ≤10
1657 activations (22.8)
5:53 (3:43 − 8:29)
· 11–24
1396 activations (19.2)
5:39 (3:49 − 8:01)
· 25–49
1586 activations (21.8)
5:44.5 (3:49 − 8:00)
· 50–99
1548 activations (21.3)
5:07 (3:38 − 7:26)
· ≥100
1086 activations (14.9)
4:46 (3:14 − 7:32)
* An asterisk represents significant results with a p-value of less than 0.0001. * An asterisk represents significant results with a p-value of less than 0.0001. While the number of CFRs roughly remained the same, the number of experienced CFRs increased and
the number of less experienced CFRs declined over the study period (Fig. 3). A Spearman’s correlation Results Page 6/18 Page 6/18 Table 2
Experience groups
CFR
activations
0–10
11–24
25–49
50–99
100+
All
activations
p-
value
All CFR
5:53 (IQR
3:49 −
8:29) n =
1657
5:39 (IQR
3:49 −
8:01) n =
1396
5:45 (IQR
3:49 −
8:00) n =
1586
5:07 (IQR
3:38 −
7:26) n =
1548
4:46 (IQR
3:14 −
7:32) n =
1086
5:29 (IQR
3:41 −
7:54) n =
7273
0.0001
CFR with
an AED
6:12 (IQR
4:25 −
8:44) n =
505
6:00 (IQR
4:17 −
8:30) n =
458
5:59 (IQR
4:11 −
8:03) n =
521
5:23 (IQR
3:52 −
7:35) n =
582
5:01 (IQR
3:21 −
7:43.5) n
= 524
5:46 (IQR
3:59 −
8:05) n =
2590
0.0001
CFR
without an
AED
5:42 (IQR
3:34.5-
8:22) n =
1152
5:22 (IQR
3:37 −
7:47) n =
938
5:32 (IQR
3:35 −
7:55) n =
1065
5:02 (IQR
3:26 −
7:18) n =
966
4:35 (IQR
3:04–
7:25) n =
562
5:18 (IQR
3:30 −
7:45) n =
4683
0.0001
Work
hours
(7:00 to
15:00)
5:37 (IQR
3:34 −
8:15) n =
686
5:19 (IQR
3:28 −
7:38) n =
617
5:44 (IQR
3:54 −
7:41) n =
643
4:51 (IQR
3:18 −
7:06) n =
635
4:28 (IQR
3:02–
6:57) n =
469
5:13 (IQR
3:27 −
7:31) n =
3050
0.0001
Spare time
(15:00 to
23:00)
5:27 (IQR
3:41 −
7:34) n =
686
5:12 (IQR
3:39 −
7:16) n =
530
5:01 (IQR
3:23 −
7:04) n =
624
4:40 (IQR
3:24 −
7:00) n =
559
4:40 (IQR
3:02–
6:58) n =
371
5:03 (IQR
3:27 −
7:13) n =
2770
0.0002
Nighttime
(23:00 to
7:00)
7:45 (IQR
5:40 −
10:06) n
= 285
7:40 (IQR
5:26 −
10:19) n
= 249
7:11 (IQR
5:03–
9:42) n =
319
6:31 (IQR
4:39 −
8:51) n =
354
6:02 (IQR
3:56 −
9:15) n =
246
7:07 (IQR
4:52 −
9:35) n =
1453
0.0001
Spring
5:58 (IQR
3:49 −
8:20) n =
393
5:06 (IQR
3:25 −
7:02) n =
339
5:35 (IQR
3:41 −
7:40) n =
455
5:09 (IQR
3:48 −
7:33) n =
341
4:58 (IQR
3:24 −
7:42) n =
234
5:26 (IQR
3:40 −
7:44) n =
1762
0.0038
Summer
5:56 (IQR
3:54 −
8:44) n =
498
6:05 (IQR
4:02–
8:36) n =
357
5:46 (IQR
3:55 −
8:04) n =
396
5:07 (IQR
3:30 −
7:40) n =
481
5:12 (IQR
3:31 −
8:23) n =
270
5:40 (IQR
3:47 −
8:12) n =
2002
0.0131
Fall
5:44 (IQR
3:49 −
8:25) n =
447
5:39 (IQR
4:03–
8:26) n =
370
5:49 (IQR
3:43 −
8:12) n =
386
5:09 (IQR
3:42 −
7:28) n =
368
4:23 (IQR
3:01–
7:11) n =
315
5:28 (IQR
3:42 −
7:59) n =
1886
0.0001
Winter
5:53 (IQR
3:41 −
8:20) n =
319
5:47 (IQR
3:51 −
7:53) n =
330
5:45 (IQR
3:57 −
8:11) n =
349
5:05 (IQR
3:31 −
6:47) n =
358
4:23 (IQR
3:04–
6:54) n =
267
5:23 (IQR
3:36 −
7:38) n =
1623
0.0001
While the number of CFRs roughly remained the same, the number of experienced CFRs increased and While the number of CFRs roughly remained the same, the number of experienced CFRs increased and
the number of less experienced CFRs declined over the study period (Fig. Page 7/18 Page 7/18 Results 3). A Spearman’s correlation was conducted to assess the relationship between experience and response times and a significant
negative correlation was found (p < 0.001, Spearman’s rho=-0.0914). was conducted to assess the relationship between experience and response times and a significant
negative correlation was found (p < 0.001, Spearman’s rho=-0.0914). A multiple regression was run to predict CFR response time from experience, AED, year, time of day,
season, Rudkoebing vs. the rest of Langeland. These variables statistically significantly predicted CFR
response time, F (13, 7259) = 17,71, p < 0.001, R2 = 0,031 (Table 3). Page 8/18 Table 3 Table 3
– Results of the regression analysis – Results of the regression analysis
Number of obs
=
7,273
F(13, 7259)
=
17.71
Prob > F
=
0.0000
R-squared
=
0.0307
Adj R-squared
=
0.0290
Root MSE
=
317.8
CFR time in seconds
Coef. Std. Err. t
P > t
[95% Conf. Interval]
Experience of CFR
-0.612
0.0918
-6.67
0.000
-0.791
-0.432
Bringing an AED
19.851
7.884
2.52
0.012
4.397
35.306
Year of 2012
0
(omitted)
Year of 2013
-33.798
16.538
-2.04
0.041
-66.219
-1.378
Year of 2014
-25.963
16.326
-1.59
0.112
-57.967
6.041
Year of 2015
22.440
16.540
1.36
0.175
-9.983
54.863
Year of 2016
65.395
16.223
4.03
0.000
33.593
97.198
Year of 2017
21.472
17.767
1.21
0.227
-13.357
56.301
Spare time
0
(omitted)
Work time
3.950
8.351
0.47
0.636
-12.421
20.320
Night time
104.934
10.331
10.16
0.000
84.682
125.185
Spring
0
(omitted)
Summer
25.066
10.434
2.40
0.016
4.613
45.518
Fall
15.897
10.635
1.49
0.135
-4.950
36.744
Winter
6.728
10.962
0.61
0.539
-14.761
28.216
City of Rudkoebing
-33.749
8.107
-4.16
0.000
-49.642
-17.856
Constant
375.640
16.041
23.42
0.000
344.196
407.084
al average ambulance response time on Langeland was 11:18 min and 12.5% of ambulances
d after more than 20 minutes, for comparison the official average ambulance response time in
on of Southern Denmark was 7:54 min and 1 2% arrived after 20 minutes11 Official average ambulance response time on Langeland was 11:18 min and 12.5% of ambulances
arrived after more than 20 minutes, for comparison the official average ambulance response time in the
Region of Southern Denmark was 7:54 min and 1.2% arrived after 20 minutes11. Discussion Results from this study of response time in CFRs show a significant correlation between experience and
response times. While it seems that there is no substantial difference in median response times for CFRs
with less than 50 calls accepted and arrived, the most experienced CFRs reduced their median response
times significantly by more than one minute when compared to the group with minor experience (Fig. 2). The effect of increasing experience generally reduced response times when analysed according to
picking up an AED or time of day through the experience groups. The significant reduction in CFR
response time with increasing experience could be replicated when using multiple regression (Table 3). Picking up an AED on the way to the emergency site increased median response times by 20–40
seconds. The effect of increased experience had an impact on median response time depending on the
time of day thus activations at night was reduced by 1:43 minutes, while working hours was decreased
with 1:08 minutes and spare time with only by 46 seconds when comparing the lowest experienced group
with the most experienced. Only the season of summer did impact response times significantly, which
may be due to increased number of tourists and therefore more traffic or because some CFRs are on
vacation leaving fewer CFRs on the island to accept the calls. The number of CFR activations is
approximately the same (spring 108 pr. month vs. summer 111 pr. month vs fall 105 pr. month vs. winter
101 pr. month). The city of Rudkoebing had a significant shorter CFR response time compared to the rest
of the more rural Langeland. The multiple regression had a low r-squared, indicating that more variables
are needed to fully verify the effect of experience on response times. More experience could mean that CFRs establish routines to quickly get to the emergency site as they
probably are better prepared with established routines and could have an increased local knowledge of
AED placements and local addresses. Another possible cause for the decrease in response times with
increasing numbers of activations could be that the most dedicated CFRs are also the ones with the
fastest response times. As they are more dedicated, they would probably stay in the project for a longer
period meaning that the shorter response times found could be a result of the less dedicated CFRs
leaving the project before getting experienced. Results Official average ambulance response time on Langeland was 11:18 min and 12.5% of ambulances
arrived after more than 20 minutes, for comparison the official average ambulance response time in the
Region of Southern Denmark was 7:54 min and 1.2% arrived after 20 minutes11. Page 9/18 Discussion In comparison the median response
time from when the system alerts the CFR to CFR arrival at the emergency site, the Swedish study from
Berglund et al. reported 2:22 min for their lay responders to arrive on-site14 compared to 5:29 min in our
study, although we found a median response time of 4:05 min if only counting the first CFR on-site. For a
lay responder to arrive with an AED, Berglund et al. reported 5:17 min in median response time14 while an
AED arrived on-site after 5:45.5 min in our study. A possible explanation for their faster response times
could be their higher population density (347 inhabitants/km2 in Stockholm County vs 48
inhabitants/km2 on Langeland). Our results also support this hypothesis as Rudkoebing which is the
biggest city on Langeland has shorter response times compared to the rest of Langeland. We believe a
higher population density results in shorter response times caused by shorter CFR travel distances as
more CFRs are available in a higher populated area. Another possible explanation for our longer response
times is a bigger search radius in our application, as their system used a search radius of 1200 meters
without AED and 2400 meters with AED14, compared to the Langeland GPS system with a maximum
search radius of 5000 meters. Thirdly, their system did not activate during nighttime which both our study
and another study have shown to increase response time4. inhabitants/km2 on Langeland). Our results also support this hypothesis as Rudkoebing which is the
biggest city on Langeland has shorter response times compared to the rest of Langeland. We believe a
higher population density results in shorter response times caused by shorter CFR travel distances as
more CFRs are available in a higher populated area. Another possible explanation for our longer response
times is a bigger search radius in our application, as their system used a search radius of 1200 meters
without AED and 2400 meters with AED14, compared to the Langeland GPS system with a maximum
search radius of 5000 meters. Thirdly, their system did not activate during nighttime which both our study
and another study have shown to increase response time4. On Langeland in the period of 2012 to 2017 a CFR arrived before the ambulance on the emergency site in
85% of cases10. Discussion However, we do not believe this is the cause, as a steady
decline in unexperienced CFRs over the study period makes it unlikely that a large amount of CFRs left
and entered the program (Fig. 3). Also, the total number of CFRs did not change much either while the
number of experienced CFRs on Langeland increased over the years which further makes this unlikely
(Fig. 3). Page 10/18
Leadership, training, quality improvement and culture of excellence are four important elements deemed
essential to support the chain of survival. These four elements are described as the frame of survival and
made by the global resuscitation alliance as a tool to define a high-quality EMS system8. The frame of
survival is also reflected in the Utstein Formula for survival, which states that medical science,
educational efficiency and local implementation together equal survival12. GPS-based programs like the
one initiated on Langeland are a way to provide and improve these four elements from the frame of
survival. Strong leadership is shown by implementation of systems to reduce response times which
multiple countries have done over the last years13. The CFRs on Langeland are trained by experienced professionals before they can be activated as part of the system. Leadership and training are
fundamentals of the frame of survival that could reduce response time to increase the survivability of
critically ill patients. No study has explored the correlation between the experience of CFRs and their response times but
multiple studies have been made with application-based systems2–7. In comparison the median response
time from when the system alerts the CFR to CFR arrival at the emergency site, the Swedish study from
Berglund et al. reported 2:22 min for their lay responders to arrive on-site14 compared to 5:29 min in our
study, although we found a median response time of 4:05 min if only counting the first CFR on-site. For a
lay responder to arrive with an AED, Berglund et al. reported 5:17 min in median response time14 while an
AED arrived on-site after 5:45.5 min in our study. A possible explanation for their faster response times
could be their higher population density (347 inhabitants/km2 in Stockholm County vs 48 No study has explored the correlation between the experience of CFRs and their response times but
multiple studies have been made with application-based systems2–7. Discussion Since studies have highlighted the importance of early CPR15–17, this study found that
the first CFR was on-sight after 4:05 min describes an effective way to counter the effect of a long EMS
response time. We also found it as a reliable system since a CFR arrived in 98.1% of the calls but this
could be extraordinary as a result of the island’s community since Berglund et al. reported 58%14 and
Derkenne et al. 18%5 of CFRs to arrive at the emergency site. Further studies are needed to enlighten the
attendance of CFRs to arrive on-site in GPS based systems, but GPS based systems could be efficient at
providing community first responders at emergency sites. Our data suggest that CFR experience is an important factor in improving response times. This could
potentially increase survival after a time-critical incident such as an out of hospital cardiac arrest,
especially in a rural area with prolonged ambulance response times like Langeland. More experienced
CFRs would probably also possess better first aid skills and thereby improve the outcome. Continued
training and focus on keeping the CFRs active should therefore not be underestimated. Limitations Since the island of Langeland is a small rural part of Denmark, data may not be applicable to other
regions or countries. Given no other studies to our knowledge have explored the correlation between
experience and response times more studies are needed to substantiate our theory. Some CFRs arrived Page 11/18 Page 11/18 further than 50 meters away from the destination resulting in the system not registering them as arrived
despite them helping at the location18. If a first responder for example, stayed at the cross-section to
indicate the direction for the ambulance, they may not register as arrived at the destination. Some data
may also have been lost as the system does not register the CFR arrival time if the GPS signal was
missing. This could result in some data missing, shown the system as less reliable than in reality and
affected CFRs experience and made some CFRs less experienced than they actually were. Consent for publication Not applicable. Declarations
Ethics approval and consent to participate
Not applicable. Not applicable. Abbreviations CFR – Community first responder GPS – Global positioning system Conclusion This retrospective study found a significant effect of CFR experience on response times. Using the
Langeland GPS system, at least one CFR arrived on-site in 98.1% of all calls and the response time for the
first arriving CFR was 4:05 min. To our knowledge, no other studies have explored the correlation between
CFR response times and their experience, thus further research is needed to confirm our findings as it
could lead to increased survival after a time-critical incident. Authors contributions Jonas Junghans Jensen, MD: Conceptualization, Methodology, Validation, Formal analysis, Investigation,
Data Curation, Writing - Original Draft, Writing - Review & Editing, Visualization, Project administration Simon Michael Starck, MD: Conceptualization, Methodology, Validation, Formal analysis, Investigation,
Data Curation, Writing - Original Draft, Writing - Review & Editing, Visualization, Project administration Laura Sarkisian, MD, PhD: Conceptualization, Methodology, Formal analysis, Investigation, Writing -
Review & Editing, Supervision Laura Sarkisian, MD, PhD: Conceptualization, Methodology, Formal analysis, Investigation, Writing -
Review & Editing, Supervision Henrik Schakow, EMT: Methodology, Investigation, Writing - Review & Editing Henrik Schakow, EMT: Methodology, Investigation, Writing - Review & Editing Claus Andersen, MD, PhD: Conceptualization, Methodology, Writing - Review & Editing, Supervision Claus Andersen, MD, PhD: Conceptualization, Methodology, Writing - Review & Editing, Supervision Claus Andersen, MD, PhD: Conceptualization, Methodology, Writing - Revie Finn Lund Henriksen, MD, PhD: Conceptualization, Methodology, Validation, Investigation, Writing -
Review & Editing, Visualization, Supervision, Project administration Availability of data and materials Page 12/18 The data that support the findings of this study are available from Langeland AED Association but
restrictions apply to the availability of these data, which were used under license for the current study,
and so are not publicly available. Data are however available from the authors upon reasonable request
and with permission of Langeland AED Association. Acknowledgements We would like to thank the Langeland AED Association and all community first responders for
contribution in data collection. Furthermore, we would like to thank FirstAED for making the data
available. Funding No funding was received for this study. Competing interests The co-investigators and co-authors Finn Lund Henriksen and Henrik Schakow have stock ownership in
the GPS-tracking system, FirstAED, which has been used in this study. All other authors have no conflicts
of interest to declare. References Page 13/18 1. Ringh M, Rosenqvist M, Hollenberg J, Jonsson M, Fredman D, Nordberg P, et al. Mobile-phone
dispatch of laypersons for CPR in out-of-hospital cardiac arrest. N Engl J Med. 2015
Jun;11(24):2316–25. 372(. 2. Pijls RWM, Nelemans PJ, Rahel BM, Gorgels APM. A text message alert system for trained volunteers
improves out-of-hospital cardiac arrest survival. Resuscitation. 2016;105:182–7. 3. Zijlstra JA, Stieglis R, Riedijk F, Smeekes M, van der Worp WE, Koster RW. Local lay rescuers with
AEDs, alerted by text messages, contribute to early defibrillation in a Dutch out-of-hospital cardiac
arrest dispatch system. Resuscitation. 2014 Nov;85(11):1444–9. 4. Caputo ML, Muschietti S, Burkart R, Benvenuti C, Conte G, Regoli F, et al. Lay persons alerted by
mobile application system initiate earlier cardio-pulmonary resuscitation: A comparison with SMS-
based system notification. Resuscitation. 2017;114:73–8. 5. Derkenne C, Jost D, Roquet F, Dardel P, Kedzierewicz R, Mignon A, et al. Mobile Smartphone
Technology Is Associated With Out-of‐hospital Cardiac Arrest Survival Improvement: The First Year
“Greater Paris Fire Brigade” Experience. Gerhardt RT, editor. Acad Emerg Med. 2020 May
23;acem.13987. 6. Smith CM, Wilson MH, Ghorbangholi A, Hartley-Sharpe C, Gwinnutt C, Dicker B, et al. The use of
trained volunteers in the response to out-of-hospital cardiac arrest – the GoodSAM experience. Resuscitation. 2017 Dec 1;121:123–6. 7. Brooks SC, Simmons G, Worthington H, Bobrow BJ, Morrison LJ. The PulsePoint Respond mobile
device application to crowdsource basic life support for patients with out-of-hospital cardiac arrest:
Challenges for optimal implementation. Resuscitation. 2016 Jan 1;98:20–6. 8. Eisenberg M, Lippert F, Castren M, Moore F, Ong M, Rea T, et al. Acting on the Call [Internet]. [cited
2020 Jan 14]. Available from: https://www.globalresuscitationalliance.org/wp-
content/pdf/acting_on_the_call.pdf. 8. Eisenberg M, Lippert F, Castren M, Moore F, Ong M, Rea T, et al. Acting on the Call [Internet]. [cited
2020 Jan 14]. Available from: https://www.globalresuscitationalliance.org/wp-
content/pdf/acting_on_the_call.pdf. 9. Henriksen FL, Schorling P, Hansen B, Schakow H, Larsen ML. FirstAED emergency dispatch, global
positioning of community first responders with distinct roles - a solution to reduce the response
times and ensuring an AED to early defibrillation in the rural area Langeland. Int J Netw Virtual
Organ. 2016 Jan 1;16(1):86–102. 9. Henriksen FL, Schorling P, Hansen B, Schakow H, Larsen ML. FirstAED emergency dispatch, global
positioning of community first responders with distinct roles - a solution to reduce the response
times and ensuring an AED to early defibrillation in the rural area Langeland. References Int J Netw Virtual
Organ. 2016 Jan 1;16(1):86–102. 10. Sarkisian L, Mickley H, Schakow H, Gerke O, Jørgensen G, Larsen ML, et al. Global positioning system
alerted volunteer first responders arrive before emergency medical services in more than four out of
five emergency calls. Resuscitation. 2020;152:170–6. 10. Sarkisian L, Mickley H, Schakow H, Gerke O, Jørgensen G, Larsen ML, et al. Global positioning system
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five emergency calls. Resuscitation. 2020;152:170–6. 11. Præhospital indsats - responstider // Region Syddanmark [Internet]. [cited 2019 Oct 9]. Available
from: https://www.regionsyddanmark.dk/wm337767. 11. Præhospital indsats - responstider // Region Syddanmark [Internet]. [cited 2019 Oct 9]. Available
from: https://www.regionsyddanmark.dk/wm337767. 12. Nadarajan GD, Tiah L, Ho AFW, Azazh A, Castren MK, Chong SL, et al. Global resuscitation alliance
utstein recommendations for developing emergency care systems to improve cardiac arrest survival. Resuscitation. 2018;132:85–9. 12. Nadarajan GD, Tiah L, Ho AFW, Azazh A, Castren MK, Chong SL, et al. Global resuscitation alliance
utstein recommendations for developing emergency care systems to improve cardiac arrest survival. Resuscitation. 2018;132:85–9. 13. Oving I, Masterson S, Tjelmeland IBM, Jonsson M, Semeraro F, Ringh M, et al. First-response
treatment after out-of-hospital cardiac arrest: a survey of current practices across 29 countries in 13. Oving I, Masterson S, Tjelmeland IBM, Jonsson M, Semeraro F, Ringh M, et al. First-response
treatment after out-of-hospital cardiac arrest: a survey of current practices across 29 countries in Page 14/18 Page 14/18 Europe. Scand J Trauma Resusc Emerg Med. 2019 Dec;16(1):112. 27(. 14. Berglund E, Claesson A, Nordberg P, Djärv T, Lundgren P, Folke F, et al. A smartphone application for
dispatch of lay responders to out-of-hospital cardiac arrests. Resuscitation. 2018;126:160–5. 14. Berglund E, Claesson A, Nordberg P, Djärv T, Lundgren P, Folke F, et al. A smartphone application for
dispatch of lay responders to out-of-hospital cardiac arrests. Resuscitation. 2018;126:160–5. 15. Perkins GD, Handley AJ, Koster RW, Castrén M, Smyth MA, Olasveengen T, et al. European
Resuscitation Council Guidelines for Resuscitation 2015. Resuscitation. 2015 Oct;95:81–99. 15. Perkins GD, Handley AJ, Koster RW, Castrén M, Smyth MA, Olasveengen T, et al. European
Resuscitation Council Guidelines for Resuscitation 2015. Resuscitation. 2015 Oct;95:81–99. 16. Larsen MP, Eisenberg MS, Cummins RO, Hallstrom AP. Predicting survival from out-of-hospital
cardiac arrest: A graphic model. Ann Emerg Med. 1993 Nov;22(11)(1):1652–8. 16. Larsen MP, Eisenberg MS, Cummins RO, Hallstrom AP. References Predicting survival from out-of-hospital
cardiac arrest: A graphic model. Ann Emerg Med. 1993 Nov;22(11)(1):1652–8. 17. Doan TN, Schultz BV, Rashford S, Bosley E. Surviving out-of-hospital cardiac arrest: The important
role of bystander interventions. Australas Emerg Care [Internet]. 2020 Jan 8 [cited 2020 Jan 16];
Available from: http://www.sciencedirect.com/science/article/pii/S2588994X19300892. 17. Doan TN, Schultz BV, Rashford S, Bosley E. Surviving out-of-hospital cardiac arrest: The important
role of bystander interventions. Australas Emerg Care [Internet]. 2020 Jan 8 [cited 2020 Jan 16];
Available from: http://www.sciencedirect.com/science/article/pii/S2588994X19300892. 18. Ganter J, Pooth JS, Damjanovic D, Trummer G, Busch HJ, Baldas K, et al. Association of GPS-Based
Logging and Manual Confirmation of the First Responders’ Arrival Time in a Smartphone Alerting
System: An Observational Study. Prehosp Emerg Care. 2021 Nov;3:1–9. 18. Ganter J, Pooth JS, Damjanovic D, Trummer G, Busch HJ, Baldas K, et al. Association of GPS-Based
Logging and Manual Confirmation of the First Responders’ Arrival Time in a Smartphone Alerting
System: An Observational Study. Prehosp Emerg Care. 2021 Nov;3:1–9. Figures Page 15/18 gure 1 Figure 1
Flow-chart showing the inclusion-exclusion process for CFRs experience. Figure 1 Flow-chart showing the inclusion-exclusion process for CFRs experience. Flow-chart showing the inclusion-exclusion process for CFRs experience. Flow-chart showing the inclusion-exclusion process for CFRs experience. Page 16/18 Figure 2
Median CFR time for each experience group
Median community first responder response time in seconds for each of the five experience groups. The
red spikes indicate the 25th interquartile to the 75th interquartile. The black line is a linear prediction plot
for the median community first responder response time. p<0.001 Figure 2 Median CFR time for each experience group Median CFR time for each experience group Median community first responder response time in seconds for each of the five experience groups. The
red spikes indicate the 25th interquartile to the 75th interquartile. The black line is a linear prediction plot
for the median community first responder response time. p<0.001 Median community first responder response time in seconds for each of the five experience groups. The
red spikes indicate the 25th interquartile to the 75th interquartile. The black line is a linear prediction plot
for the median community first responder response time. p<0.001 Page 17/18 Page 17/18 Figure 3
Annual number of CFRs in each experience group
The annual number of VFRs in each experience group. The grey line represents the total Figure 3
Annual number of CFRs in each experience group Figure 3 The annual number of VFRs in each experience group. The grey line represents the total annual number of active CFRs on Langeland. The green line represents the group with 10 or less
activations, the yellow line represents the group with 11 to 24 activations, the red line represents the
group with 25 to 49 activations, the purple line represents the group with 50 to 99 activations, and the
blue line represents the group with 100 or more activations. Page 18/18
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Radical condensation between benzylic alcohols and acetamides to form 3-arylpropanamides
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Downloaded from orbit.dtu.dk on: Oct 24, 2024 Downloaded from orbit.dtu.dk on: Oct 24, 2024 Downloaded from orbit.dtu.dk on: Oct 24, 2024 Citation (APA):
Azizi, K., & Madsen, R. (2020). Radical condensation between benzylic alcohols and acetamides to form 3-
arylpropanamides. Chemical Science, 11(30), 7800-7806. https://doi.org/10.1039/d0sc02948c General rights
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If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately
and investigate your claim. Radical condensation between benzylic alcohols and acetamides to form 3-
arylpropanamides Azizi, Kobra; Madsen, Robert Document Version
Publisher's PDF, also known as Version of record 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.
Users may download and print one copy of any publication from the public portal for the purpose of private study or research Department of Chemistry, Technical University of Denmark, 2800 Kgs. Lyngby,
Denmark. E-mail: rm@kemi.dtu.dk
† Electronic supplementary information (ESI) available: Experimental procedures,
deuterium labelling studies, EPR spectra, compound characterization data and
copies of 1H and 13C NMR spectra. See DOI: 10.1039/d0sc02948c Link back to DTU Orbit Link back to DTU Orbit Citation (APA):
Azizi, K., & Madsen, R. (2020). Radical condensation between benzylic alcohols and acetamides to form 3-
arylpropanamides. Chemical Science, 11(30), 7800-7806. https://doi.org/10.1039/d0sc02948c this document breaches copyright please contact us providing details, and we will remove access to the work immediate
ur claim. Kobra Azizi and Robert Madsen
* A new radical condensation reaction is developed where benzylic alcohols and acetamides are coupled to
generate 3-arylpropanamides with water as the only byproduct. The transformation is performed with
potassium tert-butoxide as the only additive and gives rise to a variety of 3-arylpropanamides in good
yields. The
mechanism
has
been
investigated
experimentally
with
labelled
substrates,
trapping
experiments and spectroscopic measurements. The findings indicate a radical pathway where potassium
tert-butoxide is believed to serve a dual role as both base and radical initiator. The radical anion of the
benzylic alcohol is proposed as the key intermediate, which undergoes coupling with the enolate of the
amide to form the new C–C bond. Subsequent elimination to the corresponding cinnamamide and
olefin reduction then affords the 3-arylpropanamides. Received 25th May 2020
Accepted 24th June 2020
DOI: 10.1039/d0sc02948c
rsc.li/chemical-science Received 25th May 2020
Accepted 24th June 2020
DOI: 10.1039/d0sc02948c
rsc.li/chemical-science Received 25th May 2020
Accepted 24th June 2020 dihydrogen is therefore not formed during the process.4,5 An
example is the b-alkylation of secondary alcohols and a-alkyl-
ation of ketones with primary alcohols, which can be mediated
by alkali metal hydroxides and tert-butoxides.6 Another example
is the Guerbet reaction where primary alcohols are dimerized
into higher alcohols.7 Depending on the catalyst, the hydride
transfer in the Guerbet reaction can occur either by acceptorless
dehydrogenation
or
by
a
Meerwein–Ponndorf–Verley/
Oppenauer redox process.7 The advantage of these trans-
formations with alcohols is that a stoichiometric amount of
waste is not produced since water is oen formed as the only
byproduct. Chemical
Science View Article Online
View Journal | View Issue Introduction Reactions for the formation of C–C bonds belong to the
cornerstone of organic synthesis. Since the turn of the century,
an increasing interest has been devoted to C–C bond forming
reactions with abundantly available alcohols as the substrates. A particular important development has been acceptorless
dehydrogenative transformations1,2 where a transition metal
catalyst removes dihydrogen from the alcohol to form the cor-
responding carbonyl compound, which then reacts with a C-
nucleophile, such as an enolate, to form the C–C bond. Subse-
quently, dehydration usually occurs to generate a C]C bond,
which may then be saturated by reduction with the liberated
dihydrogen. The metal catalyst can be a complex with a plat-
inum group metal1 or with a more abundantly available rst-
row transition metal.2 The mechanism for the alcohol dehy-
drogenation can involve either a metal-hydride or a metal-
dihydride species,1,2 and in some cases, the ligand also
actively
participates
through
metal–ligand
bifunctional
pathways.3 Radical mechanisms can also be involved in C–C bond
forming reactions with alcohols. a-Hydroxyalkyl radicals can be
generated photochemically from alcohols and dimerized into
1,2-diols or perform conjugate addition to olens with electron-
withdrawing groups.8 Activation of benzylic alcohols with stoi-
chiometric titanium or phosphorus reagents in the presence of
a metal catalyst has been proposed to form benzylic radicals,
which can perform cross coupling reactions with aryl halides
and conjugate addition to electron-decient olens.9 Recently,
benzylic alcohols were coupled with arylacetylenes in the pres-
ence of potassium tert-butoxide to form 1,3-diarylpropan-1-
ones.10 In this transformation, a ketyl radical is believed to be
formed from the alcohol based on experimental evidence and
the radical then reacts with the alkyne followed by several
hydrogen transfer reactions to generate the product.10 Although,
potassium tert-butoxide strictly serves as a base in this coupling,
the alkoxide has also been shown to promote radical coupling
reactions such as the formation of biaryls from aryl halides and
arenes.11 Another and mechanistically different alcohol dehydroge-
nation method is the Oppenauer oxidation,4 which aer an
aldol reaction and a Meerwein–Ponndorf–Verley reduction5 can
lead to similar products as the metal-catalyzed acceptorless
dehydrogenation. In this case, a transition metal is not required
since the transformation can be mediated by a main group
metal hydroxide or alkoxide. Radical condensation between benzylic alcohols
and acetamides to form 3-arylpropanamides† Cite this: Chem. Sci., 2020, 11, 7800 This journal is © The Royal Society of Chemistry 2020 Open Access Article. Published on 25 June 2020. Downloaded on 8/20/2020 12:00:11 PM.
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. All publication charges for this article
have been paid for by the Royal Society
of Chemistry Kobra Azizi and Robert Madsen
* Open Access Article. Published on 25 June 2020. Downloaded on 8/20/2020 12:00:11 PM.
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Thus, it was decided to optimize this condensation reaction
by investigating the inuence of the solvent and the base. When
the transformation was performed in reuxing toluene, a 1 : 1
mixture of the two amides were formed leading to a low yield of
propanamide 2 (entry 3). Lowering the amount of potassium
tert-butoxide led to even lower yields due to incomplete
conversion of benzyl alcohol (entries 4 and 5). However,
increasing the amount of potassium tert-butoxide to 2 equiv. gave full conversion of the alcohol already aer 6 h in mesity-
lene solution and afforded 2 as the sole amide product in 85%
yield (entry 6). The same experiment in toluene furnished a 9 : 1
mixture of the two amides resulting in a lower yield of 2 (entry
7). The outcome did not improve by using 3 equiv. of potassium
tert-butoxide in toluene solution although 2 was again formed
as the sole amide product (entry 8). Changing the solvent to Herein, we describe the radical coupling between benzylic
alcohols and acetamides to form 3-arylpropanamides with
water as the only stoichiometric byproduct.14,15 We are not
aware of a similar radical condensation with the release of water
and the reaction illustrates the importance of mechanistic
investigations when developing new dehydrogenative trans-
formations with alcohols. This journal is © The Royal Society of Chemistry 2020 Results and discussion Benzyl alcohol and N,N-dimethyl acetamide were selected as the
substrates for the initial investigations. In the rst experiment,
a 1 : 2 molar ratio of the alcohol and the amide were treated
with 5% of manganese(III) 5,10,15,20-tetraphenylporphyrin
chloride and 1 equiv. of potassium tert-butoxide in reuxing Table 1
Optimization of base-mediated coupling between benzyl alcohol and N,N-dimethyl acetamidea
Entry
Base
Solvent
BnOH conversionb (%)
1 : 2 ratio
Yield of 2c (%)
1d
KOtBu (1 equiv.)
Mesitylene
100
100 : 0
0
2
KOtBu (1 equiv.)
Mesitylene
85
10 : 90
68
3
KOtBu (1 equiv.)
Toluene
80
50 : 50
30
4
KOtBu (0.2 equiv.)
Mesitylene
65
40 : 60
20
5
KOtBu (0.2 equiv.)
Toluene
60
90 : 10
<5
6e
KOtBu (2 equiv.)
Mesitylene
100
0 : 100
85
7
KOtBu (2 equiv.)
Toluene
85
10 : 90
70
8
KOtBu (3 equiv.)
Toluene
90
0 : 100
62
9
KOtBu (2 equiv.)
Dioxane
60
20 : 80
35
10
KOtBu (2 equiv.)
Acetonitrile
50
30 : 70
20
11
KOtBu (2 equiv.)
DMF
35
80 : 20
<5
12
KOtBu (2 equiv.)
None
50
70 : 30
10
13
None
Mesitylene
0
—
0
14
NaOtBu (2 equiv.)
Mesitylene
80
0 : 100
75
15
KOH (2 equiv.)
Mesitylene
0
—
0
16
Cs2CO3 (2 equiv.)
Mesitylene
30
100 : 0
0
17
Na2CO3 (2 equiv.)
Mesitylene
0
—
0
18f
K2CO3 (2 equiv.)
Mesitylene
20
—
0
19f
NaOH (2 equiv.)
Mesitylene
90
70 : 30
10
20
NaH (2 equiv.)
Mesitylene
15
18 : 82
<5
21
KH (2 equiv.)
Mesitylene
20
10 : 90
10
a Conditions: BnOH (1 mmol), CH3CONMe2 (2 mmol), base, 1,3,5-trimethoxybenzene (0.5 mmol, internal standard), solvent (3 mL), reux, 12 h. b Determined by GC using the internal standard. c NMR yield with 1,3,5-trimethoxybenzene as the internal standard. d With 5% of
manganese(III) 5,10,15,20-tetraphenylporphyrin chloride added and reaction time 48 h. e Reaction time 6 h. f With 2 mL of N,N-dimethyl acetamide. Table 1
Optimization of base-mediated coupling between benzyl alcohol and N,N-dimethyl acetamidea a Conditions: BnOH (1 mmol), CH3CONMe2 (2 mmol), base, 1,3,5-trimethoxybenzene (0.5 mmol, internal standard), solvent (3 mL), reux, 12 h. b Determined by GC using the internal standard. c NMR yield with 1,3,5-trimethoxybenzene as the internal standard. d With 5% of
manganese(III) 5,10,15,20-tetraphenylporphyrin chloride added and reaction time 48 h. Introduction The mechanism involves hydride
transfer
through
a
six-membered
transition
state
and Potassium tert-butoxide is a very commonly employed base
for dehydrogenative transformations with alcohols where the This journal is © The Royal Society of Chemistry 2020 7800 | Chem. Sci., 2020, 11, 7800–7806 Chemical Science
View Article Online Chemical Science
View Article Online Chemical Science
View Article Online Edge Article mechanism either involves metal-catalyzed acceptorless dehy-
drogenation
or
Meerwein–Ponndorf–Verley/Oppenauer
processes.1,2,12
We
recently
developed
a
manganese(III)
porphyrin-catalyzed coupling between alcohols and amines to
form imines, tertiary amines and quinolines where potassium
tert-butoxide was employed in some cases.13 Attempts were
made to extend the reaction to a-alkylation of acetamides with
alcohols to form 3-substituted propanamides. Surprisingly, the
best results were obtained with potassium tert-butoxide as the
only additive, and more surprisingly, mechanistic studies
indicated the condensation to take place by a radical pathway. Herein, we describe the radical coupling between benzylic
alcohols and acetamides to form 3-arylpropanamides with
water as the only stoichiometric byproduct.14,15 We are not
aware of a similar radical condensation with the release of water
and the reaction illustrates the importance of mechanistic
investigations when developing new dehydrogenative trans-
formations with alcohols. mechanism either involves metal-catalyzed acceptorless dehy-
drogenation
or
Meerwein–Ponndorf–Verley/Oppenauer
processes.1,2,12
We
recently
developed
a
manganese(III)
porphyrin-catalyzed coupling between alcohols and amines to
form imines, tertiary amines and quinolines where potassium
tert-butoxide was employed in some cases.13 Attempts were
made to extend the reaction to a-alkylation of acetamides with
alcohols to form 3-substituted propanamides. Surprisingly, the
best results were obtained with potassium tert-butoxide as the
only additive, and more surprisingly, mechanistic studies
indicated the condensation to take place by a radical pathway. mesitylene (Table 1, entry 1). Under these conditions, full
conversion of the alcohol occurred as determined by GC and
N,N-dimethyl cinnamamide (1) was formed in 28% yield as
measured by NMR with 1,3,5-trimethoxybenzene as the internal
standard. The main product, however, was benzaldehyde
resulting from dehydrogenation with the manganese catalyst. Notably, when the same experiment was performed in the
absence of the manganese complex, the major product was N,N-
dimethyl 3-phenylpropanamide (2) and only minor amounts of
the corresponding cinnamamide was formed (entry 2). Results and discussion e Reaction time 6 h. f With 2 mL of N,N-dimethyl acetamide. This journal is © The Royal Society of Chemistry 2020 Chem. Sci., 2020, 11, 7800–7806 | 7801 Edge Article
View Article Online Edge Article
View Article Online This journal is © The Royal Society of Chemistry 2020 Chemical Science Chemical Science propanamide 6 in a similar 83% yield while 2,4,6-trime-
thylbenzyl alcohol gave amide 7 in 75% yield. o-Methyl- and o-
methoxybenzyl alcohol produced amides 8 and 9 in 85 and 72%
yield, respectively, while alcohols with additional aromatic
moieties
such
as
p-phenylbenzyl
alcohol
and
1-naph-
thylmethanol gave rise to the corresponding amides 10 and 11
in 74–75% yield. p-Fluoro-, p-chloro- and p-bromobenzyl alcohol
were poor substrates for the condensation under the optimized
conditions since dehalogenation product 2 was obtained in 50,
70 and 100% yield, respectively, according to GC analysis. Less
dehalogenation occurred when potassium tert-butoxide was
replaced
with
sodium
tert-butoxide
where
p-chlorobenzyl
alcohol could be converted into amide 12 in 64% isolated yield. Several other para-substituted benzyl alcohols also failed to give
a good yield of the condensation product under the optimized
conditions. p-Methylthiobenzyl alcohol afforded desulfuriza-
tion product 2 in 58% yield according to GC analysis while
almost no conversion occurred with p-nitrobenzyl alcohol. p-
Cyano- and p-(triuoromethyl)benzyl alcohol gave complex
mixtures of products, which were not further characterized. dioxane, acetonitrile or DMF or performing the condensation
under neat conditions all led to low yields of 2 due to moderate
conversion of the alcohol and formation of amide mixtures
(entries 9–12). No conversion of the alcohol occurred in the
absence of potassium tert-butoxide (entry 13). Replacing the
base with sodium tert-butoxide again gave exclusive formation
of amide 2 although in a slightly lower yield due to some
unreacted alcohol remaining (entry 14). A number of other
bases were also included in the study, but very poor results were
obtained in all cases (entries 15–21). Consequently, the
optimum conditions for the condensation uses 2 equiv. of
potassium tert-butoxide in reuxing mesitylene solution. Open Access Article. Published on 25 June 2020. Downloaded on 8/20/2020 12:00:11 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. The optimized procedure was then applied to a variety of
alcohols and amides to investigate the substrate scope of the
transformation. The products were isolated by ash chroma-
tography, which furnished compound 2 in 78% yield (Table 2). Electron-donating methyl, methoxy and benzyloxy groups in the
para position of the benzyl alcohol were well tolerated and all
led to a higher 84–86% yield of N,N-dimethyl 3-arylpropana-
mides
3,
4
and
5. Open Access Article. Published on 25 June 2020. Downloaded on 8/20/2020 12:00:11 PM.
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. cheme 1
Cinnamamide as a reaction intermediate. with N,N-dimethyl acetamide, but no cinnamamide or prop-
anamide was generated in this case and the aldehyde was
mainly converted into benzyl benzoate by a Tishchenko reac-
tion. No reaction occurred when the same experiment was
performed with benzyl methyl ether revealing the importance of
the hydroxy group in the condensation. N-Acetyl piperidine and morpholine were also subjected to
the base-mediated condensation reaction (Table 2). With benzyl
alcohol and 1-naphthylmethanol, the corresponding prop-
anamides 21–24 were isolated in 78–80% yield. p-Methox-
ybenzyl alcohol gave amide 25 in the reaction with N-acetyl
piperidine (85% yield) while p-methylbenzyl alcohol afforded
amide 26 with N-acetyl morpholine (80% yield). In addition, N-
methyl acetamide could be employed as a substrate where 72–
80% yield of amides 27–29 were isolated in the reaction with
benzyl
alcohol,
p-benzyloxybenzyl
alcohol
and
1-naph-
thylmethanol. a-Substituted acetamides were poor substrates
for the condensation since no conversion was observed with
N,N-dimethyl 2-methylpropanamide in the reaction with benzyl
alcohol while N,N-dimethyl propanamide gave very little
conversion in the same reaction. The cyclic amide N-methyl d-
valerolactam, although, did participate in the condensation and
was converted in to the a-benzylated amide 30 in 70% yield. No
reaction occurred when the aliphatic alcohols hexan-1-ol and
heptan-1-ol were submitted to the optimized conditions in the
reaction with N,N-dimethyl acetamide. Thus, a new potassium
tert-butoxide-mediated condensation reaction between benzylic
alcohols and acetamides has been developed to form a variety of
3-arylpropanamides in good yields. To probe the inuence of traces of dioxygen, an experiment
was carried out with mesitylene that had not been subjected to
degassing, but a signicantly lower 38% yield of propanamide 2
was observed in this case due to incomplete conversion of the
alcohol. Repeating the reaction under an atmosphere of dioxy-
gen or air resulted in the formation of benzaldehyde and cin-
namamide
1
due
to
a
base-mediated
aerobic
alcohol
oxidation.16 The condensation was also attempted in the pres-
ence of 10% of tert-butyl hydrogen peroxide, but only 30%
conversion of benzyl alcohol into benzaldehyde was observed
and none of propanamide 2 was formed. No change in the yield
occurred when the reaction in Table 1, entry 6 was performed in
the absence of light. The primary kinetic isotope effect (KIE) was determined with
both PhCD2OH and PhCH2OD against PhCH2OH by measuring
the initial rates. Chemical Science 3,4-Dimethylbenzyl
alcohol
afforded This article is licensed under a Creat Table 2
Condensation between benzylic alcohols and acetamidesa Table 2
Condensation between benzylic alcohols and acetamidesa Table 2
Condensation between benzylic alcohols and acetamides
a Conditions: alcohol (1 mmol), amide (2 mmol), KOtBu (2 mmol), mesitylene (3 mL), reux, 6 h (isolated yields). b With NaOtBu (2 mmol) instead of
KOtBu. a Conditions: alcohol (1 mmol), amide (2 mmol), KOtBu (2 mmol), mesitylene (3 mL), reux, 6 h (isolated yields). b With NaOtBu (2 mmol) instead of
KOtBu. This journal is © The Royal Society of Chemistry 2020 This journal is © The Royal Society of Chemistry 2020 7802 | Chem. Sci., 2020, 11, 7800–7806 Chemical Science
View Article Online Chemical Science
View Article Online This journal is © The Royal Society of Chemistry 2020 Edge Article Scheme 1
Cinnamamide as a reaction intermediate. Besides N,N-dimethyl acetamide, the condensation was also
carried out with other N-substituted amides (Table 2). N,N-
Diethyl acetamide was reacted with p-methyl-, p-methoxy-, p-
benzyloxy- and 3,4-dimethylbenzyl alcohol to afford N,N-diethyl
3-arylpropanamides 13, 14, 15 and 16 in 80–85% yield, which
closely resembles the results obtained in the same reactions
with
N,N-dimethyl
acetamide. The
N,N-diethyl-substituted
amide was also reacted with the ortho-substituted substrates
o-methyl- and o-methoxybenzyl alcohol to furnish amides 17
and 18 in 87 and 76% yield, respectively. p-Phenylbenzyl alcohol
gave rise to amide 19 in 78% yield while the para-chloro coun-
terpart with sodium tert-butoxide as the base afforded amide 20
in 73% yield. Open Access Article. Published on 25 June 2020. Downloaded on 8/20/2020 12:00:11 PM.
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. of several radical scavengers were added to the reaction
with benzyl alcohol and N,N-dimethyl acetamide and the reac-
tion time was extended to 48 h. No coupling product was
formed
when
cyclohexa-1,4-diene
and
4-methyl-2,4-
diphenylpent-1-ene were included in the mixture and both
scavengers underwent conversion into other products accord-
ing to GCMS analysis. The main product from cyclohexa-1,4-
diene was a-cyclohexenylbenzyl alcohol while 4-methyl-2,4-
diphenylpent-1-ene afforded 1,2-dimethyl-1,2-diphenylbutane
and small amounts of cumene. TEMPO was also employed as
a scavenger and led to a lower 50% yield of propanamide 2. During the transformation TEMPO was mainly reduced to
2,2,6,6-tetramethylpiperidine, but small amounts of 1-benzyl-
2,2,6,6-tetramethylpiperidine could also be detected. In addi-
tion, 2,6-di-tert-butyl-4-methylphenol (BHT) was added as
a scavenger and in this case complete conversion of benzyl
alcohol was observed, but the product was a 1 : 1 mixture of
cinnamamide 1 and propanamide 2, and BHT was primarily
oxidized into 2,6-di-tert-butyl-4-methylenecyclohexa-2,5-dien-1-
one. y
Accordingly, a mechanism is proposed where benzyl alcohol
is deprotonated by the base to form alkoxide A (Scheme 3). Initiation then occurs by generating radical anion B.17 In prin-
ciple, the a-hydrogen atom transfer can be mediated by traces of
dioxygen or the amide. Recently similar hydrogen atom transfer
reactions have been proposed from alkoxides to alkynes10 and
ketones18 as part of a radical initiation process. However, in our
case a more likely scenario involves potassium tert-butoxide for
abstracting the a-hydrogen atom in A. It has been debated
whether potassium tert-butoxide is able to serve as a single
electron donor to form the alkoxyl radical,11 which would
rapidly remove an a-hydrogen atom from A.19 As an example,
potassium tert-butoxide reduces benzophenone under photo-
excitation conditions to the benzophenone ketyl radical anion.20
Our transformation, however, is not affected by light and
instead potassium tert-butoxide may perform an additional
deprotonation of A to form the dianion PhCHO2. Open Access Article. Published on 25 June 2020. Downloaded on 8/20/2020 12:00:11 PM.
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. In both cases, a KIE around 1 was observed,
which shows that a hydrogen transfer is not involved in the rate-
determining step. The products from both labelling experi-
ments were isolated and analyzed by 1H and 2H NMR. In the
reaction with PhCD2OH, the product propanamide showed
deuterium incorporation in both the a and the b position or
exclusively in the b position, and the two labelled compounds
were formed in a ratio of approximately 1 : 1 (Scheme 2). In the
reaction with PhCH2OD, the product showed relatively little
deuterium incorporation in either the a or the b position. The substrate scope revealed signicant dehalogenation and
desulfurization of halogen- and sulfur-containing substrates,
which
are
unusual
side
reactions
for
transformations
proceeding by Meerwein–Ponndorf–Verley/Oppenauer redox
processes.4–6 Therefore, a number of experiments were per-
formed in order to obtain more detailed information about the
reaction mechanism. No benzaldehyde could be detected when
the optimized reaction in Table 1, entry 6 was monitored by GC. Instead, small amounts of cinnamamide 1 and N,N-dimethyl 3-
hydroxy-3-phenylpropanamide
were
observed
and
both
compounds appear to be reaction intermediates. The reduction
of a cinnamamide during the transformation was conrmed by
a control experiment where 1 equiv. of p-methoxybenzyl alcohol
was allowed to react with a mixture of 2 equiv. of N,N-diethyl
acetamide and 1 equiv. of N,N-diethyl cinnamamide (Scheme 1). GC analysis of the reaction mixture revealed a 2 : 1 : 1 : 1
mixture of compounds 4, 14, 31 and 32 and thus the conversion
of the starting cinnamamide into 31. Notably, the intermediate
cinnamamide is generated in a reversible process since an equal
mixture of propanamides 14 and 31 was formed. An experiment
was also carried out where benzaldehyde was allowed to react Based on these experiments, it is highly unlikely that the
coupling
takes
place
by
a
Meerwein–Ponndorf–Verley/
Oppenauer pathway and speculations arose whether a radical Scheme 2
Reaction with deuterium-labelled benzyl alcohol. Scheme 2
Reaction with deuterium-labelled benzyl alcohol. This journal is © The Royal Society of Chemistry 2020 Chem. Sci., 2020, 11, 7800–7806 | 7803 Edge Article
View Article Online View Article Online Edge Article
View Article Online View Article Online Chemical Science indicate that the carbon-centered radical is formed in connec-
tion with the deprotonation of benzyl alcohol. mechanism was responsible for the condensation. Therefore, 2
equiv. Open Access Article. Published on 25 June 2020. Downloaded on 8/20/2020 12:00:11 PM.
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. A number of
organic compounds have been shown to undergo deprotona-
tion or double deprotonation by potassium tert-butoxide to
generate anionic intermediates, which can serve as strong
single electron donors.21 A relevant example is the double
deprotonation of 2-pyridinemethanol to yield the dianion
capable of initiating haloarene – arene couplings.22 Thus, anion
A is most likely deprotonated by potassium tert-butoxide at the
elevated temperature to form PhCHO2, which will convert into
radical anion B by single electron transfer to the acetamide
(during the reaction) or to the aromatic moiety of the solvent or
the alcohol (in the absence of the amide). Open Access Article. Published on 25 June 2020. Downloaded on 8/20/2020 12:00:11 PM.
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. 2 (a) B. G. Reed-Berendt, K. Polidano and L. C. Morrill, Org. Biomol. Chem., 2019, 17, 1595–1607; (b) T. Irrgang and
R. Kempe,
Chem. Rev.,
2019,
119,
2524–2549;
(c)
G. A. Filonenko, R. van Putten, E. J. M. Hensen and
E. A. Pidko, Chem. Soc. Rev., 2018, 47, 1459–1483. 2 (a) B. G. Reed-Berendt, K. Polidano and L. C. Morrill, Org. Biomol. Chem., 2019, 17, 1595–1607; (b) T. Irrgang and
R. Kempe,
Chem. Rev.,
2019,
119,
2524–2549;
(c)
G. A. Filonenko, R. van Putten, E. J. M. Hensen and
E. A. Pidko, Chem. Soc. Rev., 2018, 47, 1459–1483. 3 (a) P. A. Dub and J. C. Gordon, ACS Catal., 2017, 7, 6635–
6655; (b) J. R. Khusnutdinova and D. Milstein, Angew. Chem., Int. Ed., 2015, 54, 12236–12273; (c) R. H. Morris,
Acc. Chem. Res., 2015, 48, 1494–1502. p
pp
g
Thus, the study illustrates the importance of additional
mechanistic investigations when performing transformations,
which seems to proceed by dehydrogenation of an alcohol. This
is
especially
important
when
potassium
tert-butoxide
is
employed as an additive since the salt can serve both as a base
and as a reagent for initiating radical reactions. It should be
noted that the previous transition metal-catalyzed syntheses of
3-arylpropanamides from benzylic alcohols and acetamides
have all been carried out with potassium tert-butoxide as
a stoichiometric additive.14 Although, the transformations all
use known alcohol dehydrogenation catalysts,14 it cannot be
excluded that the present radical coupling is a minor pathway in
some of these reactions. 4 (a) B. C. Roy, I. A. Ansari, S. A. Samim and S. Kundu, Chem.–
Asian
J.,
2019,
14,
2215–2219;
(b)
J. Ballester,
A.-M. Caminade, J.-P. Majoral, M. Taillefer and A. Ouali,
Catal. Commun., 2014, 47, 58–62; (c) T. Ooi, H. Otsuka,
T. Miura, H. Ichikawa and K. Maruoka, Org. Lett., 2002, 4,
2669–2672. 5 (a) T. B. Boit, M. M. Mehta and N. K. Garg, Org. Lett., 2019, 21,
6447–6451; (b) V. Polshettiwar and R. S. Varma, Green Chem.,
2009,
11,
1313–1316;
(c)
A. Ouali,
J.-P. Majoral,
A.-M. Caminade and M. Taillefer, ChemCatChem, 2009, 1,
504–509. 6 6 (a) G.-M. Zhao, W.-J. Sun, H.-L. Zhang, D.-L. Li and X. Yang, J. Catal., 2019, 373, 126–138; (b) Q. Xu, J. Chen, H. Tian,
X. Yuan, S. Li, C. Zhou and J. Liu, Angew. Chem., Int. Ed.,
2014, 53, 225–229; (c) Y.-F. Liang, X.-F. Zhou, S.-Y. Tang,
Y.-B. Huang, Y.-S. Feng and H.-J. Open Access Article. Published on 25 June 2020. Downloaded on 8/20/2020 12:00:11 PM.
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. To gain further evidence for radical intermediates, a radical
clock experiment was then performed with o-allylbenzyl alcohol
and N,N-dimethyl acetamide. No propanamide was formed in
this case, but 50% conversion of o-allylbenzyl alcohol occurred
into a 5 : 3 : 2 mixture of 2-methylindene, 2-methylindane and
o-propylbenzaldehyde according to GCMS analysis. Finally,
several EPR spectra were recorded under different conditions
with benzyl alcohol, N,N-dimethyl acetamide and potassium
tert-butoxide aer 45 min in reuxing mesitylene. With all three
reactants in the mixture, a singlet with a g value of 2.0045 was
observed indicating a carbon-centered radical bound to oxygen
(Fig. 1). The same signal, although weaker, was detected when
only benzyl alcohol and potassium tert-butoxide were subjected
to reuxing mesitylene. However, no radicals were observed
upon reuxing only the base, the acetamide and the base, or the
alcohol and the acetamide in mesitylene solution. These results Scheme 3
Proposed mechanism for radical condensation. Fig. 1
EPR spectrum of the reaction in Table 1, entry 6. Scheme 3
Proposed mechanism for radical condensation. 7804 | Chem. Sci., 2020, 11, 7800–7806 This journal is © The Royal Society of Chemistry 2020 Chemical Science
View Article Online Chemical Science
View Article Online Chemical Science
View Article Online Acknowledgements We thank the Torkil Holm Foundation for nancial support. Open Access Article. Published on 25 June 2020. Downloaded on 8/20/2020 12:00:11 PM.
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Xu, RSC Adv., 2013, 3,
7739–7742; (d) Q. Xu, J. Chen and Q. Liu, Adv. Synth. Catal.,
2013,
355,
697–704;
(e)
L. J. Allen
and
R. H. Crabtree, Green Chem., 2010, 12, 1362–1364. Edge Article With the formation of the radical anion B, a radical chain
pathway can be envisioned where B rst reacts with the enolate
of the amide, possibly through a six-membered transition state
C, to form D. This C–C bond forming reaction is most likely the
rate-determining step in the condensation. The addition of
carbon-centered radicals to enolates has previously been
described to form a-alkylated and a-arylated ketones.23 Radical
anion D is an aminoketyl radical,24 which can be converted into
the corresponding amide E by single electron transfer to an
acceptor, e.g. N,N-dimethyl acetamide. Amide E then undergoes
elimination under the basic conditions to form cinnamamide F. Finally, the olen is reduced through single electron transfer25
to generate radical anion G,26 which reacts with benzyl alcohol
to produce the product propanamide and regenerate radical
anion B. The proposed pathway is in accordance with the
experimental observations from both the deuterium-labelling
experiments and the radical trapping reactions. Conclusions In summary, we have described a new radical coupling where 3-
arylpropanamides are formed from benzylic alcohols and
acetamides in the presence of potassium tert-butoxide. A variety
of alcohols and amides have been subjected to the trans-
formation to generate the product propanamides in good yields. Experimental mechanistic studies have revealed that the
condensation takes place by a radical pathway where the radical
anion of the benzylic alcohol is proposed as the key interme-
diate. The ndings illustrate the importance of radical trapping
experiments when performing coupling reactions with alcohols,
which appear to occur by transition metal-catalyzed accept-
orless
dehydrogenation
or
Meerwein–Ponndorf–Verley/
Oppenauer pathways. This is especially important when potas-
sium tert-butoxide is employed since the salt may serve a dual
role as both base and initiator of radical reactions. The
discoveries are envisioned to spur further interest in the
development of new radical-mediated C–C bond-forming reac-
tions with alcohols. In summary, we have described a new radical coupling where 3-
arylpropanamides are formed from benzylic alcohols and
acetamides in the presence of potassium tert-butoxide. A variety
of alcohols and amides have been subjected to the trans-
formation to generate the product propanamides in good yields. 7 7 (a) D. Gabri¨els, W. Y. Hern´andez, B. Sels, P. Van Der Voort
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R. Carmieli, Y. Diskin-Posner and D. Milstein, Angew. Chem., Int. Ed., 2019, 58, 3373–3377. Conflicts of interest 11 (a) J. P. Barham, G. Coulthard, K. J. Emery, E. Doni,
F. Cumine, G. Nocera, M. P. John, L. E. A. Berlouis,
T. McGuire, T. Tuttle and J. A. Murphy, J. Am. Chem. Soc., This journal is © The Royal Society of Chemistry 2020 Notes and references 1 (a) F. Huang, Z. Liu and Z. Yu, Angew. Chem., Int. Ed., 2016,
55, 862–875; (b) Y. Obora, ACS Catal., 2014, 4, 3972–3981. There are no conicts to declare. This journal is © The Royal Society of Chemistry 2020 Chem. Sci., 2020, 11, 7800–7806 | 7805 Edge Article
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lower temperature, we consider 2.0045 a reasonable value
for radical anion B in mesitylene solution at elevated
temperature. 12 See also: (a) K. Azizi and R. Madsen, ChemCatChem, 2018, 10,
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and
acetamides
by
transition
metal-catalyzed
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2012, 14, 2384–2387. 26 Radical anion G is also proposed as an intermediate in the
homolysis of 3-aryl-2-(2,2,6,6-tetramethylpiperidine-N-oxyl)
propanamides in the presence of potassium tert-butoxide,
see: M.-M. Wang, G. H. Sui, X.-C. Cui, H. Wang, J.-P. Qu
and Y.-B. Kang, J. Org. Chem., 2019, 84, 8267–8274. 26 17 The g values for ketyl radical anions in protic solvents at
room temperature are around 2.0031–2.0039 (A. G. Davies
and A. G. Neville, J. Chem. Soc., Perkin Trans. 2, 1992, 163– 7806 | Chem. Sci., 2020, 11, 7800–7806 This journal is © The Royal Society of Chemistry 2020
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Transplantation of Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells in Macular Degeneration
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Copyright for the publications made accessible via the Edinburgh Research Explorer is retained by the author(s)
and / or other copyright owners and it is a condition of accessing these publications that users recognise and
abide by the legal requirements associated with these rights. Edinburgh Research Explorer
Transplantation of Human Embryonic Stem Cell-Derived Retinal
Pigment Epithelial Cells in Macular Degeneration Edinburgh Research Explorer Citation for published version:
Mehat, MS, Sundaram, V, Ripamonti, C, Robson, AG, Smith, AJ, Borooah, S, Robinson, M, Rosenthal, AN,
Innes, W, Weleber, RG, Lee, RWJ, Crossland, M, Rubin, GS, Dhillon, B, Steel, DHW, Anglade, E, Lanza,
RP, Ali, RR, Michaelides, M & Bainbridge, JWB 2018, 'Transplantation of Human Embryonic Stem Cell-
Derived Retinal Pigment Epithelial Cells in Macular Degeneration', Ophthalmology. https://doi.org/10.1016/j.ophtha.2018.04.037 Citation for published version:
Mehat, MS, Sundaram, V, Ripamonti, C, Robson, AG, Smith, AJ, Borooah, S, Robinson, M, Rosenthal, AN,
Innes, W, Weleber, RG, Lee, RWJ, Crossland, M, Rubin, GS, Dhillon, B, Steel, DHW, Anglade, E, Lanza,
RP, Ali, RR, Michaelides, M & Bainbridge, JWB 2018, 'Transplantation of Human Embryonic Stem Cell-
Derived Retinal Pigment Epithelial Cells in Macular Degeneration', Ophthalmology. https://doi.org/10.1016/j.ophtha.2018.04.037 Citation for published version: Digital Object Identifier (DOI):
10.1016/j.ophtha.2018.04.037
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content complies with UK legislation. If you believe that the public display of this file breaches copyright please
contact openaccess@ed.ac.uk providing details, and we will remove access to the work immediately and
investigate your claim. Download date: 24. Oct. 2024 Transplantation of Human Embryonic Stem
Cell-Derived Retinal Pigment Epithelial Cells
in Macular Degeneration We also investigated evidence of the survival of transplanted cells and measured retinal structure
and function using microperimetry and spectral-domain OCT. g
p
y
p
Results:
Focal areas of subretinal hyperpigmentation developed in all participants in a dose-dependent
manner in the recipient retina and persisted after withdrawal of systemic immunosuppression. We found no
evidence of uncontrolled proliferation or inflammatory responses. Borderline improvements in best-corrected VA
in 4 participants either were unsustained or were matched by a similar improvement in the untreated contralateral
eye. Microperimetry demonstrated no evidence of benefit at 12 months in the 12 participants. In one instance at
the highest dose, localized retinal thinning and reduced sensitivity in the area of hyperpigmentation suggested the
potential for harm. Participant-reported quality of life using the 25-item National Eye Institute Visual Function
Questionnaire indicated no significant change. Conclusions:
Subretinal hyperpigmentation is consistent with the survival of viable transplanted hESC-
derived RPE cells, but may reflect released pigment in their absence. The findings demonstrate the value of
detailed analysis of spatial correlation of retinal structure and function in determining with appropriate sensitivity
the impact of cell transplantation and suggest that intervention in early stage of disease should be approached
with caution. Given the slow rate of progressive degeneration at this advanced stage of disease, any protection
against
further
deterioration
may
be
evident
only
after
a
more
extended
period
of
observation. Ophthalmology 2018;-:1e11 Crown Copyright ª 2018 Published by Elsevier Inc. on behalf of the
American Academy of Ophthalmology. This is an open access article under the CC BY license (http://
creativecommons.org/licenses/by/4.0/). Conclusions:
Subretinal hyperpigmentation is consistent with the survival of viable transplanted hESC-
derived RPE cells, but may reflect released pigment in their absence. The findings demonstrate the value of
detailed analysis of spatial correlation of retinal structure and function in determining with appropriate sensitivity
the impact of cell transplantation and suggest that intervention in early stage of disease should be approached
with caution. Given the slow rate of progressive degeneration at this advanced stage of disease, any protection
against
further
deterioration
may
be
evident
only
after
a
more
extended
period
of
observation. Ophthalmology 2018;-:1e11 Crown Copyright ª 2018 Published by Elsevier Inc. on behalf of the
American Academy of Ophthalmology. This is an open access article under the CC BY license (http://
creativecommons.org/licenses/by/4.0/). Supplemental material available at www.aaojournal.org. Transplantation of Human Embryonic Stem
Cell-Derived Retinal Pigment Epithelial Cells
in Macular Degeneration Manjit S. Mehat, PhD, FRCOphth,1,2,3 Venki Sundaram, MD, FRCOphth,1,2,3 Caterina Ripamonti, PhD,4
Anthony G. Robson, PhD,1,2 Alexander J. Smith, PhD,1,3 Shyamanga Borooah, PhD, FRCOphth,5
Martha Robinson, PhD,3 Adam N. Rosenthal, PhD, FRCOG,6 William Innes, MRCP,7 Richard G. Weleber, MD,8
Richard W.J. Lee, PhD, FRCOphth,1,2,3 Michael Crossland, PhD,1,2,3 Gary S. Rubin, PhD,1,2,3
Baljean Dhillon, FRCS,5 David H.W. Steel, FRCOphth,7,9 Eddy Anglade, MD,10 Robert P. Lanza, PhD,10
Robin R. Ali, PhD,1,3,11 Michel Michaelides, MD, FRCOphth,1,2,3 James W.B. Bainbridge, PhD, FRCOphth1,2,3 Purpose:
Transplantation of human embryonic stem cell (hESC)-derived retinal pigment epithelial (RPE) cells
offers the potential for benefit in macular degeneration. Previous trials have reported improved visual acuity (VA),
but lacked detailed analysis of retinal structure and function in the treated area. Purpose:
Transplantation of human embryonic stem cell (hESC)-derived retinal pigment epithelial (RPE) cells
offers the potential for benefit in macular degeneration. Previous trials have reported improved visual acuity (VA),
but lacked detailed analysis of retinal structure and function in the treated area. y
Design:
Phase 1/2 open-label dose-escalation trial to evaluate safety and potential efficacy (clinicaltrials.gov
identifier, NCT01469832). y
Design:
Phase 1/2 open-label dose-escalation trial to evaluate safety and potential efficacy (clinicaltrials.gov
identifier, NCT01469832). Participants:
Twelve participants with advanced Stargardt disease (STGD1), the most common cause of
macular degeneration in children and young adults. Participants:
Twelve participants with advanced Stargardt disease (STGD1), the most common cause of
macular degeneration in children and young adults. Participants:
Twelve participants with advanced Stargardt disease (STGD1), the most common cause of
macular degeneration in children and young adults. Methods:
Subretinal transplantation of up to 200 000 hESC-derived RPE cells with systemic immunosup-
pressive therapy for 13 weeks. Methods:
Subretinal transplantation of up to 200 000 hESC-derived RPE cells with systemic immunosup-
pressive therapy for 13 weeks. O
SC y
Main Outcome Measures:
The primary end points were the safety and tolerability of hESC-derived RPE cell
administration. We also investigated evidence of the survival of transplanted cells and measured retinal structure
and function using microperimetry and spectral-domain OCT. Main Outcome Measures:
The primary end points were the safety and tolerability of hESC-derived RPE cell
administration. We also investigated evidence of the survival of transplanted cells and measured retinal structure
and function using microperimetry and spectral-domain OCT. Main Outcome Measures:
The primary end points were the safety and tolerability of hESC-derived RPE cell
administration. Crown Copyright ª 2018 Published by Elsevier Inc. on behalf of the American
Academy of Ophthalmology. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/by/4.0/). https://doi.org/10.1016/j.ophtha.2018.04.037
ISSN 0161-6420/18 Ophthalmology
Volume -, Number -, Month 2018 transplantation in the poorer-seeing eye of individuals with
advanced disease, acknowledging that any potential for
benefit would be limited in this context by established
degeneration of photoreceptor cells. Retinal degeneration in
STGD1 typically advances progressively by expansion from
the macula. We chose to administer cells to a target area,
predefined for each study eye, extending from relatively well-
preserved functional retina across a transitional zone of pro-
gressive degeneration to an area of atrophic nonfunctional
retina. This afforded us the opportunity both to determine the
safety of the transplanted cells in relatively healthy retina and
to explore the potential benefit to function and survival of
overlying photoreceptor cells in the degenerating and atrophic
areas. By defining the target zone in the retina of each study
eye before intervention, according to the distribution of dis-
ease severity and preferred locus of fixation, we were able to
evaluate in detail the structure and function of this region
before intervention using OCT, autofluorescence imaging,
full-field perimetry, and microperimetry. For assessment of
visual function, we established each participants’ testeretest
variability for each test by means of multiple testing at
baseline and controlled for the natural history of the condi-
tion, learning effects, and performance variability by com-
parison with the contralateral uninjected eye, on the basis that
disease progression in an individual is symmetrical11 and that
monocular performance of the contralateral uninjected eye is
not influenced by the intervention. We considered that for
each participant, changes in performance of both eyes
symmetrically would indicate disease progression, learning
effects, or both and that differences between the eyes may
indicate a consequence of the intervention. intervention under direct observation; its laminar structure
lends itself to the targeted delivery of cellular therapies. The highly compartmentalized structure of the globe restricts
potential dissemination locally and systemically. The intra-
ocular environment is relatively protected against systemic
immune responses that threaten allograft survival. Retinal
microstructure can be observed in detail in the living eye
noninvasively owing to the optical transparency of ocular
media, and retinal function can be mapped topographically
with exquisite sensitivity. The impact of intervention within a
defined target region of the retina can be determined with
confidence by comparison with untreated regions within the
same eye and in the contralateral eye, which offer invaluable
intraocular and intraindividual controls for the natural history
of the condition and variability in performance. Ophthalmology
Volume -, Number -, Month 2018 Stargardt disease (STGD1) is the most common cause of
macular degeneration in children and young adults. The con-
dition originates in disease-causing sequence variants in the
ABCA4 gene1 and results in progressively severe impairment
of sight. The ABCA4 gene encodes a rim protein located on
the intracellular disc membranes of light-sensitive photore-
ceptor cells that play an essential role in the retinoid cycle.2
Gene defects result in accelerated accumulation of a putative
toxic
metabolite,
Di-retinoid-pyridinium-ethanolamine,
within the underlying phagocytic retinal pigment epithelial
(RPE) cells, leading to cell dysfunction and eventual cell
death3 with progressive atrophy expanding from the central
macula.4
Retinal
pigment
epithelial
cells
support
the
function and survival of overlying photoreceptor cells by
multiple mechanisms, including recycling of visual pigment
and phagocytosis of outer segments.5 Degeneration of RPE
cells leads to secondary dysfunction and degeneration of
overlying
photoreceptor
cells,
and
consequently
progressively severe impairment of sight. Stargardt disease
currently is untreatable, but replenishment of degenerating
RPE cells with healthy cells offers the possibility of benefit
by better supporting the function and survival of overlying
photoreceptor
cells,
and
consequently
improving
or
protecting sight for a period limited by the consequences of
the underlying photoreceptor disorder. A similar approach
may benefit atrophic age-related macular degeneration,
which shares key features with STGD1, including progressive
atrophy of RPE and overlying photoreceptor cells. However,
differences in their cause and progression are likely to influ-
ence the potential benefit of subretinal administration of hu-
man
embryonic
stem
cell
(hESC)-derived
RPE
cell
suspensions. For example, age-related changes in Bruch’s
(basement) membrane and chronic inflammation in age-
related macular degeneration may influence the adhesion and
survival of donor cells. Trial Design We performed a phase 1/2 open-label multicenter dose-escalation
trial in 12 adult participants to evaluate the safety and tolerability
of subretinal transplantation of hESC-derived RPE cells. The
intervention was unmasked. The study received the approval of the
Medicines and Health Products Regulatory Authority, the United
Kingdom Gene Therapy Advisory Committee, and the Moorfields
Eye Hospital Research Governance Committee. We provided
potential candidates with detailed information, including an
explanation of the aims of the trial and the possible consequences
of participation. Participants provided their written informed con-
sent before enrolment. We performed the study in accordance with
the tenets of the Declaration of Helsinki and registered the trial at
clinicaltrials.gov (identifier, NCT01469832). Ophthalmology
Volume -, Number -, Month 2018 Transplantation of Human Embryonic Stem
Cell-Derived Retinal Pigment Epithelial Cells
in Macular Degeneration Embryonic stem cells are a potentially valuable source of
donor cells for therapeutic repair and regeneration. Their
pluripotency and innate capacity for self-renewal offer a
virtually unlimited supply of highly specialized cells for
therapeutic transplantation, but also present a potential risk
of harm from uncontrolled proliferation and unintended
differentiation. Their impact in experimental models has been investigated extensively, but evidence of their safety
and potential efficacy in humans is limited. Embryonic stem
cells have attracted attention for their potential value in a
broad range of degenerative conditions, including those
affecting the brain and eye. The eye has unique advantages
as a target organ for cell transplantation. The retina is an
extension of the brain that is readily accessible to surgical 1 https://doi.org/10.1016/j.ophtha.2018.04.037
ISSN 0161-6420/18 https://doi.org/10.1016/j.ophtha.2018.04.037
ISSN 0161-6420/18 Intervention Retinal pigment epithelial cells were generated from the hESC line
MA09 in accordance with good manufacturing practice. The hESC
bank was thawed and expanded on mitomycin Cetreated mouse
embryonic fibroblasts for 3 passages. Human embryonic stem cells
were dissociated and seeded to allow embryoid body formation. Pigmented RPE patches then were isolated with collagenase from
cellular outgrowths. After purification and trypsinization, the RPE
cells were expanded and cryopreserved at passage 2 for clinical
application. We characterized RPE cells in process and after freezing
and formulation, including karyotyping, pathogen and phagocytosis
assay testing, and differentiation and purity evaluation by morpho-
logic assessment, quantitative polymerase chain reaction, and
quantitative immunohistochemistry for RPE and hESC markers. On
the day of transplantation, cells were thawed from cryopreserved
hESC-derived RPE cell banks, formulated at the appropriate con-
centration, stained to exclude bacterial contamination, and delivered
to the operating room for administration.8 We evaluated participants at baseline and after hESC-derived RPE
cell transplantation at intervals for 12 months. We performed exami-
nation by slit-lamp biomicroscopy and indirect binocular ophthal-
moscopy, color fundus photography, FAF imaging, fundus fluorescein
angiography, and SD OCT. We measured best-corrected VA (BCVA)
and color discrimination (Universal Colour Discrimination Test) and
performed microperimetry (Nidek MP-1, NIDEK Inc, Fremont, CA)
full-field static perimetry (Octopus 900, Haag-Streit, Switzerland),
Goldmann kinetic perimetry, and electroretinography (including full-
field, pattern, and multifocal electroretinography to international
standards using gold-foil electrodes). We used standardized protocols
and a fixed sequence of test patterns. We assessed participants’ sys-
temic health by physical examination results, electrocardiography
results, cancer screening results, and hematologic and serologic re-
sults. After completion of the initial 12-month evaluation period,
participants were followed up for long-term safety assessments, the
anticipated duration of which is lifelong. The surgical procedure was conducted under general anaes-
thesia. We performed 3-port pars plana vitrectomy, with separation
of the posterior hyaloid from the posterior pole toward the equator. Administration of hESC-derived RPE cells was preceded by
subretinal injection of up to 0.2 ml Hartmann’s solution using a
41-gauge cannula to establish the target tissue plane and to mini-
mize unintended administration of cells into the vitreous or
choroid. The hESC-derived RPE cell suspension, reconstituted to
the appropriate concentration in 150 ml, was injected using a
38-gauge subretinal cannula (MedOne PolyTip Cannula 23/38;
MedOne Surgical, Sarasota, FL) into the preformed bleb of
Hartmann’s solution. Role of the Funding Source The funder of the study participated in the study design, data
analysis and interpretation, and writing of the report. The corre-
sponding author had full access to all the data in the study and had
final responsibility for the decision to submit for publication. Mehat et al
hESC-Derived RPE Cell Transplantation in STGD1 150 000, and 200 000 hESC-derived RPE cells. Each dose group
included 3 participants. For each participant, we selected the
poorer-seeing eye (according to dominance and VA) as the study
eye for intervention; the better-seeing contralateral eye served as an
untreated control for intraindividual natural history and perfor-
mance variability. prescribed a standard postvitrectomy regimen of topical antibiotics
(chloramphenicol 0.5% 4 times daily for 7 days) and topical nonste-
roidal anti-inflammatory drugs (ketorolac 0.5% 4 times daily for 4
weeks). Outcome Measures The primary end points were the safety and tolerability of hESC-
derived RPE cells as defined by the absence of any grade 2 or
more (National Cancer Institute grading system) adverse event
relating to the transplanted cells, any evidence that the cells were
contaminated with an infectious agent, any evidence that the cells
showed potential of tumorigenicity, or a combination thereof. Secondary end points included safety of the surgical procedure,
dose selection for future studies, evidence of donor cell survival
and engraftment in the target location, and retinal function as
measured by electroretinography. Exploratory end points included
VA, color discrimination, retinal sensitivity by perimetry, and
quality of life (assessed with the 25-item National Eye Institute
Visual Function Questionnaire). Intervention After subretinal injection, we performed a
washout of the vitreous cavity by continuous infusion and aspira-
tion of fluid to minimize the presence of donor cells remaining in
this compartment. Participants were advised to maintain a supine
position for 6 hours after surgery. Statistical Analysis The small sample size was not designed for statistical analysis. The
end points for efficacy were solely descriptive and were defined as
any improvement in visual function beyond the testeretest vari-
ability within the full cohort for each assessment, determined by
means of 1-way analysis of variance with the use of multiple
measurements at baseline.12 Human embryonic stem cell-derived RPE cells were delivered
into a preselected transition region of the retina, defined for each
participant as a region extending from an area of relatively pre-
served structural integrity and function to an area of atrophic
nonfunctional retina. The rationale was to investigate the safety of
hESC-derived RPE cell administration in areas of relatively well-
preserved retina and the potential benefit in areas of retinal
dysfunction and atrophy. The target region was defined before
surgery for the study eye of each participant with regard to the
distribution of the disease severity and preferred locus of fixation
using spectral-domain (SD) OCT, fundus autofluorescence (FAF)
imaging, and microperimetry. We defined target regions close to
the preferred locus of fixation to optimize the quality of structural
and functional assessment. Trial Participants In experimental models of retinal degeneration, subretinal
injection of hESC-derived RPE cell suspensions can protect
photoreceptor cells and retinal function.6,7 In human partici-
pants with STGD1, subretinal injection of up to 150 000
hESC-derived RPE cells resulted in no serious adverse events
related to the transplanted cells.8e10 However, assessment of
visual function has been limited. Herein we present the results
of a dose-escalation trial of up to 200 000 hESC-derived RPE
cells in 12 participants. To mitigate the risk of harm, we
chose to investigate the safety of hESC-derived RPE We enrolled 12 participants with clinical features of STGD1,
electroretinographic evidence of generalized rod and cone photo-
receptor cell dysfunction, and molecular characterization of
mutations in ABCA4. We excluded candidates in whom the visual
acuity (VA) of their poorer-seeing eye was 20/400 or better, those
with any history of malignant neoplasia or significant ocular
pathologic features other than STGD1, any contraindication to
systemic immunosuppression or surgery under general anaesthesia,
and women who were pregnant or lactating. We enrolled partici-
pants sequentially into escalating dose groups of 50 000, 100 000, 2 Mehat et al
hESC-Derived RPE Cell Transplantation in STGD1 Mehat et al
hESC-Derived RPE Cell Transplantation in STGD1 Effect of Human Embryonic Stem Cell-Derived
Retinal Pigment Epithelial Cell Transplantation
on Retinal Structure Within 4 weeks of administration of hESC-derived RPE cells, sub-
retinal hyperpigmentation developed within the area of injection of
the cell suspension in all 12 participants (Fig 1). The mean area of
subretinal hyperpigmentation increased progressively, at a rate that
seemed to be dose dependent during the first 3 months, and
subsequently slowed (Fig S1, available at www.aaojournal.org). A
subsequent
modest
reduction
in
area
of
subretinal
hyperpigmentation was evident in 2 participants (patients 4 and 9). The mean area of subretinal pigmentation at 12 months was
correlated directly with the dose of hESC-derived RPE cells
administered (R2 ¼ 0.981; Fig S2, available at www.aaojournal.org). We investigated retinal sensitivity in detail using mesopic
microperimetry (NidekMP-1). We measured retinal sensitivity at loci
in the macular area (central 20) in both the study eye (including a
variable portion of the transplanted area) and contralateral eye (in all
except patient 12) and calculated the change in sensitivity from
baseline (Fig S5, available at www.aaojournal.org). A borderline
improvement in retinal sensitivity of the study eye in 1 participant
(patient 7) was evident at 3 months only. The mean retinal
sensitivity in all 4 dose groups remained within the limits of the
testeretest variability. Areas of subretinal hyperpigmentation corresponded on SD OCT
to a hyperreflective signal located between the photoreceptor cell
layer and the underlying Bruch’s membrane that persisted for 12
months in all eyes. Focal changes in FAF, which may be an indirect
indication of RPE cell function, were evident within the region of
cell administration. A relative predilection for the development of
hyperpigmentation at sites of retinal atrophy within the injected area
was evident. In 1 patient (patient 4), a circular region of atrophic
retina, which on SD OCT appeared to be devoid of RPE, developed
progressively confluent hyperpigmentation with increasing sub-
retinal hyperreflectivity on SD OCT and masking of choroidal
hyperreflectivity (Fig 2A). However, recovery of FAF in this area
was undetectable at 12 months (Fig 2Axii). Hyperpigmentation in
areas overlying surviving host RPE typically was associated with
reduced FAF, consistent with masking of the autofluorescence of
underlying host RPE (Fig 2B). Ophthalmology
Volume -, Number -, Month 2018 in the 3 participants (patients 8, 9, and 10) whose fixation stability
enabled reliable serial analysis; in 2 participants (patients 8 and 9),
we identified no clear relationship; in 1 participant (patient 10),
who was administered the highest dose, retinal thinning was
evident in the hyperpigmented areas at 12 months (Fig S3,
available at www.aaojournal.org). in 10 eyes and superior to the superotemporal arcade in 2 eyes
(patients
5
and
11;
Fig
1). We
performed
intraoperative
prophylactic retinopexy for pre-existing pigmented retinal breaks
in 2 participants (patients 3 and 5) and for atrophic retinal holes in
2 participants (patients 10 and 11). In 1 participant (patient 12),
cryoretinopexy and injection of sulphahexafluouride gas were
performed for 2 small superonasal retinal tears and a small infe-
rotemporal retinal dialysis with localized avulsion of pars plana
epithelium. In all eyes, the bleb of subretinal fluid resorbed clini-
cally within 24 hours, with no persistent or unplanned retinal
detachment. Small subretinal hemorrhages at the injection sites
were evident in 2 participants (patients 1 and 12). Mild post-
operative vitreous cavity hemorrhage in 1 participant (patient 5),
arising from a pars plana entry site, cleared spontaneously within 4
weeks. A mild asymptomatic posterior subcapsular lens opacity
was evident in 1 participant (patient 6) at month 9. Adverse events
related to immunosuppression in 5 participants (patients 5, 6, 7, 10,
and 12) included lethargy, headache, nausea, and herpes simplex
virus 1 (HSV1) reactivation. All participants completed 12 weeks
of immunosuppression after transplantation. Focal preretinal pigmentation (on the inner surface of the retina)
developed in 2 participants (Fig 1). In 1 participant (patient 1),
preretinal pigmentation was associated with limited adherence to
advice to maintain a supine position after surgery; in this instance,
the area of preretinal pigmentation increased progressively during
the 12-month period, but subsequently stabilized (data not shown). One participant (patient 12) demonstrated a nonpigmented and
noncontractile epiretinal membrane over the injection site at month
6; this subsequently stabilized in extent with no measurable effect on
visual function. Focal pigmented deposits were evident in the vit-
reous cavity in 2 participants (patients 6 and 7), both of whom
reported visual floaters. Effect of Human Embryonic Stem Cell-Derived
Retinal Pigment Epithelial Cell Transplantation
on Retinal Function We identified no evidence of acute immune rejection such as
vitreitis, retinitis, retinal exudates, retinal edema, or vascular
hyperpermeability. In no instance was intraretinal fluid evident on
OCT, and no change of clinical concern was apparent on fundus
fluorescein angiography. We found no evidence of adverse pro-
liferation of transplanted hESC-derived RPE cells either intraocu-
larly or on systemic screening by clinical examination (including
testicular and prostate examination in men and breast and pelvic
examination in women) or investigation (including fecal occult
blood test, chest radiography, thyroid function analysis, prostate-
specific
antigen
analysis,
and
cervical
smear
analysis,
as
appropriate). We measured no clinically significant decline or improvement in
retinal function by electroretinography in any of the participants
during the 12-month period. Participant-reported quality of life using
the 25-item National Eye Institute Visual Function Questionnaire
indicated no significant change. A transient reduction in BCVA of
operated eyes was evident in 7 participants (patients 2, 3, 4, 5, 8, 11,
and 12) after surgery and resolved within 96 days (range, 3e21
days; Fig S4, available at www.aaojournal.org). Borderline
improvements in BCVA were evident in 4 participants, although
these were either unsustained (patients 1, 7, and 10) or matched by
a similar improvement in the contralateral eye (patient 12). The
mean BCVA of the 150 000-cell dose improved to the limit of
baseline variability; for the other dose groups, the mean BCVA
remained within the limits of testeretest variability during the 12-
month period. The participants’ preferred loci of fixation were
unaltered (data not shown). Ophthalmology
Volume -, Number -, Month 2018 Administration of Human Embryonic Stem Cell-
Derived Retinal Pigment Epithelial Cell
Suspension To manage the risk of immune rejection of the transplanted hESC-
derived RPE cells, participants were prescribed oral immunosup-
pression in the perioperative period. Low-dose tacrolimus (0.1 mg/kg)
was divided into 2 daily doses from 1 week before transplantation,
titrated to achieve trough serum levels of 3 to 7 ng/ml, and maintained
until week 6 after transplantation, when it was discontinued. Myco-
phenolate was commenced on the day of surgery (day 0) in an esca-
lating dose regimen: 0.25 g twice daily (day 0 to day 1); 0.5 g twice
daily (day 2 to day 3), and 1 g twice daily from day 4 until 12 weeks
after transplantation, when it was discontinued. Participants also were We administered hESC-derived RPE cells to 12 participants (age
range, 34e53 years) with molecularly proven advanced STGD1
(Table 1). The hESC-derived RPE cells were reconstituted and
administered subretinally by 2 of the authorsdpatients 1 through
11 by J.W.B.B. and patient 12 by D.S.dto the predefined target
regions, which were inferior to the inferotemporal vascular arcade 3 Table 1. Participants and Dosing
Participant
number
Age (yr)
hESC-RPE dose
Sex
Study eye
Baseline Visual Acuity ETDRS LogMAR
Genotype Pathogenic
variants on ABCA4 gene
Study eye
Control eye
P1
34
50 000
M
OD
1.29
1.23
c.768G>T (p.?)
c.5461-10T>C (p.?)
P2
44
M
OS
1.36
1.39
c.4918C>T (p.Arg1640Trp)
c.6079C>T (p.Leu202Phe)
P3
46
M
OS
1.33
1.33
c.2588G>C (p.Gly863Ala)
c.5461-10T (p.?)
c.2828G>A (p.Arg943Gln)
c.5603A>T (p.Asn1868Ile)
P4
41
100 000
M
OS
1.39
1.35
c.4139C>T (p.Pro1380Leu)
c.5461-10C>T (p.?)
P5
51
M
OD
1.37
1.39
c.454C>T (p.Arg152*)
c.1715G>A (p.Arg572Gln)
c.2588G>C (p.Gly863Ala)
c.6148G>C (p.Val2050Leu)
P6
44
F
OD
1.84
1.84
c.161G>A (p.Cys54Try)
c.4462T>C (p.Cys1488Arg)
P7
40
150 000
M
OD
1.84
1.36
c.666_678del (p.Lys223fs)
c.5461-10T>C (p?)
P8
53
M
OS
1.27
1.23
c.2588G>C (p.Gly863Ala)
c.2828G>A (p.Arg943Gln)
c.5603A>T (p.Asn1868Ile)
P9
40
M
OS
1.39
1.33
c.634C>T (p.Arg212Cys)
c.4319T>C (p.Phe1440Ser)
P10
50
200 000
M
OS
1.25
1.19
c.1906C>T (p.Gln636*)
c.2588G>C (p.Gly863Ala)
P11
45
M
OS
1.39
1.39
c.5461-10T>C
c.5929G>A (p.Gly1977Ser)
P12
45
M
OS
1.09
1.10
c.2588G>C (p.Gly863Ala)
c.4469G>A (p.Cys1490Tyr)
Mehat et al
hESC-Derived RPE Cell Transplantation in STGD1 Mehat et al
hESC-Derived RPE Cell Transplantation in STGD1 Table 1. Participants and Dosing
Mehat et al
hESC-Derived RPE Cell Transplantation in STGD1 localized changes in retinal sensitivity, we derived corresponding
topographic contour maps using Visual Field Modelling and
Analysis software13 (Fig 3). Localized areas of improved retinal
sensitivity (for example, in patients 2, 3, 8, 9, and 11) were not
hyperpigmented,
whereas
retinal
sensitivity
in
areas
of
hyperpigmentation appeared unchanged or reduced (for example,
in patients 8 and 10). of the compromised or degenerate RPE by transplantation
of healthy RPE cells offers the possibility of protecting or
improving sight by supporting the function or survival of
overlying photoreceptor cells. The results of the trial
satisfied its primary end point for safety, with no evidence
of inflammatory responses or uncontrolled intraocular or
systemic proliferation, after subretinal administration of up
to 200 000 hESC-derived RPE cells. There were no
serious adverse events related to the surgical procedure;
intraoperative adverse events resulted in no harm to visual
function beyond the immediate postoperative period. A brief
decline in BCVA in the operated eye after administration of
hESC-derived RPE cells was a predictable temporary
consequence of intraocular surgery. The development of
pigmented epiretinal membranes after injection of hESC-
derived RPE cells has been reported previously9 and is
consistent
with
reflux
of
cell
suspension
from
the
subretinal space. Although we measured no associated
adverse impact on visual function at 12 months, the
longer-term significance has yet to be determined. Adverse
events
related
to
the
immunosuppressive
medications
occurred during the period of their administration, but had
no impact subsequently. localized changes in retinal sensitivity, we derived corresponding
topographic contour maps using Visual Field Modelling and
Analysis software13 (Fig 3). Localized areas of improved retinal
sensitivity (for example, in patients 2, 3, 8, 9, and 11) were not
hyperpigmented,
whereas
retinal
sensitivity
in
areas
of
hyperpigmentation appeared unchanged or reduced (for example,
in patients 8 and 10). We calculated the mean full-field retinal sensitivity in both the
study eyes and control eyes of each dose cohort (in all participants
except patients 6 and 12) by full-field static perimetry (Octopus
900). Deterioration in full-field sensitivity was evident in both the
study and control eyes of 2 participants (patients 2 and 4), and an
associated reduction in both the study and control eyes of the
100 000-cell dose cohort to the lower limit of testeretest variability
(Fig S7, available at www.aaojournal.org). The individual and
mean sensitivities otherwise were maintained within the limits of
variability for the 12-month period. Because participants in the early stages of the trial described
subjective improvements after intervention in their sensitivity of
their study eye to color, we measured color vision in a subset of 4
participants (patients 8e11) using the Universal Colour Discrimi-
nation Test. A single assessment at baseline demonstrated poor
color discrimination in both eyes. Serial assessments after inter-
vention indicated no improvement in color vision that was specific
to the study eye (Fig S7). The development of subretinal hyperpigmentation within
the injected area in all 12 participants was dose dependent and
associated in many instances with a hyperreflective signal on
OCT, suggesting survival of injected donor hESC-derived
RPE cells. The high density of pigmentation in these areas
may reflect a greater melanin content, higher density, Effect of Human Embryonic Stem Cell-Derived
Retinal Pigment Epithelial Cell Transplantation
on Retinal Structure We investigated the relationship
between hyperpigmentation and retinal thickness using SD OCT For the study eyes of 8 participants (all except patients 1, 6, 7, and
12, for whom the assessment was unavailable or unreliable) we
measured in greater detail, using a stimulus grid of high density, the
sensitivity of the area of retina specifically targeted with the hESC-
derived RPE cell suspension. The total retinal sensitivities of each
eye and the mean sensitivities of each dose group remained within the
limits of testeretest variability during the 12-month period (Fig S6,
available
at
www.aaojournal.org). To
quantify
the
spatial
correlation of retinal sensitivity with hyperpigmentation, we
measured the change in retinal sensitivity at 12 months at
hyperpigmented and nonhyperpigmented loci (Fig 3). The retinal
sensitivity at most loci remained within the limits of variability,
whether within or outside the transplanted area, and regardless of
the presence of hyperpigmentation (Fig 4); in 2 participants
administered higher doses (patients 8 and 10), retinal sensitivity at
hyperpigmented loci tended to be reduced. To demonstrate highly 4 Discussion Stargardt disease causes progressive and irreversible mac-
ular degeneration and severe impairment of sight.14 Repair 5 1. Fundus photographs of recipient eyes at 12 months after transplantation. In each image, the retinotomy site is indicated by the gree
subretinal administration is outlined by the dotted black line. P ¼ patient. Ophthalmology
Volume -, Number -, Month 2018 Ophthalmology
Volume -, Number -, Month 2018 Ophthalmology
Volume -, Number -, Month 2018 Figure 1. Fundus photographs of recipient eyes at 12 months after transplantation. In each image, the retinotomy site is indicated by the green dot, and the
area of subretinal administration is outlined by the dotted black line. P ¼ patient. areas devoid of host RPE is plausible given the anchorage-
dependent properties of RPE cells18,19 and similarly has
been described in experimental in vivo studies.20 The
progressive local extension of hyperpigmentation during the
first 3 months and the predilection for areas of atrophy in
the recipient retina are consistent with local migration of
surviving donor cells, but may reflect subretinal migration
of released pigment. hypertrophy,15 or multilayering of surviving donor hESC-
derived RPE cells. The presence of pigmented subretinal
material is not definitive evidence of donor cell survival and
alternatively may reflect persistence of melanin pigment after
their death,16,17 a consequence of transplantation that equally
could be dose dependent. We found no definitive evidence of
local accumulation of lipofuscin on FAF imaging to
demonstrate physiologic activity, and hence viability, of the
cells, although FAF may take longer than 12 months to
develop. Preferential engraftment of RPE cell transplants to To mitigate the risk of immune rejection of injected allo-
genic hESC-derived RPE cells, we administered systemic 6 Fundus photographs, OCT images, and fundus autofluorescence images in (A) patient 4 and (B) patient 10. A, In patient 4, fundus im
ting the time-course of hyperpigmentation (Ai, Aiv, and Avii) are presented with corresponding OCT line scans, at lower and higher ma
A progressive increase in optical signal evident in the outer retina consistent with the continued presence of transplanted cells. B, In patient
monstrating the time-course of hyperpigmentation (Bi, Bii, and Biii) are presented with the associated fundus autofluorescence images dem
duced signal consistent with masking of endogenous autofluorescence. Mehat et al
hESC-Derived RPE Cell Transplantation in STGD1 Mehat et al
hESC-Derived RPE Cell Transplantation in STGD1 Mehat et al
hESC-Derived RPE Cell Transplantation in STGD1 Figure 2. Fundus photographs, OCT images, and fundus autofluorescence images in (A) patient 4 and (B) patient 10. A, In patient 4, fundus images
demonstrating the time-course of hyperpigmentation (Ai, Aiv, and Avii) are presented with corresponding OCT line scans, at lower and higher magni-
fication. A progressive increase in optical signal evident in the outer retina consistent with the continued presence of transplanted cells. B, In patient 10,
images demonstrating the time-course of hyperpigmentation (Bi, Bii, and Biii) are presented with the associated fundus autofluorescence images demon-
strating reduced signal consistent with masking of endogenous autofluorescence. 7 Ophthalmology
Volume -, Number -, Month 2018 roperimetry images showing topography of retinal sensitivity. The retinal sensitivities at test loci in each study eye, m
ek MP-1) at baseline and 12 months, are superimposed on the respective fundus image. Each is presented with a correspon
o illustrate the hill of vision of retinal sensitivity, constructed by interpolation of sensitivities at the test loci using Visual Fi
MA) software. The blue line outlines the area of recipient retina administered human embryonic stem cell-derived retinal p
. P ¼ patient; K ¼ thousand. Figure 3. Microperimetry images showing topography of retinal sensitivity. The retinal sensitivities at test loci in each study eye, measured by micro-
perimetry (Nidek MP-1) at baseline and 12 months, are superimposed on the respective fundus image. Each is presented with a corresponding topographic
contour map to illustrate the hill of vision of retinal sensitivity, constructed by interpolation of sensitivities at the test loci using Visual Field Modelling and
Analysis (VFMA) software. The blue line outlines the area of recipient retina administered human embryonic stem cell-derived retinal pigment epithelial
cell suspension. P ¼ patient; K ¼ thousand. 8 Mehat et al
hESC-Derived RPE Cell Transplantation in STGD1 Figure 4. Boxplots showing the change in retinal sensitivity from the baseline (mean of 3 baseline tests) as measured by microperimetry (Nidek MP-1) for
each test locus at month 12. Loci were stratified into 3 groups: outside the transplantation area (solid green dots), within the transplantation and associated
with subretinal hyperpigmentation (solid blue dots), or within the transplantation area and without subretinal hyperpigmentation (solid orange dots). Using
a selection tool on the Visual Field Modelling and Analysis (VFMA) software, volumetric sensitivity within regions of retinal pigmentation were extracted
from regions of pigmentation and nonpigmentation. The boxplots show the median value, quartiles, and range. hESC ¼ human embryonic stem cell; K ¼
thousand; P ¼ patient; RPE ¼ retinal pigment epithelial. Mehat et al
hESC-Derived RPE Cell Transplantation in STGD1 l
h
h
h
l d b
i
i
(Nid k MP 1) f h b
l
(
b
l
) Figure 4. Boxplots showing the change in retinal sensitivity from the baseline (mean of 3 baseline tests) as measured by microperimetry (Nidek MP-1) for
each test locus at month 12. Loci were stratified into 3 groups: outside the transplantation area (solid green dots), within the transplantation and associated
with subretinal hyperpigmentation (solid blue dots), or within the transplantation area and without subretinal hyperpigmentation (solid orange dots). Using
a selection tool on the Visual Field Modelling and Analysis (VFMA) software, volumetric sensitivity within regions of retinal pigmentation were extracted
from regions of pigmentation and nonpigmentation. The boxplots show the median value, quartiles, and range. hESC ¼ human embryonic stem cell; K ¼
thousand; P ¼ patient; RPE ¼ retinal pigment epithelial. such cells nonetheless may be subject to rejection, and the
process of cell derivation specific to an individual presents
considerable logistical challenges.22 immunosuppressive medications to all participants for 13
weeks. We identified no overt clinical signs to suggest acute
or delayed rejection. Specifically, we noted no vitreitis, retinal
exudation or retinitis, vascular leakage, choroiditis, or cystoid
macular edema, although features of rejection may not always
be readily evident.21 We identified no change on clinical
examination
or
imaging
after
withdrawal
of
immunosuppression, 12 weeks after the injection procedure. Ophthalmology
Volume -, Number -, Month 2018 The aim of intervention is to protect or improve aspects
of sight. In the current trial, all participants showed
advanced disease before intervention, with established
retinal degeneration and severe impairment of visual func-
tion. We measured no consistent progression of disease
during the 12-month trial; deterioration in the visual fields of
both eyes of 2 participants (patients 2 and 4) was apparent
on full-field perimetry, but was not evident on micro-
perimetry. Given the slow rate of progressive degeneration
at this advanced stage of disease, any protection against
further deterioration may be evident only after a more
extended period of observation. 4. McBain VA, Townend J, Lois N. Progression of retinal
pigment epithelial atrophy in Stargardt disease. Am J Oph-
thalmol. 2012;154:146e154. 5. Sparrow JR, Hicks D, Hamel CP. The retinal pigment
epithelium in health and disease. Curr Mol Med. 2010;10:
802e823. 6. Lund RD, Wang S, Klimanskaya I, et al. Human embryonic
stem cell-derived cells rescue visual function in dystrophic
RCS rats. Cloning Stem Cells. 2006;8:189e199. 7. Lu B, Malcuit C, Wang S, et al. Long-term safety and function of
RPE from human embryonic stem cells in preclinical models of
macular degeneration. Stem Cells. 2009;27:2126e2135. 8. Schwartz SD, Hubschman JP, Heilwell G, et al. Embryonic
stem cell trials for macular degeneration: a preliminary report. Lancet. 2012;379:713e720. We also sought to determine whether the function of
photoreceptor cells that are surviving but compromised
may improve after the provision of healthy supporting RPE
cells. In a previous trial of up to 150 000 hESC-derived
RPE cells in STGD1, variable improvements in VA were
measured in 3 of 8 eyes without secondary cataract,
although the mean difference in VA between the recipient
and contralateral eyes was not statistically significant.9 In a
subsequent
trial
in
2
participants
with
STGD1,
improvements in VA were reported in both the recipient
and
contralateral
eyes.10
In
the
present
trial,
we
administered up to 200 000 hESC-derived RPE cells and
measured retinal structure and function in detail using
microperimetry and SD OCT. Our findings demonstrate the
value of detailed analysis of spatial correlation of retinal
structure and function in determining with appropriate
sensitivity the impact of cell transplantation. We found no
significant benefit to retinal function at 12 months in the 12
participants. 1. Allikmets R, Singh N, Sun H, et al. A photoreceptor cell-
specific ATP-binding transporter gene (ABCR) is mutated in
recessive Stargardt macular dystrophy. Nat Genet. 1997;15:
236e246. Ophthalmology
Volume -, Number -, Month 2018 Although RPE cell transplantation theoreti-
cally may improve the function of overlying photoreceptor
cells, the potential for improvement is limited in this
particular population of participants by the severity of
established retinal degeneration. 9. Schwartz SD, Regillo CD, Lam BL, et al. Human embryonic
stem cell-derived retinal pigment epithelium in patients with
age-related macular degeneration and Stargardt’s macular
dystrophy: follow-up of two open-label phase 1/2 studies. Lancet. 2015;385:509e516. 10. Song WK, Park KM, Kim HJ, et al. Treatment of macular
degeneration
using
embryonic
stem
cell-derived
retinal
pigment epithelium: preliminary results in Asian patients. Stem
Cell Reports. 2015;4:860e872. 11. Fujinami K, Lois N, Davidson AE, et al. A longitudinal study
of Stargardt disease: clinical and electrophysiologic assess-
ment, progression, and genotype correlations. Am J Oph-
thalmol. 2013;155:1075e1088.e13. 12. Bland JM, Altman DG. Measurement error. BMJ. 1996;312:
1654. 13. Weleber RG, Smith TB, Peters D, et al. VFMA: topographic
analysis of sensitivity data from full-field static perimetry. Transl Vis Sci Technol. 2015;4:14. 14. Tanna P, Strauss RW, Fujinami K, Michaelides M. Stargardt
disease: clinical features, molecular genetics, animal models
and therapeutic options. Br J Ophthalmol. 2017;101:25e30. 15. McKay DL. Congenital hypertrophy of the retinal pigment
epithelium and familial adenomatous polyposis. Aust N Z J
Ophthalmol. 1993;21:3e6. The evidence of safety broadly supports the rationale for
further studies to explore the impact of intervention at an
earlier stage of degeneration when surviving photoreceptors
cells may stand to benefit with improved function and sur-
vival. However, instances of focally reduced sensitivity and
thinning in hyperpigmented retina at higher doses of hESC-
derived RPE cells suggest the potential for harm and indi-
cate that intervention at earlier stages of degeneration should
be approached with caution. 16. Carr AJ, Vugler AA, Hikita ST, et al. Protective effects of
human iPS-derived retinal pigment epithelium cell trans-
plantation in the retinal dystrophic rat. PloS One. 2009;4:
e8152. 17. Engelhardt M, Tosha C, Lopes VS, et al. Functional and
morphological analysis of the subretinal injection of retinal
pigment epithelium cells. Vis Neurosci. 2012;29:83e93. 18. Abercrombie M. Contact inhibition: the phenomenon and its
biological implications. Natl Cancer Inst Monogr. 1967;26:
249e277. 19. Frisch SM, Francis H. Disruption of epithelial cell-matrix in-
teractions induces apoptosis. J Cell Biol. 1994;124:619e626. 3. Molday RS, Zhong M, Quazi F. The role of the photoreceptor
ABC transporter ABCA4 in lipid transport and Stargardt
macular degeneration. Biochim Biophys Acta. 2009;1791:
573e583. A reduction in the pigmentation density at the site of
transplantation, evident in 2 participants (at month 6 in
patient 9 and at month 12 in patient 4) may reflect rejection
of pigmented donor hESC-derived RPE cells, although no
associated change in retinal function was apparent. The risk of
immune rejection may be reduced by use of autologous RPE
cells derived from induced pluripotent stem cells, although Reports of vision loss after intravitreal injection of
autologous adipose tissue-derived stem cells highlight the
potential for harm resulting from transplantation of cell
populations that are poorly characterized and controlled.23
In
our
study,
intraocular
administration
of
carefully
obtained hESC-derived RPE cells resulted in the develop-
ment of pigmented foci in the vitreous cavity in 2 partici-
pants and on the surface of the inner retina in 2 participants,
suggesting reflux of donor hESC-derived RPE cells from the
subretinal compartment into the vitreous cavity. Despite the
presence of preretinal or intravitreal pigmentation, no
associated adverse effect was evident. 9 Footnotes and Financial Disclosures Originally received: January 4, 2018. United Kingdom; The Wellcome Trust (grant no.: 099173/Z/12/Z); Moor-
fields Eye Charity, London, United Kingdom; Moorfilelds Eye Hospital
Special Trustees, London, United Kingdom; and RP Fighting Blindness. James Bainbridge is an NIHR Research Professor. The sponsor participated
in the design of the study and approval of the manuscript. Originally received: January 4, 2018. Final revision: April 23, 2018. Final revision: April 23, 2018. Accepted: April 25, 2018. Accepted: April 25, 2018. References 20. Lopez R, Gouras P, Brittis M, Kjeldbye H. Transplantation of
cultured rabbit retinal epithelium to rabbit retina using a
closed-eye method. Invest Ophthalmol Vis Sci. 1987;28:
1131e1137. 1. Allikmets R, Singh N, Sun H, et al. A photoreceptor cell-
specific ATP-binding transporter gene (ABCR) is mutated in
recessive Stargardt macular dystrophy. Nat Genet. 1997;15:
236e246. 21. Grisanti S, Szurman P, Jordan J, et al. Xenotransplantation of
retinal pigment epithelial cells into RCS rats. Jpn J Oph-
thalmol. 2002;46:36e44. 2. Illing M, Molday LL, Molday RS. The 220-kDa rim protein of
retinal rod outer segments is a member of the ABC transporter
superfamily. J Biol Chem. 1997;272:10303e10310. 22. Mandai M, Watanabe A, Kurimoto Y, et al. Autologous
induced stem-cell-derived retinal cells for macular degenera-
tion. N Engl J Med. 2017;376:1038e1046. 3. Molday RS, Zhong M, Quazi F. The role of the photoreceptor
ABC transporter ABCA4 in lipid transport and Stargardt
macular degeneration. Biochim Biophys Acta. 2009;1791:
573e583. 23. Kuriyan AE, Albini TA, Townsend JH, et al. Vision loss after
intravitreal injection of autologous “stem cells” for AMD. N Engl J Med. 2017;376:1047e1053. 10 Available online: ---. hESC ¼ human embryonic stem cell; STGD1 ¼ Stargardt disease;
RPE ¼ retinal pigment epithelial; SD ¼ spectral-domain; VA ¼ visual
acuity. D.H.W.S.: Consultant - Alcon, Bayer. D.H.W.S.: Consultant - Alcon, Bayer. E.A.: Employee - Astellas Institute for Regenerative Medicine. Available online: ---. Available online: ---. 1 NIHR Biomedical Research Centre for Ophthalmology, Moorfields Eye
Hospital and University College London, London, United Kingdom. 1 NIHR Biomedical Research Centre for Ophthalmology, Moorfields Eye
Hospital and University College London, London, United Kingdom. 1 NIHR Biomedical Research Centre for Ophthalmology, Moorfields Eye
Hospital and University College London, London, United Kingdom. HUMAN SUBJECTS: This study included human subjects or tissues. No
animals were used in this study. Study protocol was approved by the
Medicines and Health Products Regulatory Authority, the UK Gene Ther-
apy Advisory Committee, and the Moorfields Eye Hospital Research
Governance Committee. Informed consent was obtained from all human
subjects. This adhered to the tenets of the Declaration of Helsinki. 2 Moorfields Eye Hospital NHS Foundation Trust, London, United
Kingdom. 2 Moorfields Eye Hospital NHS Foundation Trust, London, United
Kingdom. 3 Institute of Ophthalmology, University College London, London, United
Kingdom. 4 Cambridge Research Systems Ltd., Rochester, United Kingdom. 5 University of Edinburgh, Edinburgh, United Kingdom. Conception and design: Mehat, Sundaram, Ripamonti, Weleber, Lee,
Rubin, Dhillon, Steel, Anglade, Lanza, Michaelides, Bainbridge 6 University College Hospital, London, United Kingdom. 6 University College Hospital, London, United Kingdom. 7 Newcastle Eye Centre, Newcastle upon Tyne, United Kingdom. Analysis and interpretation: Mehat, Sundaram, Ripamonti, Robson, Smith,
Borooah, Robinson, Innes, Weleber, Lee, Crossland, Rubin, Dhillon, Steel,
Anglade, Lanza, Ali, Michaelides, Bainbridge 8 Casey Eye Institute, University of Oregon Health & Science University,
Portland, Oregon. 9 Institute of Genetic Medicine, Newcastle University, Newcastle upon
Tyne, United Kingdom. Data collection: Mehat, Sundaram, Ripamonti, Robson, Borooah, Rosen-
thal, Innes, Crossland, Rubin, Dhillon, Steel, Michaelides, Bainbridge 10 Astellas
Institute
for
Regenerative
Medicine,
Marlborough,
Massachusetts. 10 Astellas
Institute
for
Regenerative
Medicine,
Marlborough,
Massachusetts. Obtained funding: not applicable Overall responsibility: Mehat, Sundaram, Ripamonti, Robson, Smith,
Borooah, Robinson, Rosenthal, Weleber, Lee, Crossland, Rubin, Dhillon,
Steel, Anglade, Lanza, Ali, Michaelides, Bainbridge 11 Kellogg Eye Center, University of Michigan, Ann Arbor, Michigan. Kellogg Eye Center, University of Michigan, Ann Arbor, Michigan. Financial Disclosure(s):
The author(s) have made the following disclosure(s): A.N.R.: Consultant -
Abcodia, Myriad Genetics. Financial Disclosure(s): The author(s) have made the following disclosure(s): A.N.R.: Consultant -
Abcodia, Myriad Genetics. y
BCVA ¼ best-corrected visual acuity; FAF ¼ fundus autofluorescence;
hESC ¼ human embryonic stem cell; STGD1 ¼ Stargardt disease;
RPE ¼ retinal pigment epithelial; SD ¼ spectral-domain; VA ¼ visual
acuity. R.G.W.: Scientific Advisory Board - Foundation Fighting Blindness,
AGTC; Patent - Visual Field Modelling and Analysis software. Correspondence: Correspondence: James W.B. Bainbridge, PhD, FRCOphth, Institute of Ophthalmology,
University College London, Bath Street, London EC1V 9EL, United
Kingdom. E-mail: j.bainbridge@ucl.ac.uk. R.P.L.: Patents relevant to the generation and use of RPE cells derived from
pluripotent stem cells. J.W.B.B.: Consultant - Astellas Institute for Regenerative Medicine. Supported by Advanced Cell Technology; Ocata Therapeutics; Astellas
Institute for Regenerative Medicine; NIHR Biomedical Research Centre for
Ophthalmology at Moorfields and University College London, London, 11
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Behavioral repertoire of high‐shore littorinid snails reveals novel adaptations to an extreme environment
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Ecology and evolution
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cc-by
| 9,475
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Received: 9 February 2021 | Revised: 28 March 2021 | Accepted: 31 March 2021 Received: 9 February 2021 | Revised: 28 March 2021 | Accepted: 31 March 2021
DOI: 10.1002/ece3.7578 N AT U R E N O T E S Abstract Abstract
Species that inhabit high-shore environments on rocky shores survive prolonged pe-
riods of emersion and thermal stress. Using two Hong Kong high-shore littorinids
(Echinolittorina malaccana and E. radiata) as models, we examined their behavioral
repertoire to survive these variable and extreme conditions. Environmental tempera-
tures ranged from 4°C in the cool season to 55.5°C in the hot season, with strong
seasonal and daily fluctuations. In the hot season, both species allocated >35% of
their activity budgets to stress-mitigating thermoregulatory behaviors (e.g. stand-
ing, towering) and relatively small proportions to foraging (<20%) and reproduction
(<10%). In the assumedly benign cool season, greater proportions (>70%) of activity
budgets were allocated to stress mitigation behaviors (crevice occupation, aggre-
gation formation). Both species exhibited multifunctional behaviors that optimized
time use during their tidally-constrained activity window in the hot season. Females
mated while foraging when awash by the rising tide, and some males crawled on
top of females prior to ceasing movement to form 'towers', which have both ther-
moregulatory benefits and reduce searching time for mates during subsequent activ-
ity. The function of such behaviors varies in a state-dependent manner, for example,
the function of trail following changes over an activity cycle from mate searching on
rising tides, to stress mitigation on falling tides (aiding aggregation formation), and to
both functions through tower formation just before movement stops. Many of these
behavioral responses are, therefore, multifunctional and can vary according to local
conditions, allowing snails in this family to successfully colonize the extreme high-
shore environment. 3Department of Life and Environmental
Sciences, Bournemouth University, Poole,
UK Terence P. T. Ng1 | Sarah L. Y. Lau1 | Mark S. Davies2
| Richard Stafford3 |
Laurent Seuront4,5,6 | Neil Hutchinson7 | Tommy T. Y. Hui1 | Gray A. Williams1 Terence P. T. Ng1 | Sarah L. Y. Lau1 | Mark S. Davies2
| Richard Stafford3 |
Laurent Seuront4,5,6 | Neil Hutchinson7 | Tommy T. Y. Hui1 | Gray A. Williams1 Terence P. T. Ng1 | Sarah L. Y. Lau1 | Mark S. Davies2
| Richard Stafford3 |
Laurent Seuront4,5,6 | Neil Hutchinson7 | Tommy T. Y. Hui1 | Gray A. Williams1 1The Swire Institute of Marine Science and
School of Biological Sciences, The University
of Hong Kong, Hong Kong SAR, China
2Applied Sciences, University of Sunderland,
Sunderland, UK
3Department of Life and Environmental
Sciences, Bournemouth University, Poole,
UK
4Laboratoire d’Océanologie et de
Géosciences (LOG), UMR 8187, CNRS, Univ. Lille, Univ. Littoral Côte d’Opale, Wimereux,
France
5Department of Marine Resource and
Energy, Tokyo University of Marine Science
and Technology, Minato, Japan
6Department of Zoology and Entomology,
Rhodes University, Grahamstown, South
Africa
7Tropical Futures Institute/TropWATER—
Centre for Tropical Water and Aquatic
Ecosystem Research, James Cook University,
Singapore
Correspondence
Mark S. Davies, Applied Sciences, University
of Sunderland, Sunderland, SR1 3SD, UK. Email: Mark.davies@sunderland.ac.uk
Funding information
French Ministère de l'Enseignement
Supérieur et de la Recherche; Research
Grants Council of the Hong Kong SAR
Government, Grant/Award Number:
17121914 M 1The Swire Institute of Marine Science and
School of Biological Sciences, The University
of Hong Kong, Hong Kong SAR, China
2Applied Sciences, University of Sunderland,
Sunderland, UK 1The Swire Institute of Marine Science and
School of Biological Sciences, The University
of Hong Kong, Hong Kong SAR, China
2Applied Sciences, University of Sunderland,
Sunderland, UK
3Department of Life and Environmental
Sciences, Bournemouth University, Poole,
UK
4Laboratoire d’Océanologie et de
Géosciences (LOG), UMR 8187, CNRS, Univ. Lille, Univ. Littoral Côte d’Opale, Wimereux,
France
5Department of Marine Resource and
Energy, Tokyo University of Marine Science
and Technology, Minato, Japan
6Department of Zoology and Entomology,
Rhodes University, Grahamstown, South
Africa
7Tropical Futures Institute/TropWATER—
Centre for Tropical Water and Aquatic
Ecosystem Research, James Cook University,
Singapore
Correspondence
Mark S. Davies, Applied Sciences, University
of Sunderland, Sunderland, SR1 3SD, UK. Email: Mark.davies@sunderland.ac.uk
Funding information
French Ministère de l'Enseignement
Supérieur et de la Recherche; Research
Grants Council of the Hong Kong SAR
Government, Grant/Award Number:
17121914 M 1 | INTRODUCTION To address this, we investigated
variation in the behavioral repertoire, in terms of activity budget and
utilization of multifunctional behaviors, of two high-shore species,
Echinolittorina malaccana and E. radiata, in Hong Kong. Hong Kong
has a seasonal, monsoonal climate with a benign cool (and dry) win-
ter season and a stressful hot (and wet) summer (Chan et al., 2006;
Kaehler & Williams, 1996; Nagarkar & Williams, 1999). On account of
this strong seasonal shift in environmental conditions, we hypothe-
sized that the activity budget of snails would vary between seasons;
specifically that snails would allocate proportionately more time to
foraging and reproduction in thermally benign conditions (the cool
season), and to stress-mitigating behaviors (e.g. standing; towering;
seeking refuge in crevices; aggregating) in thermally stressful condi-
tions (the hot season). Due to the constrained duration of potential
activity periods, we also assessed how different behaviors are uti-
lized dependent on environmental conditions and considered how
they may potentially contribute to survival in extreme and strongly
fluctuating thermal environments. A suite of behavioral adaptations, however, enable mobile high-
shore species to maintain a wider thermal safety margin, maintaining
their body temperatures below lethal thermal limits, than species
found lower on the shore (Ng et al., 2017). High-shore littorinids,
for instance, have exceptional thermal tolerances (Liao et al., 2017;
McMahon, 2001; Ng et al., 2017); can depress metabolism during
severe heat stress (Marshall & McQuaid, 2011; Marshall, McQuaid,
et al., 2010); and also adopt various stress-mitigating, thermoreg-
ulatory behaviors that enhance their ability to withstand extreme
thermal environments (Garrity, 1984; Hayford et al., 2015; Marshall
& Chua, 2012; Seuront & Ng, 2016). Marshall et al. (2015), in partic-
ular, noted that these snails have a potentially narrow thermal safety
margin but behavioral thermoregulation can provide considerable
buffering capacity. Stress-mitigating behaviors play a key role in the survival of
mobile intertidal species when thermal and desiccation stresses
become severe during emersion periods (Garrity, 1984; Harper &
Williams, 2001; Hayford et al., 2015; Reid & Harley, 2021; Williams
& Morritt, 1995). Most species are adapted to time their periods of
activity and inactivity to minimize exposure to thermal stress. In the
tropics this usually involves moving to feed and reproduce while
awash by the tide and then adopting various stress-mitigating behav-
iors such as sheltering in cool refuges while emersed (Garrity, 1984;
Hutchinson & Williams, 2001; Williams & Little, 2007; Williams &
Morritt, 1995). 1 | INTRODUCTION Snails in the family Littorinidae, being abundant and important
herbivores in high-shore environments worldwide (reviewed by
McMahon, 1990, 2001; McQuaid, 1996; Reid, 1989), are excellent
models to investigate the strategic partitioning of behaviors that
allow successful utilization of these extreme habitats. These snails
exhibit various behaviors common to many mobile intertidal ecto-
therms such as trail following, refuge selection, aggregation, and
as they undergo internal fertilization, mating and fighting for mates
(see Cartwright & Williams, 2012; Ng et al., 2013, 2016; Stafford
et al., 2007). They also perform shell-posturing behaviors, for ex-
ample standing and towering (Marshall & Chua, 2012; Marshall,
Mustafa, et al., 2010; Seuront & Ng, 2016). Some of these behaviors
are known to have multiple functions; trail following, for example,
is associated with foraging, mating, energy saving, and aggregation
formation (reviewed by Ng et al., 2013). 1 Physiological tolerance plays a critical role in the distribution lim-
its of species in the intertidal zone. Porcelain crabs living high on
the shore, for example, have greater upper thermal tolerances than
lower shore species (Stickle et al., 2017; Stillman, 2002; Stillman &
Somero, 2000). Marine species living at the upper levels of the inter-
tidal zone ('high-shore species' hereafter) often persist in what are
essentially terrestrial conditions for days or even weeks (Marshall &
McQuaid, 2011; Marshall, McQuaid, et al., 2010; McMahon, 2001;
McQuaid, 1996; Ng et al., 2017). On tropical rocky shores these
high-shore species often experience environmental (rock) tempera-
tures >55°C in the hot season (Marshall et al., 2013; Williams &
Morritt, 1995) and are remarkably tolerant to heat stress (e.g. Lee &
Lim, 2009; Liao et al., 2017; Marshall et al., 2011; McMahon, 1990). High-shore species are also assumed to live close to their upper ther-
mal limits (Stillman, 2002; Stillman & Somero, 2000) and seem to
have evolved high heat tolerance at the expense of their physiologi-
cal and biochemical acclimation capacities, making them vulnerable
to increasing temperatures (Somero, 2010; Stillman, 2003). Most previous studies (e.g. Cartwright & Williams, 2012; Ng
et al., 2013, 2016; Seuront & Ng, 2016; Stafford et al., 2007) have,
however, only investigated the importance of a single function at a
given time and have not considered how these snails can vary their
behavioral repertoires to optimize the partitions between energy
gain, reproduction, and amelioration of thermal stress in the ex-
treme conditions of the high shore. 1 | INTRODUCTION The time individuals allocate to stress mitigation is,
therefore, strongly influenced by environmental variation which
can impose a limit on the time allocated to other key activities (e.g. foraging and reproduction), with subsequent consequences for fit-
ness (Dunham et al., 1989; Frid & Dill, 2002). The ability to maxi-
mize fitness through strategic partitioning of different activities can
be crucial in determining species’ life-history traits and hence suc-
cess in a given environment (Dunham et al., 1989; Frid & Dill, 2002;
Gunderson & Leal, 2015). K E Y W O R D S K E Y W O R D S
activity budget, Echinolittorina malaccana, Echinolittorina radiata, intertidal, snail, thermal
stress, thermoregulation, trail following 7114 |
Ecology and Evolution. 2021;11:7114–7124. www.ecolevol.org
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium,
provided the original work is properly cited. © 2021 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium,
provided the original work is properly cited. © 2021 Th
A th
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Ltd Ecology and Evolution. 2021;11:7114–7124. Ecology and Evolution. 2021;11:7114–7124. NG et al. 7115 NG et al. individuals m-2. At each site three waterproofed (embedded in
Scotchcast model 2131 resin (3M, USA) within the cap of a Falcon
tube, following Marshall et al., 2013) loggers were fixed to the rock
using epoxy resin at 2.2–2.3 m above Chart Datum (CD) in the hot
season (June to August 2015) and at 2.6–2.7 m above CD in the cool
season (January to March 2016). As these loggers were separated
from the rock by a thin layer of epoxy, they provided an index of the
rock temperature experienced on the shore. (June to October, see Kaehler & Williams, 1996), the vertical ranges
of the species largely overlap, forming a high-density band with E. malaccana slightly higher on the shore than E. radiata and exhibiting
a correspondingly higher thermal tolerance (LT50 in air during sum-
mer = 56.5 and 55.5°C, respectively, Li, 2012). In contrast, in the
cool season (December to April), when temperatures may lead to
reduced speed but are not cold enough to limit activity of the snails
(SLY Lau, unpublished data), there is less overlap, and although both
species migrate up-shore, E. malaccana is found consistently higher
on the shore than E. radiata (Mak, 1996). 2.1 | Study site The behavioral patterns of the littorind snails Echinolittorina malac-
cana and E. radiata were investigated between 2014 and 2016 on
semi-exposed rocky shores in the Cape d’Aguilar Marine Reserve,
Hong Kong (22°12′27″N, 114°15′36″E), at four (data pooled across
sites to ensure adequate sample sizes) sites (each 6–20 m long; sep-
arated by barriers such as sand or boulders, or located more than
10 m apart) where these snails co-occur and are abundant in the
high shore (Mak, 1996; Mak & Williams, 1999). In the hot season 7116 NG et al. NG et al. 2.2 | Thermal environment Hourly surveys lasting 24 hr were conducted during spring tides
to investigate the proportion of snails exhibiting various behav-
iors (Table 1; Figure 1) associated with different fitness-associated
activities (i.e. foraging, reproduction, and stress mitigation) on
days with typical thermal patterns in the hot (August 2015, hot
and without rain with average daily maximum rock tempera-
tures >40°C) and cool (February 2016, cool and without rain with
average daily maximum rock temperatures <30°C) seasons at one
site. There was no within-season replication of these surveys, During Hong Kong's hot season, spring low tides occur in the after-
noon, when solar irradiation is high; whereas in the cool season they
occur in the early morning (Kaehler & Williams, 1996). To measure
the thermal environment, temperature loggers (Thermochron iBut-
ton DS 1922L, Maxim Integrated, USA) sampling at hourly intervals
were deployed in the resting zone (areas that the snails occupied dur-
ing emersion) of the two species at two sites with abundances >400 Fitness-associated
activities
Behaviors
Description
Reproduction
Mating
An individual (male) mounts the other individual's
(female's) shell in an anticlockwise manner,
eventually reaching the right-hand side where the
male copulates with the female by inserting its
penis into the female's mantle cavity (Figure 1a)a
Fighting
When two individuals (males) simultaneously
mount the same individual (female) and the males
push against each other, or when one male (the
defender) is copulating with a female and another
(the challenger) encounters the pair and attempts to
push away the defenderb
Foraging
Moving
In <5 s observation, snails crawled
Turning
In <5 s observation, snails rotated in-place without
any distance traveled
Stress mitigation
In crevices
Snails residing in pits and holes in rock (Figure 1c)
Towering
Snails climbing and settling on others to form a stack
(Figure 1d)c
Standing
A snail whose shell is attached to the substratum
via a mucus holdfast, so that the body is lifted
from the substratum with the aperture positioned
perpendicular to the substratum (Figure 1e)d
Aggregating
Three or more snails in direct contact with each
other (Figure 1f)e
Inactive
Stationary
In >5 s observation, snails showing no motion
or other activity, and not exhibiting any stress
mitigation behaviors
aGibson (1965), Saur (1990), Ng and Williams (2014). bGibson (1965), Ng et al. (2016). cMarshall, Mustafa, et al. (2010)), Seuront and Ng (2016). dLim (2008), Marshall and Chua (2012), Seuront and Ng (2016). eChapman (1995), Stafford and Davies (2004). 2.2 | Thermal environment In
cases where animals were trail following in a chain of three or more,
only the two leading animals were sampled. Snails were transferred
to the laboratory and sexed, based on the presence of a penis. We
considered it extremely unlikely that we had mistaken female snails
for immature males because: juveniles recruit in the cool season and
usually reach sexual maturity (with shell length ~>3 mm) before the
hot season (Mak, 1996); and mating males of E. malaccana have been
recorded at 3.2 mm and of E. radiata at 2.5 mm (Ng et al., 2019), and
all the females we identified as such in this study were >4 mm, ex-
cept one E. malaccana at 2.9 mm. Sampling was not conducted at
high tide, because animals were either stationary or moving slowly
and trail following was not apparent. Repeated sampling on each
shore on both rising and falling tides might have affected snail be-
havior, but as densities were so high we were able to sample snails
without affecting other individuals and so considered this potential
effect to be negligible. which limits interpretation of variation within seasons owing to
small-scale weather patterns, but does allow assessment of larger-
scale differences between seasons. Each hour ten 25 × 25 cm
quadrats were haphazardly placed in the zone where the two
species occurred and the number of individuals exhibiting differ-
ent behaviors recorded (Table 1; Figure 1). Behaviors scored and
their assumed fitness-associated activities were based on previ-
ous studies of species in the same family (Littorinidae, Table 1). Individuals performing multiple behaviors simultaneously related
to the same fitness-associated activity (e.g. aggregation and shel-
tering in crevices, Figure 1c; or standing and towering, Figure 1d)
were only scored once to avoid bias toward any particular activity. Since it was not possible to observe when individuals were grazing
on the shore biofilm, we ascribed any movement to foraging (after
Hartnoll & Wright, 1977; Little et al., 1988; since in general snails
rasp the rock surface when they move). Due to both the difficulty
in simultaneously scoring multiple behaviors when snails were
awash in the hot season and in identifying the multifunctional
nature of trail-following behavior (Ng et al., 2013), we did not re-
cord trail-following behavior in awash quadrats, but conducted a
separate study on this behavior (see below). 2.2 | Thermal environment TA B LE 1 Descriptions of various
fitness-associated activities and behaviors
of Echinolittorina malaccana and E. radiata | 7117
(b)
(c)
(e)
(f) | 7117
(b)
(c)
(e)
(f) NG et al. 7117 | 7117
NG et al. FI G U R E 1 Behavioral repertoire
of Echinolittorina malaccana (EM) and
E. radiata (ER): a mating pair of ER (a);
trail-following behavior in ER (b); both
species sheltering in a crevice (c); towering
behavior in ER (inset: thermal image
showing the cooling effect of the tower)
(d); standing behavior in EM (e); and an EM
aggregation that retains water (f)
(a)
(b)
(c)
(e)
(f)
(d) rtoire
M) and
ER (a);
b); both
(c); towering
mage
he tower)
); and an EM
(f)
(a)
(b)
(c)
(e)
(f)
(d) FI G U R E 1 Behavioral repertoire
of Echinolittorina malaccana (EM) and
E. radiata (ER): a mating pair of ER (a);
trail-following behavior in ER (b); both
species sheltering in a crevice (c); towering
behavior in ER (inset: thermal image
showing the cooling effect of the tower)
(d); standing behavior in EM (e); and an EM
aggregation that retains water (f)
(a)
(b
(c
(e
(d) (c) (d) (e) another (a marker) individual for >5 s (Davies & Beckwith, 1999). In
the hot season this behavior in most cases involved two individu-
als during rising tides and multiple individuals during falling tides. Snails involved in trail following were sampled at all four sites to test
whether the function of the behavior changed from finding a mate
on the rising tide (i.e. males following females; see Ng et al., 2011,
2013, for more details), to finding and forming an aggregation on the
falling tide (i.e. trail following regardless of sex and species; Stafford
et al., 2007). At each site in the hot season of 2014 (July and August),
a minimum of 30 trail-following pairs of each species along differ-
ent trails were haphazardly collected on both the rising (tidal height:
~1.7 to 2.0 m +CD) and the falling tide (~2.0 to 1.7 m +CD). For
E. malaccana, the pairs were collected during one rising tide and one
falling tide within the same day, whereas those for E. radiata were
collected during one rising tide and one falling tide within 2 days. 2.2 | Thermal environment In the hot season, the
height of the high-density band of snails moving while awash was
measured hourly, recording the height above CD at the middle of
the band using a cross-staff (see Little et al., 2009). FI G U R E 1 Behavioral repertoire
of Echinolittorina malaccana (EM) and
E. radiata (ER): a mating pair of ER (a);
trail-following behavior in ER (b); both
species sheltering in a crevice (c); towering
behavior in ER (inset: thermal image
showing the cooling effect of the tower)
(d); standing behavior in EM (e); and an EM
aggregation that retains water (f) 2.4 | Multifunctional behavior: trail following for
mating and/or for stress mitigation Trail-following behavior occurred when snails were awash by the
tide and involved an individual (a tracker) moving along the trail of 7118 |
NG et al. FI G U R E 2 Examples of seasonal fluctuations (79 days in the hot, summer season and 73 days in the cool, winter season) in temperature
(mean of three iButton data loggers at two sites, S1 and S2) in Cape d' Aguilar Marine Reserve, Hong Kong. The Arrhenius Breakpoint
Temperature for heart rate (thin line) and LT50 (bold line) values (averaged of the two study species, after Li, 2012) are indicated by the
horizontal lines 7118 | 7118 | NG et al. FI G U R E 2 Examples of seasonal fluctuations (79 days in the hot, summer season and 73 days in the cool, winter season) in temperature
(mean of three iButton data loggers at two sites, S1 and S2) in Cape d' Aguilar Marine Reserve, Hong Kong. The Arrhenius Breakpoint
Temperature for heart rate (thin line) and LT50 (bold line) values (averaged of the two study species, after Li, 2012) are indicated by the
horizontal lines FI G U R E 3 Tidal height (a and c) and hot season (summer) vertical movement (a) of snails across a 24-hr tidal cycle with the percentage
of each species performing different behaviors (see Table 1) each hour (b and d, note time axes are different and midnight is marked by
asterisks) in the hot (summer) and cool (winter) seasons. Note the total number of behaviors can exceed the total number of snails if snails
perform multiple behaviors (hence maximum % individuals can exceed 100%) FI G U R E 3 Tidal height (a and c) and hot season (summer) vertical movement (a) of snails across a 24-hr tidal cycle with the percentage
of each species performing different behaviors (see Table 1) each hour (b and d, note time axes are different and midnight is marked by
asterisks) in the hot (summer) and cool (winter) seasons. 2.5 | Multifunctional behavior: towering for
mating and/or for stress mitigation (standing, towering, aggregation, and crevice occupation) occurred
mainly during the falling tide and emersion periods (Figure 3). When
the tide was rising in the hot season, individuals engaged in foraging
and reproductive activities (i.e. mating and fighting, Figure 3). Fewer
E. radiata showed reproductive activities than E. malaccana and E. radiata also stopped these activities earlier than E. malaccana during
the awash phase, even though both species allocated a similar pro-
portion of their activity budget to foraging (17%–19%; Figure 3). Sampling was conducted during the hot season of 2015 (June) to
investigate whether towering behavior functioned as a strategy to
facilitate mating in addition to aiding thermoregulation (Marshall,
Mustafa, et al., 2010; Seuront & Ng, 2016). Over 5 days when tower-
ing occurred, >40 conspecific towers of each species were collected
from the four sites during daytime both during emersion before
activity began on the rising tide (E. malaccana, N = 41; E. radiata,
N = 40) and when activity ceased on emersion on the falling tide
(E. malaccana, N = 64; E. radiata, N = 43). The position (top or bot-
tom) of individuals in each tower was noted prior to collection, and
each individual was sexed on return to the laboratory. Data were
pooled across sites and days. Only conspecific towers that consisted
of two individuals were sampled because these towers were by far
the most abundant on the shore (Seuront & Ng, 2016; Figure 1). For
sampling conducted before the rising tide, individuals were collected
after they were wetted by the tide. As towers were wetted, the top-
most individual dismounted the other and it was noted whether this
individual then initiated mating (defined as when it had maintained
the mating position for >20 s) or moved away from the other animal. Individuals often performed more than one behavior associated with
more than one fitness-associated activity (resulting in sums of >100%
activity for individuals at certain times, especially in the hot season;
Figure 3). Such cases included (a) females mating (reproduction) and
moving/turning (foraging) simultaneously (these females moved notice-
ably more slowly than unladen females), (b) individuals mating (reproduc-
tion) while forming aggregations (stress mitigation), and (c) individuals
aggregating (stress mitigation) while turning (foraging). 2.5 | Multifunctional behavior: towering for
mating and/or for stress mitigation Overall, our hypothesis that snails would allocate proportion-
ately more time to foraging and reproduction in thermally benign
conditions (the cool season), and to stress-mitigating behaviors in
thermally stressful conditions (the hot season) was not supported. While snail behavior was consistent with individuals remaining
largely inactive in crevices and aggregations in the cool season, in
the hot season the picture was more complex, whereby some be-
haviors, specifically trail following and towering, appeared to serve
more than one function. To test whether the towers were dominated by a particular sex
combination (male on top of male, male on top of female, etc.), chi-
squared tests were performed to test the observed frequencies of
the four sex combinations against the expected frequencies, which
were calculated assuming random pairing (expected numbers calcu-
lated as described above). 3.3 | Multifunctional behaviors During the rising tide, trail-following pairs were not randomly
formed, with the most dominant marker–tracker combination being
males following females (54% and 51% in E. malaccana and E. radiata,
respectively; Table 2; Figure 4). During the falling tide, however,
the sex of trail-following pairs was randomly distributed (Table 2;
Figure 4). 3.1 | Thermal environment Ambient temperatures ranged from 24.1 to 55.5°C in the hot season
with ~25% and 42% of day temperatures exceeding 45°C in Sites 1 and
2, respectively, and from 4.0 to 49.1°C in the cool season with ~8% and
3% of days temperatures exceeding 40°C in Sites 1 and 2 (Figure 2). Towers of both species collected before the rising tide and after
the falling tide were also not randomly formed, consisting mainly
of a male on top of a female (overall ~60%, Table 3). This phenom-
enon was more pronounced in E. radiata and before the rising tide
(Table 3). All pairs of E. malaccana, and 55.6% of E. radiata, in con-
specific towering pairs that consisted of a male on top and a female
at the bottom, mated after becoming awash on the rising tide. No
mating was observed when the individual on the top was a female or
when the bottom individual was a male. Both trail-following behavior
and towering behavior, therefore, appear to function both in repro-
duction and in stress mitigation in a tidal state-dependent manner. 3.2 | Activity budget Both species spent most of their time 'inactive' (sensu Table 1) or ex-
hibiting stress mitigation behaviors, especially in the cool, assumedly
benign, season, where these behaviors accounted for >90% of activ-
ity budgets (Figure 3). In the cool season, both species stayed close
to the corresponding daily high water level, consistently aggregating
in crevices, with E. radiata spending more time inactive on bare rock
than E. malaccana (Figure 3). No shell-posturing behaviors (standing
or towering) or reproductive activities and almost no foraging (<1%
activity budget) were recorded for either species (Figure 3). 2.4 | Multifunctional behavior: trail following for
mating and/or for stress mitigation Note the total number of behaviors can exceed the total number of snails if snails
perform multiple behaviors (hence maximum % individuals can exceed 100%) FI G U R E 3 Tidal height (a and c) and hot season (summer) vertical movement (a) of snails across a 24-hr tidal cycle with the percentage
of each species performing different behaviors (see Table 1) each hour (b and d, note time axes are different and midnight is marked by
asterisks) in the hot (summer) and cool (winter) seasons. Note the total number of behaviors can exceed the total number of snails if snails
perform multiple behaviors (hence maximum % individuals can exceed 100%) being either a marker or follower, the expected numbers of FM, MM,
MF, and FF (where XY denotes Y follows X; e.g. MF = male followed
by a female) are n*0.5*(m/(n + m − 1)), m*0.5*((m − 1)/(n + m − 1)),
m*0.5*(n/(n + m − 1)), and n*0.5*((n − 1)/(n + m − 1)), respectively. Data from all four sites were pooled for each species on rising
(E. malaccana: N = 126 pairs. E. radiata: N = 120 pairs) and falling
tides (E. malaccana: N = 120 pairs. E. radiata: N = 120 pairs). To test whether trail-following pairs were dominated by a par-
ticular marker–tracker combination (male followed by a male, female
followed by a male, etc.) on rising and falling tides, expected numbers
of these different combinations were calculated assuming random
pairing and tested against the observed numbers using a chi-squared
test. Specifically, if there are m males and n females and, based on a
random pairing scenario where each individual has a 50% chance of 7119 NG et al. NG et al. TA B LE 3 Number of towers with each sex at the different TA B LE 3 Number of towers with each sex at the different
positions (top or bottom) sampled before the rising tide and after
the falling tide in Echinolittorina malaccana (N = 41 before rising tide
and N = 64 after falling tide) and E. radiata (N = 40 before rising tide
and N = 43 after falling tide) TA B LE 2 Number of each different male–female marker–tracker
combination in trail-following pairs sampled on rising tides and
falling tides for Echinolittorina malaccana (N = 126 pairs on rising
tides and N = 120 pairs on falling tides) and E. radiata (N = 120 pairs
on rising tides and N = 120 pairs on falling tides) TA B LE 3 Number of towers with each sex at the different
positions (top or bottom) sampled before the rising tide and after
the falling tide in Echinolittorina malaccana (N = 41 before rising tide
and N = 64 after falling tide) and E. radiata (N = 40 before rising tide
and N = 43 after falling tide) TA B LE 3 Number of towers with each sex at the different
positions (top or bottom) sampled before the rising tide and after
the falling tide in Echinolittorina malaccana (N = 41 before rising tide
and N = 64 after falling tide) and E. radiata (N = 40 before rising tide
and N = 43 after falling tide) B LE 3 Number of towers with each sex at the differe Note: Chi-squared tests (df = 3 in all cases) report observed frequencies
against expected frequencies where trail-following pairs form at
random. Note: Chi-squared tests (df = 3 in all cases) report observed frequencies
against expected frequencies where trail-following pairs form at
random. Note: Chi-squared tests (df = 3 in all cases) report observed frequencies
against expected frequencies where towers are formed by random
pairing. Note: Chi-squared tests (df = 3 in all cases) report observed frequencies
against expected frequencies where towers are formed by random
pairing. seasonal differences in both phenology of the species, which are re-
productively active in summer (Mak, 1996), and physiological accli-
matization, with species showing slightly greater thermal tolerance
(Li, 2012) in summer than winter. 4 | DISCUSSION Although there was no assessment of within season variation in
this study, there were strong seasonal and tidal variations in the be-
havioral budgets of the two high-shore littorinids. There are clear In the hot season, individuals of both species moved up and down
the shore with the tide. Inactivity and stress mitigation behaviors 7120 NG et al. 7120 |
TA B LE 2 Number of each different male–female marker–tracker
combination in trail-following pairs sampled on rising tides and
falling tides for Echinolittorina malaccana (N = 126 pairs on rising
tides and N = 120 pairs on falling tides) and E. radiata (N = 120 pairs
on rising tides and N = 120 pairs on falling tides)
Marker
Tracker
Observed
Expected
E. malaccana
On rising tides
Female
Male
68
31.6
Male
Male
26
34.4
Female
Female
20
28.4
Male
Female
12
31.6
χ2 = 58.79, p < .001
On falling tides
Female
Male
38
30.1
Male
Male
29
29.4
Female
Female
30
30.4
Male
Female
23
30.1
χ2 = 3.75, n.s. E. radiata
On rising tides
Female
Male
61
29.7
Male
Male
15
22.8
Female
Female
30
37.8
Male
Female
14
29.7
χ2 = 45.72, p < .001
On falling tides
Female
Male
32
30.0
Male
Male
25
26.0
Male
Female
30
30.0
χ2 = 0.20, n.s. Note: Chi-squared tests (df = 3 in all cases) report observed frequencies
against expected frequencies where trail-following pairs form at
random. TA B LE 3 Number of towers with each sex at the different
positions (top or bottom) sampled before the rising tide and after
the falling tide in Echinolittorina malaccana (N = 41 before rising tide
and N = 64 after falling tide) and E. radiata (N = 40 before rising tide
and N = 43 after falling tide)
Top
Bottom
Observed
Expected
E. malaccana
Before rising tide
Male
Female
31
10.3
Male
Male
2
8.7
Female
Male
3
10.3
Female
Female
5
11.7
χ2 = 55.58, p < .001
After falling tide
Male
Female
23
16.1
Male
Male
17
15.9
Female
Male
7
16.1
Female
Female
17
15.9
χ2 = 8.25, p < .05
E. radiata
Before rising tide
Male
Female
36
10.1
Male
Male
2
9.9
Female
Male
0
10.1
Female
Female
2
9.9
χ2 = 88.79, p < .001
After falling tide
Male
Female
25
10.7
Male
Male
10
13.8
Female
Male
4
10.7
Female
Female
4
7.8
χ2 = 26.37,
p < .001
Note: Chi-squared tests (df = 3 in all cases) report observed frequencies
against expected frequencies where towers are formed by random
pairing. for activity at this shore level, species should partition behav-
iors efficiently in time to maximize their fitness (Hughes, 1988). Nevertheless, trade-offs may occur among decisions to perform
various behaviors. Trade-offs between stress mitigation and other
fitness-associated activities may, for example, have important
sublethal consequences, as they will alter energy acquisition and
expenditure (Gunderson & Leal, 2015, 2016). The reproductive activities of both species were also temporally
partitioned: in the hot season mating occurred when snails were
awash during the rising tide and stopped when the tide started to
fall. E. malaccana allocated more time to reproduction (they stopped
mating and moved down the shore slightly later) than E. radiata
(Stafford et al., 2012), which may reflect their higher thermal toler-
ance (Li, 2012), reducing the requirements for stress mitigation. The
overall available window for mating is, therefore, severely restricted
to rising tides that are high enough to reach the snails during the
hot season (Ng et al., 2016). Stress mitigation activity (trail following
for aggregation formation) as the tide falls, it would seem, overrides
reproductive activity. One of the most parsimonious strategies in stressful environ-
ments is to utilize traits or behaviors that can serve more than one
function, and therefore minimize time and/or energetic trade-offs
between functions (Hui et al., 2019; Sherbrooke et al., 2007; Song
et al., 2013; Yao et al., 2010). Female littorinids in this study, for ex-
ample, were able to maintain feeding while mating and so avoided
a possible trade-off in times spent between these behaviors during
their constrained activity period (although there may be some cost
as females that carried males appeared to move at a slower speed
than unladen females). Similarly, towering behavior blurs the choice
between finding a thermal refuge and finding a mate, achieving both
and reducing the need for any trade-off as on hot days the topmost
snail gains a reproductive advantage and reduces its thermal stress. Indeed, since males are the active partner seeking females to mate
(Figure 4; Ng et al., 2013), there may be an advantage to females and
little apparent cost in allowing themselves to be at the base of towers
(for example, we observed no dislodgement of the towers by waves). Towering has previously been identified as a thermoregulatory be-
havior in littorinid snails (Marshall, Mustafa, et al., 2010; Seuront &
Ng, 2016), but also facilitates reproduction as shown in this study
(see also Marshall & Ng, 2013). Males on top of the tower, for exam-
ple, benefit from being cooler (by up to 10.3°C, Seuront & Ng, 2016)
during emersion while reducing the time required to search for a
mate when wetted by the rising tide. Clearly there is an evolutionary
advantage to adopting a trait that can serve multiple functions with
minimal costs (Friedman et al., 2019; Liao et al., 2019; Ng et al., 2017). A significant proportion (E. malaccana, ~33% in summer and ~9%
in winter; E. radiata, ~44% in summer and ~28% in winter) of the
activity budget of both species was categorized as 'inactive' (sta-
tionary). While true in the sense of not moving, such 'inactivity' in
snails can be regarded as a stress mitigation, especially during emer-
sion. During emersion, these snails generally retract their foot and
seal their shells with mucus, often attaching themselves to the rock
with a mucus thread to reduce water loss and heat transfer from the
rock surface (Miller & Denny, 2011; Vermeij, 1971), and may enter a
phase of metabolic depression, which can function in energy saving
(Marshall & McQuaid, 2011). This 'inactive' behavior, therefore, may
in fact be associated with an active physiological mechanism to min-
imize energy expenditure. In terms of the prevalence of stress-mitigating behaviors, con-
trary to our preconceptions, the cool season appeared more 'stress-
ful' for these snails than the hot season, as they spent most of the
cool season sheltering in specific microhabitats (aggregations and
crevices). We considered this 'sheltering' rather than a temperature-
based lack of function, since although these snails have reduced
metabolism (20%–60% of maximum) in the cool season, their
locomotory function is not greatly diminished (SLY Lau, unpub-
lished data). Further, both food availability (Mak, 1996; Nagarkar
& Williams, 1999) and the opportunity to feed (i.e. their potential
window of activity, SLY Lau, unpublished data) are reduced in the
cool season, due to the snails’ higher location on the shore. In this
scenario, it is likely that energy maximization would be paramount,
and consequently, moving and foraging would be expected, unless
prevented by other factors. During the hot season, snails of
both species performed 'multifunctional' behaviors, such as trail fol-
lowing and towering behaviors (see below), to optimize their use of
time during the constrained activity window they experience living
on the high shore. During the cool season however, there was little
movement or foraging and snails of both species mainly aggregated
in crevices. A high proportion (E. malaccana: ~67% in summer and ~91%
in winter; E. radiata: ~56% in summer and ~72% in winter) of the
activity budgets of both species was allocated to stress mitiga-
tion, reproduction, and foraging, with the remainder classified as
inactive (Table 1). The allocations of these behaviors are context-
dependent, varying between season and with the tidal cycle,
presumably as strategies to accommodate the dynamic and chal-
lenging high-shore environment. Given the limited time window FI G U R E 4 Percentage trail-following
pairs of the four marker–tracker
combinations observed on rising and
falling tides in E. malaccana and E. radiata
pooled from four sites. FM, female
followed by male; MM, male followed by
male; MF, male followed by female; FF,
female followed by female FI G U R E 4 Percentage trail-following
pairs of the four marker–tracker
combinations observed on rising and
falling tides in E. malaccana and E. radiata
pooled from four sites. FM, female
followed by male; MM, male followed by
male; MF, male followed by female; FF,
female followed by female NG et al. 7121 ACKNOWLEDGMENTS The summer helpers and postgraduates from the Hard Rock Ecology
Laboratory at SWIMS are thanked for their assistance in fieldwork. This work was supported by the Research Grants Council of the Hong
Kong SAR Government (grant number: 17121914 M) and is a contri-
bution to the CPER research project CLIMIBIO. We thank the French
Ministère de l'Enseignement Supérieur et de la Recherche, the Hauts
de France Region, and the European Fund for Regional Economic
Development for their financial support. We also thank two anony-
mous referees for suggestions that improved the manuscript. Davies, M. S., & Blackwell, J. (2007). Energy saving through trail
following in a marine snail. Proceedings of the Royal Society B:
Biological Sciences, 274, 1233–1236. https://doi.org/10.1098/
rspb.2007.0046 Dunham, A. E., Grant, B. W., & Overall, K. L. (1989). Interfaces between
biophysical and physiological ecology and the population ecology of
terrestrial vertebrate ectotherms. Physiological Zoology, 62, 335–355. https://doi.org/10.1086/physzool.62.2.30156174 Frid, A., & Dill, L. M. (2002). Human-caused disturbance stimuli as a
form of predation risk. Conservation Ecology, 6(1), 11. https://doi. org/10.5751/ES-00404-060111 Friedman, N. R., Miller, E. T., Ball, J. R., Kasuga, H., Remeš, V., & Economo,
E. P. (2019). Evolution of a multifunctional trait: Shared effects of
foraging ecology and thermoregulation on beak morphology, with
consequences for song evolution. Proceedings of the Royal Society
B: Biological Sciences, 286, 20192474. https://doi.org/10.1098/
rspb.2019.2474 Mark S. Davies
https://orcid.org/0000-0003-1900-1022 Mark S. Davies
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Structural constraints in current stomatal conductance models preclude accurate estimation of evapotranspiration and its partitions
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Structural constraints in current stomatal
conductance models preclude accurate estimation
of evapotranspiration and its partitions
Pushpendra Raghav
The University of Alabama https://orcid.org/0000-0002-2982-069X
Mukesh Kumar ( mkumar4@eng.ua.edu )
University of Alabama https://orcid.org/0000-0001-7114-9978
Yanlan Liu
The Ohio State University https://orcid.org/0000-0001-5129-6284
Article
Keywords: Stomatal conductance, Flux partitioning, Plant hydraulics, Machine learning
Posted Date: March 28th, 2023
DOI: https://doi.org/10.21203/rs.3.rs-2694012/v1
License: This work is licensed under a Creative Commons Attribution 4.0 International License.
Read Full License
Additional Declarations: There is NO Competing Interest. Structural constraints in current stomatal
conductance models preclude accurate estimation
of evapotranspiration and its partitions
Pushpendra Raghav
The University of Alabama https://orcid.org/0000-0002-2982-069X
Mukesh Kumar ( mkumar4@eng.ua.edu )
University of Alabama https://orcid.org/0000-0001-7114-9978
Yanlan Liu
The Ohio State University https://orcid.org/0000-0001-5129-6284
Article
Keywords: Stomatal conductance, Flux partitioning, Plant hydraulics, Machine learning
Posted Date: March 28th, 2023
DOI: https://doi.org/10.21203/rs.3.rs-2694012/v1
License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License
Additional Declarations: There is NO Competing Interest. Article Additional Declarations: There is NO Competing Interest. Additional Declarations: There is NO Competing Interest. Structural constraints in current stomatal conductance models preclude accurate
estimation of evapotranspiration and its partitions Pushpendra Raghav1, Mukesh Kumar1,*, Yanlan Liu2
1Department of Civil, Construction, and Environmental Engineering, University of Alabama,
Tuscaloosa, AL, USA
2School of Earth Sciences, The Ohio State University, Columbus, OH, USA
*Corresponding author. Email address: mkumar4@eng.ua.edu (M. Kumar) 2School of Earth Sciences, The Ohio State University, Columbus, OH, USA
*Corresponding author. Email address: mkumar4@eng.ua.edu (M. Kumar) 1 1 Abstract Evaporation (E) and Transpiration (T) play a critical role in water and energy budgets at regional
to global scales. T is regulated via stomatal conductance (𝑔𝑠𝑐), which depends on a multitude of
plant physiological processes and hydrometeorological forcings. In recent years, significant
advances have been made toward estimating 𝑔𝑠𝑐 using a variety of models, ranging from relatively
simple empirical models to more complex and data-intensive plant hydraulics-based models. However, a detailed assessment of the ability of these models for predicting evapotranspiration
components (E and T) remains lacking. Using machine learning and eddy covariance flux tower
data of 642 years, distributed across 84-sites and ten land covers globally, here we show that
structural constraints in both empirical and plant hydraulics-based models of 𝑔𝑠𝑐 limit their
effectiveness for predicting evapotranspiration (ET) and its components, i.e., E and T. Notably,
even when the current generation 𝑔𝑠𝑐 models are calibrated locally, their limiting structures don’t
allow them to use the information contained in the data optimally. Performance of empirical
models, which are still widely used for ET estimation, is observed to be especially underwhelming
for partitioning T from ET. While the plant hydraulics-based model structure is relatively effective
because of its ability to capture the inextricably-linked stomatal response to soil moisture (SM)
and vapor pressure deficit (VPD), we show that there still is a significant room for improvement
in the structure of these models. These results underscore the need to prioritize improvements in
models of 𝑔𝑠𝑐 to constraint estimates of E and T, and thus to reduce uncertainties in assessments
of plants’ role in regulating the earth’s climate. Keywords: Stomatal conductance, Flux partitioning, Plant hydraulics, Machine learning Main ET plays a critical role in regulating water and energy balance over the land surface. In fact, around
60% of water falling over the land surface as precipitation is returned back to the atmosphere by
evapotranspiration (ET)1. ET is comprised of two distinct processes, namely direct evaporation (E,
i.e., evaporation from bare soil and other open surfaces, and canopy interception loss) and plant
transpiration (T)2. Partitioning ET into its primary components (i.e., E and T) is challenging3 but
critical to understand the role of plants in regulating earth’s climate4-9. This is especially needed
as relative contributions of E and T vary in space and time, in part due to the varied controls
on them10,11. Ongoing and anticipated changes in forested12-15 and cropped area16-18 further
underscore the need to individually obtain the E and T fluxes. Despite this need, there still exists
a large uncertainty in ET partition estimates3. T is regulated by stomatal conductance (𝑔𝑠𝑐), which in turn depends on plant physiological
processes and hydrometeorological forcings. To obtain accurate and robust estimates of ET and
its partitions, it is important to accurately estimate 𝑔𝑠𝑐. Over the past years, numerous efforts have
been made to model 𝑔𝑠𝑐 using a range of approaches ranging from relatively simple empirical
models19-21 to more sophisticated but oftentimes data-intensive plant hydraulic models22-24. Irrespective of the approach taken, each model has some inherent structural assumptions to
simplify the response of stomatal kinetics to hydrometeorological conditions. Specifically, most 2 empirical approaches assume the influence of hydrometeorological condition on 𝑔𝑠𝑐 to be
independent of each other. In contrast, plant hydraulic models account for the coupled impacts of
soil moisture and meteorological forcings on water transport from the roots to the leaves, which
determines leaf water status and stomatal kinetics. While plant hydraulic models mechanistically
represent stomatal kinetics, they also rely on a range of simplifications, such as prescribed stomatal
response function to leaf water status25,26, neglected/simplified xylem network27, and usage of a
simplified whole-plant vulnerability curve28. The extent to which these structural simplifications
limit the capability of these models for estimating ET and ET partitions remain unknown. Several studies have focused on assessing the performance of the two aforementioned approaches
for modeling ET24,29-31. However, studies focused on assessing whether and to what extent the two
approaches can accurately partition ET are lacking. Main Notably, a few recent studies have indicated
improved capabilities of plant hydraulic models to capture ET, though primarily during specific
hydroclimatic periods such as periods that are dry32 or have high vapor pressure deficit (VPD)33. Given that model performance and ET partition changes with hydroclimatic regimes34-36 different
model representations likely result in distinct ET partitions. Here we evaluate the limitation of
model structures on accurately estimating ET and ET partitioning by comparing the performances
of empirical and plant hydraulic models with a machine-learning (ML) based model, which is not
restricted by assumptions of model structure or pre-defined relations between hydroclimatic
conditions and stomatal response. To this end, we use a generalized form of empirical and
hydraulic models that has been tested at flux tower sites across ecosystem types33,
𝑔𝑠𝑐= 𝑔𝑠
∗(1 −𝑚∙𝑙𝑛(𝑉𝑃𝐷)) (1) empirical approaches assume the influence of hydrometeorological condition on 𝑔𝑠𝑐 to be
independent of each other. In contrast, plant hydraulic models account for the coupled impacts of
soil moisture and meteorological forcings on water transport from the roots to the leaves, which
determines leaf water status and stomatal kinetics. While plant hydraulic models mechanistically
represent stomatal kinetics, they also rely on a range of simplifications, such as prescribed stomatal
response function to leaf water status25,26, neglected/simplified xylem network27, and usage of a
simplified whole-plant vulnerability curve28. The extent to which these structural simplifications
limit the capability of these models for estimating ET and ET partitions remain unknown. Several studies have focused on assessing the performance of the two aforementioned approaches
for modeling ET24,29-31. However, studies focused on assessing whether and to what extent the two
approaches can accurately partition ET are lacking. Notably, a few recent studies have indicated
improved capabilities of plant hydraulic models to capture ET, though primarily during specific
hydroclimatic periods such as periods that are dry32 or have high vapor pressure deficit (VPD)33. Given that model performance and ET partition changes with hydroclimatic regimes34-36 different
model representations likely result in distinct ET partitions. Here we evaluate the limitation of
model structures on accurately estimating ET and ET partitioning by comparing the performances
of empirical and plant hydraulic models with a machine-learning (ML) based model, which is not
restricted by assumptions of model structure or pre-defined relations between hydroclimatic
conditions and stomatal response. Main To this end, we use a generalized form of empirical and
hydraulic models that has been tested at flux tower sites across ecosystem types33,
𝑔
= 𝑔∗(1 −𝑚∙𝑙𝑛(𝑉𝑃𝐷))
(1) 𝑔𝑠𝑐= 𝑔𝑠
∗(1 −𝑚∙𝑙𝑛(𝑉𝑃𝐷)) (1) 𝑔𝑠𝑐= 𝑔𝑠
∗(1 −𝑚∙𝑙𝑛(𝑉𝑃𝐷)) (1) where m is the VPD sensitivity parameter and 𝑔𝑠
∗ is reference stomatal conductance at a VPD of 1
kPa, which reduces with soil moisture or plant water status. In the empirical model (referred as
EMP henceforth), m is held constant and 𝑔𝑠
∗ is a function of root-zone soil moisture. In the plant
hydraulic model, both m and 𝑔𝑠
∗ depend upon leaf water potential, and change over time with soil
moisture and VPD25,37 (see Methods for more details). We use two versions of plant hydraulic
models in this study. The first version, hereafter referred as PHexp, explicitly prescribes a function
that reduces stomatal conductance 𝑔𝑠𝑐 based on leaf water potential, as a result of hydraulic
response to hydroclimatic conditions15,26. The second is a hybrid approach representing plant
hydraulics implicitly (referred as PHimp henceforth), wherein the dependence of stomatal
conductance on leaf water potential and thus hydroclimatic conditions is substituted with a data-
driven approach. PHimp acts as a transition between PHexp and the last model under consideration
i.e., a machine learning (ML) based model, which is not bound by model structure or pre-defined
relations, as is the case in both empirical and plant hydraulics models. We test if the ML model
outperforms empirical and plant-hydraulic models, which would indicate prescribed model
structures are sub-optimal at predicting ET and its partitions. All these four stomatal conductance
models, i.e., EMP, PHexp, PHimp, and ML, are then incorporated within the two-source
Shuttleworth-Wallace (SW) ET model38 to estimate ET and its partitions (see Methods). We apply the four models at 84 flux sites (Fig. 1). All the models are trained for each site
individually to ensure that each model performs optimally, and any difference in model We apply the four models at 84 flux sites (Fig. 1). All the models are trained for each site
individually to ensure that each model performs optimally, and any difference in model 3 performance arises only from structural difference instead of poor parameterization. Training and
validation accuracies show that all four models are performing comparably well in predicting ET
and partition (Figs. S1 and S2), indicating the models are not overfitting the data. Main Notably, all four
models here are trained for two different configurations of loss functions, Configuration-1 and
Configuration-2. Configuration-1 trains the model by minimizing the mean squared error (MSE)
of ET, whereas Configuration-2 minimizes the sum of MSE of both ET and the partitioned
transpiration (T). Given the inherent uncertainty in ET partitioning methods39-41, to ensure the
robustness of the findings, partitioned T that is used to evaluate the two modeling approaches is
obtained from multiple methods. Specifically, we tested T obtained from multiple partitioning
methods, including the Transpiration Estimation Algorithm (TEA)42, Underlying Water Use
Efficiency (uWUE)43, and leaf and ecosystem water use efficiency (Yu22)44. performance arises only from structural difference instead of poor parameterization. Training and
validation accuracies show that all four models are performing comparably well in predicting ET
and partition (Figs. S1 and S2), indicating the models are not overfitting the data. Notably, all four
models here are trained for two different configurations of loss functions, Configuration-1 and
Configuration-2. Configuration-1 trains the model by minimizing the mean squared error (MSE)
of ET, whereas Configuration-2 minimizes the sum of MSE of both ET and the partitioned
transpiration (T). Given the inherent uncertainty in ET partitioning methods39-41, to ensure the
robustness of the findings, partitioned T that is used to evaluate the two modeling approaches is
obtained from multiple methods. Specifically, we tested T obtained from multiple partitioning
methods, including the Transpiration Estimation Algorithm (TEA)42, Underlying Water Use
Efficiency (uWUE)43, and leaf and ecosystem water use efficiency (Yu22)44. We compare the performance of all four models under the two configurations for ET and T:ET
(the ratio of T over ET) predictions. The results (Fig. 2) indicate that ML model is most accurate,
while both empirical and hydraulic models are subpar, indicating the structures of EMP and plant
hydraulic models are limiting their efficacy for the prediction of ET and partition. ML model
generally outperforms empirical and plant hydraulics models for estimating T even when the
models are trained against ET (i.e., Configuration-1) only. We compare the performance of all four models under the two configurations for ET and T:ET
(the ratio of T over ET) predictions. The results (Fig. 2) indicate that ML model is most accurate,
while both empirical and hydraulic models are subpar, indicating the structures of EMP and plant
hydraulic models are limiting their efficacy for the prediction of ET and partition. Main ML model
generally outperforms empirical and plant hydraulics models for estimating T even when the
models are trained against ET (i.e., Configuration-1) only. Fig. 1. Fluxnet sites used in this study. Constraining the ML model with T improves the ET partition over the entire record period (Fig. 2b,d,f) and also the ET partitions under extremes of soil moisture (SM) and VPD (Figs. S4b,d,f
and S5b,d,f). To diagnose the stomatal kinetics represented in the four models under different SM
and VPD regimes, the modeled 𝑔𝑠𝑐 is compared to that derived from observation via inversion of
the SW model using observed ET data (see Methods for further details). The ML model captures
the observed 𝑔𝑠𝑐 dynamics under Configuration-2 (Fig. 3 and Fig. S21). The results are consistent
across different ET partitioning methods and biomes (see Figs. S7-S20). Fig 1 Fluxnet sites used in this study Fig. 1. Fluxnet sites used in this study. Fig. 1. Fluxnet sites used in this study. Constraining the ML model with T improves the ET partition over the entire record period (Fig. 2b,d,f) and also the ET partitions under extremes of soil moisture (SM) and VPD (Figs. S4b,d,f
and S5b,d,f). To diagnose the stomatal kinetics represented in the four models under different SM
and VPD regimes, the modeled 𝑔𝑠𝑐 is compared to that derived from observation via inversion of
the SW model using observed ET data (see Methods for further details). The ML model captures
the observed 𝑔𝑠𝑐 dynamics under Configuration-2 (Fig. 3 and Fig. S21). The results are consistent
across different ET partitioning methods and biomes (see Figs. S7-S20). Constraining the ML model with T improves the ET partition over the entire record period (Fig. 2b,d,f) and also the ET partitions under extremes of soil moisture (SM) and VPD (Figs. S4b,d,f
and S5b,d,f). To diagnose the stomatal kinetics represented in the four models under different SM
and VPD regimes, the modeled 𝑔𝑠𝑐 is compared to that derived from observation via inversion of
the SW model using observed ET data (see Methods for further details). The ML model captures
the observed 𝑔𝑠𝑐 dynamics under Configuration-2 (Fig. 3 and Fig. S21). The results are consistent
across different ET partitioning methods and biomes (see Figs. S7-S20). 4 Fig 2. Main Performance statistics of different models (i.e., EMP (empirical), PHexp (explicit
representation of plant hydraulics), PHimp (implicit representation of plant hydraulics) and
machine learning (ML)) for the predictions of evapotranspiration (ET), plant transpiration (T),
and transpiration to evapotranspiration ratio (T:ET) for all the sites. Results are only shown for
the validation period. The left (right) column shows the performance statistics for Configuration-
1 (Configuration-2). 𝜌 is the Pearson’s correlation coefficient. Gray, pink, blue, and green boxes
correspond to EMP, PHimp, PHexp, and ML models, respectively. Fig 2. Performance statistics of different models (i.e., EMP (empirical), PHexp (explicit
representation of plant hydraulics), PHimp (implicit representation of plant hydraulics) and
machine learning (ML)) for the predictions of evapotranspiration (ET), plant transpiration (T),
and transpiration to evapotranspiration ratio (T:ET) for all the sites. Results are only shown for
the validation period. The left (right) column shows the performance statistics for Configuration-
1 (Configuration-2). 𝜌 is the Pearson’s correlation coefficient. Gray, pink, blue, and green boxes
correspond to EMP, PHimp, PHexp, and ML models, respectively. To explore why both empirical and plant hydraulics models show subpar performance than the
ML model, we examine the modeled response of 𝑔𝑠𝑐 to VPD and SM with respect to the observed. For this heat maps showing the co-regulation pattern of 𝑔𝑠𝑐 with VPD and SM are developed. To
develop these maps, we calculated the average values of 𝑔𝑠𝑐 for each 0.1 (kPa)-VPD and 0.01
(𝑚3𝑚−3)-SM bins. Results in Fig. 3 (also see Figs. S21-31 for the co-regulation patterns in
different biomes) for EMP shows a co-regulation pattern reflective of the prescribed independent
functional response of 𝑔𝑠𝑐 to VPD and SM, with 𝑔𝑠𝑐 reducing with high VPD and low SM45,46. In
contrast, the observed 𝑔𝑠𝑐 varies non-monotonically with SM and VPD. This expressed pattern of
observed 𝑔𝑠𝑐 is attributable to the influence of other meteorological variables (Table S2) and non-
linear lagged (Fig. S32) or hysteretic responses47,48 on 𝑔𝑠𝑐. Notably, plant hydraulic models,
overall, better capture the coupled response of SM and VPD than EMP model. Plant hydraulic
models are also generally less sensitive to SM and more sensitive to VPD than the EMP model,
consistent with the observation. Co-regulation pattern of ML-based 𝑔𝑠𝑐 closely resembles the
observed co-regulation pattern. 5 e contrast between 𝑔𝑠𝑐 variations from plant hydraulic models and that from the ML model or
g. 3. Stomatal conductance (𝑔𝑠𝑐) co-regulated by VPD and soil moisture (SM). Main Co-regulation
ttern for 𝑔𝑠𝑐 obtained from (a) the empirical model (EMP), (b) the implicit representation of
ant hydraulics model (PHimp), (c) the explicit representation of plant hydraulics model (PHexp),
) the pure machine learning model (ML), and (e) observations. Data from all the sites were
ed. Note that the results here are shown for the case when model calibration is performed
ainst both LE and TEA-obtained T (i.e., Configuration-2 with TEA). Fig. 3. Stomatal conductance (𝑔𝑠𝑐) co-regulated by VPD and soil moisture (SM). Co-regulation
pattern for 𝑔𝑠𝑐 obtained from (a) the empirical model (EMP), (b) the implicit representation of
plant hydraulics model (PHimp), (c) the explicit representation of plant hydraulics model (PHexp),
(d) the pure machine learning model (ML), and (e) observations. Data from all the sites were
used. Note that the results here are shown for the case when model calibration is performed
against both LE and TEA-obtained T (i.e., Configuration-2 with TEA). The contrast between 𝑔𝑠𝑐 variations from plant hydraulic models and that from the ML model or
observations could be due to differential response of 𝑔𝑠𝑐 in plant hydraulic model to additional 6 influencing variables (Table S2) other than SM and VPD (Fig. S35). To assess this, a random
forest regressor is fitted to observed and modeled 𝑔𝑠𝑐, individually, as a function of a range of
climate variables, viz. VPD, SM, atmospheric 𝐶𝑂2 concentration (𝐶𝑂2), solar radiation (SRad),
plant’s biophysical state (LAI in this study), and air temperature (𝑇𝑎) (see Methods for more
details). Partial dependence49 of 𝑔𝑠𝑐 on different variables for all the models and observations are
obtained. Notably, the sensitivity of 𝑔𝑠𝑐 to different variables are similar for observations and the
ML model, although this is not always the case for PHemp, and PHimp (Fig. 4). Specifically, plant
hydraulic models capture the observed sensitivity of 𝑔𝑠𝑐 to VPD and SM reasonably well, but they
show discrepancies in senstivity to other variables (e.g., solar radiation, atmospheric 𝐶𝑂2
concentration, LAI, and air temperature). In contrast to plant hydraulic model, 𝑔𝑠𝑐 from empirical
model is overall oversensitive to SM (Fig. S34). Notably, 𝑔𝑠𝑐 from the empirical model is much
smaller at low soil moisture (Fig. S34), a result that agrees with findings in Liu, et al. 33. ML-
derived 𝑔𝑠𝑐 closely resembles the obseved sensitivites for Configuration-2, although it is not the
case for Configuration-1 (Fig. S33). Main Calibrating models with T:ET results in better representaton
of 𝑔𝑠𝑐 response to different hydroclimatological variables in plant hydraulic and ML models. Discussion Discussion Given the large uncertainty in ET partitioning estimates3, which has implications for assessing
vegetation-atmosphere feedback and the changes in water budget resulting from alterations in land
cover and climate, there is an urgent need to pin-point and henceforth ameleorate the causes of
poor ET partition estimates. To this end, this study investigated the efficacy of the two class of
models of stomatal conductance, 𝑔𝑠𝑐, a salient variable that controls plant water use and hence the
ET partition into evaporation (E) and transpiration (T). The experimental design made the best use
of available flux tower data, machine learning, and physical modeling to understand the structural
errors in current generation 𝑔𝑠𝑐 models. Specifically, the study evaluated the ability of an empirical
and two plant hydraulics-based models (one with explicit functional definition of water potential
gradient along the root-xylem-leaf continuum, and the other with an implicit data-based
representation of the relation between soil moisture and leaf water potential) of 𝑔𝑠𝑐 to predict ET
and T. A machine learning model of 𝑔𝑠𝑐, which was not bound by any model structure or pre-
defined relations between cause and response, was also developed. The thesis was that if the ML
model outperforms empirical and plant-hydraulics based models, that would indicate that
empirical and plant hydraulics-based models are sub-optimal at predicting ET and its partitions
due to their restrictive structure. All these four stomatal conductance models were then
incorporated within the two-source Shuttleworth-Wallace (SW) ET model38 that models ET and
its components as a function of 𝑔𝑠𝑐 (see Methods: Modeling stomatal conductance, ET, and T). All the models were trained for each flux site individually to ensure that any error in the model
output is from the model structure and not from model parameterization. While previous studies have reported a superior proficiency of ML model with respect to the
empirical model for ET predictions50,51, results from this analysis at 84 flux towers (located in 10 While previous studies have reported a superior proficiency of ML model with respect to the
empirical model for ET predictions50,51, results from this analysis at 84 flux towers (located in 10
different biomes) revealed for the first time that structural constraints in both empirical and plant
hydraulics-based models of 𝑔𝑠𝑐 limit their effectiveness for predicting both ET and its 7 components. Discussion Pure ML model, which is minimally ceiled by any structural constraints, predicted
both ET and T more accurately than any other model. On the contrary, the structure of empirical
and plant hydraulics-based models prevented an effective utilization of input data. This indicates
current generation models of 𝑔𝑠𝑐 cannot be used to obtain robust and accurate prediction of ET
and its components over different biomes even if models are trained locally. Notably, even though
the ML model outperformed other models overall, in Configuration-1 (i.e., when models are
trained only for ET only), PHimp model consistently outperformed EMP and sometimes even the
ML model for prediction of ET partitions during SM and VPD extremes. This result indicates that
for certain periods such as when SM and VPD extremes occur, if either the ET is small or the
training sample size is small, ML model training and subsequent performance may not be as robust. Notably, performance of all models in general and ML model in particular improves with training
against T:ET data. This highlights that better and more robust models can be developed at sites
with additional information regarding T:ET partitioning. Given that observation data of T:ET
partitions from different partitioning methods continue to be uncertain39-41, the study underscores
the need to further refine such methods to develop more robust ET models. To delve deep into the factors responsible for the inadequate performance of current generation
𝑔𝑠𝑐 models for the prediction of ET and its components, we assessed the co-regulation of stomatal
conductance not only by soil moisture and VPD but also by additional climate factors including
plant’s biophysical state (LAI), air temperature (𝑇𝑎), solar radiation (SRad), and atmospheric 𝐶𝑂2
concentration (𝐶𝑂2). Using a random forest regressor, we found that these models don’t represent
the correct sensitivities of 𝑔𝑠𝑐 to different hydroclimatic variables. Specifically, the empirical
model exhibited discrepancy in sensitivity to SM and VPD, with 𝑔𝑠𝑐 from the empirical model
being oversensitive to SM. As noted in a previous study Liu, et al. 33, this is attributable to a
restrictive independent functional dependence of 𝑔𝑠𝑐 on SM and VPD, as is prescribed in the EMP
model. The plant hydraulic model also fell short in capturing 𝑔𝑠𝑐 sensitivities to 𝐶𝑂2, SRad, and
LAI. Discrepancy in the influence LAI on 𝑔𝑠𝑐 could, in part, be due to the big-leaf assumption in
the SW model. Discussion This issue could be alleviated by increasing the number of canopy layers and/or by
explicit accounting of shaded and sunlit fractions in the SW model52,53. Limitations in the
photosynthesis model (e.g., Farquhar model54) could partly be responsible for discrepancy in 𝑔𝑠𝑐’s
dependence on solar radiation and atmospheric 𝐶𝑂2 concentration in the plant hydraulic model. To reduce the discrepancy, the structural limiations of the photosynthesis model may need to be
addressed. It is also possible that calibration of parameters for air temperature and solar radiation
responses in the photosynthesis model may reduce this discrepancy. This indicates the room for
further improvement in a plant hydraulic model is not just restricted to improving only plant-water
relations but also the coupled light and temperature responses. Overall, aforementioned
discrepancies in 𝑔𝑠𝑐’s sensitivity to hydrometeorological variables highlight the need for future
research on incorporating these relationships into improved versions of EMP and plant hydraulic
models. Notably, for plant hydraulic models, there is a need to improve both the plant-water
relations and the photosynthetic response to light and/or temperature. 8 The structural limitations of current generation 𝑔𝑠𝑐 models as identified in this study, if amended,
may provide numerous benefits. For instance, the consequent improved assessment of ET
partitioning will have significant implications on fidelity of earth system models especially in
regards to improving the dynamics of the plant transpiration in response to rising atmospheric CO2
and changing climate conditions55. Accurate estimation of plant transpiration will enhance the
understanding of vegetation’s biophysical cooling effect, the strength of which is highly
underestimated in current LSMs in part because of uncertain ET partitioning in current LSMs3,4,56. Under future climate where weather extremes are expected to intensify due to increased
temperature57, a better understanding of plant’s cooling effect may help in modulating the earth’s
temperature. Transpiration from plants also plays a significant role in recycling the terrestrial water
and thus reducing the runoff. Thus any error in the estimation of T will reflect in runoff estimation
that directly or indirectly affects the floods/droughts estimation3. In addition, plants also modulate
the energy balance of the land suface. Any change in transpiration will affect the land-atmosphere
exchange of water that may change the atmospheric water demand. Furthermore, accurate
estimation of T:ET (and thus actual water used by plants) will help in optimizing the precision
irrigation scheduling. 1. Modeling stomatal conductance, ET, and T 1. Modeling stomatal conductance, ET, and T ET is the sum of soil evaporation (𝐸𝑠) and plant transpiration (𝑇𝑐 or T). It can be formulated using
t
ET
d l b
d
Sh ttl
th
d W ll
38 ET is the sum of soil evaporation (𝐸𝑠) and plant transpiration (𝑇𝑐 or T). It can be of soil evaporation (𝐸𝑠) and plant transpiration (𝑇𝑐 or T). It can be formulated using e ET model based on Shuttleworth and Wallace 38 as: o-source ET model based on Shuttleworth and Wallace 38 as: a two-source ET model based on Shuttleworth and Wallace 38 as: ET = Es + Tc = 𝑓𝑠∙𝑃𝑀𝑠+ 𝑓𝑐∙𝑃𝑀𝑐 (1𝑎)
𝑃𝑀𝑠= 𝑠∙𝐴+ (𝜌𝑎∙𝐶𝑝∙𝑉𝑃𝐷−𝑠∙𝑟𝑎𝑠(𝐴−𝐴𝑠))/(𝑟𝑎𝑎+ 𝑟𝑎𝑠)
𝑠+ 𝛾(1 + 𝑟𝑠𝑠/(𝑟𝑎𝑎+ 𝑟𝑎𝑠))
(1𝑏)
𝑃𝑀𝑐= 𝑠∙𝐴+ (𝜌𝑎∙𝐶𝑝∙𝑉𝑃𝐷−𝑠∙𝑟𝑎𝑐𝐴𝑠)/(𝑟𝑎𝑎+ 𝑟𝑎𝑐)
𝑠+ 𝛾(1 + 𝑟𝑠𝑐/(𝑟𝑎𝑎+ 𝑟𝑎𝑐))
(1𝑐)
𝑓𝑐=
1
1 + 𝑅𝑐∙𝑅𝑎/(𝑅𝑠(𝑅𝑐+ 𝑅𝑎)) (1𝑑)
𝑓𝑠=
1
1 + 𝑅𝑠∙𝑅𝑎/(𝑅𝑐(𝑅𝑠+ 𝑅𝑎)) (1𝑒)
𝑅𝑐= (𝑠+ 𝛾)𝑟𝑎𝑐+ 𝛾∙𝑟𝑠𝑐 (1𝑓)
𝑅𝑠= (𝑠+ 𝛾)𝑟𝑎𝑠+ 𝛾∙𝑟𝑠𝑠 (1𝑔)
𝑅𝑎= (𝑠+ 𝛾)𝑟𝑎𝑎 (1ℎ) (1ℎ) In the above equations, subscripts s and c represent soil and canopy components, respectively. 𝑓 is
a weighting factor, 𝑃𝑀 is a term similar to that in the Penman-Monteith model 58. 𝑠 is the slope
of the saturation vapor pressure curve (𝑃𝑎 𝐾−1), A is the total available energy for sensible and
latent heat flux (i.e., 𝐴= 𝑅𝑛−𝐺; where 𝑅𝑛 is net radiation and G is the ground heat flux)
(𝑊 𝑚−2), 𝐴𝑠 is the part of 𝐴 available for soil (𝑊 𝑚−2), 𝜌𝑎 is the density of the air (𝑘𝑔 𝑚−3), 𝐶𝑝
is the specific heat of dry air at constant pressure (𝐽 𝑘𝑔−1 𝐾−1), 𝛾 is the psychrometric constant
(𝑃𝑎 𝐾−1), 𝑉𝑃𝐷 is the vapor pressure deficit (𝑃𝑎), 𝑟𝑎𝑠 is the resistance to vapor transfer between
the soil surface and the canopy layer (𝑠 𝑚−1), 𝑟𝑎𝑎 is the aerodynamic resistance between the
canopy source and a reference height (𝑠 𝑚−1), 𝑟𝑎𝑐 is the canopy boundary layer resistance (𝑠 𝑚−1),
𝑟𝑠𝑠 is the soil surface resistance for vapor transfer (𝑠 𝑚−1), and 𝑟𝑠𝑐 is the stomatal resistance
(𝑠 𝑚−1). Additionally, ditionally,
𝐴= 𝑅𝑛−𝐺 (1𝑖)
𝐴𝑠= 𝑅𝑛𝑠−𝐺 (1𝑗)
𝑅𝑛𝑠= 𝑅𝑛exp(−𝑘𝑟∙𝐿𝐴𝐼) (1𝑘) 𝐴= 𝑅𝑛−𝐺
𝐴𝑠= 𝑅𝑛𝑠−𝐺 𝐴𝑠= 𝑅𝑛𝑠−𝐺 𝑅𝑛𝑠= 𝑅𝑛exp(−𝑘𝑟∙𝐿𝐴𝐼) (1𝑘) 𝑅𝑛𝑠= 𝑅𝑛exp(−𝑘𝑟∙𝐿𝐴𝐼) (1𝑘) where 𝑘𝑟 is the canopy extinction coefficient of net radiation, and LAI is the leaf area index. Readers are encouraged to consult Shuttleworth and Wallace 38 for an in-depth understanding of
the aforementioned equations. Discussion Although, our study have limitations, including restricting the analysis to
no-interception-loss periods, and use of inverted 𝑔𝑠𝑐 as observations, overall, our findings still
provide important insights into the strengths and limitations of different modeling approaches of
stomatal conductances for prediction of ET and its components. The study also emphasizes the
need to enhance the structure of current generation 𝑔𝑠𝑐 models for advancing our understanding
of plant water use dynamics and land-atmosphere feedback under changing land cover and climate. 9 Fig. 4. Partial dependance plots: Stomatal conductance (𝑔𝑠𝑐) as a function of different variables
including vapor pressure deficit (VPD), soil moisture (SM), atmospheric 𝐶𝑂2 concentration
(𝐶𝑂2), solar radiation (SRad), leaf area index (LAI), and air temperature (𝑇𝑎) in the observations
and different models for Configuration-2. Fig. 4. Partial dependance plots: Stomatal conductance (𝑔𝑠𝑐) as a function of different variables
including vapor pressure deficit (VPD), soil moisture (SM), atmospheric 𝐶𝑂2 concentration
(𝐶𝑂2), solar radiation (SRad), leaf area index (LAI), and air temperature (𝑇𝑎) in the observations
and different models for Configuration-2. 10 Methods Methods ng stomatal conductance, ET, and T 1. Modeling stomatal conductance, ET, and T The calculation procedure of the different resistance terms in the above equations is well
documented elsewhere. For example, to obtain aerodynamic resistances (i.e., 𝑟𝑎𝑎, 𝑟𝑎𝑠, 𝑎𝑛𝑑 𝑟𝑎𝑐), one
can refer to Shuttleworth and Gurney 59 and Brutsaert 60 where these terms are defined as the
functions of canopy height, leaf structure, wind speed, friction velocity, LAI, etc. 11 The calculation of 𝑟𝑠𝑠 is presented in Sellers, et al. 61 where 𝑟𝑠𝑠 is defined as the function of
soil wetness of the top soil layer. The calculation of 𝑟𝑠𝑠 is presented in Sellers, et al. 61 where 𝑟𝑠𝑠 is defined as the function of
soil wetness of the top soil layer. p
y
𝑟𝑠𝑠= exp (8.206 −4.225𝜃/𝜃𝑠) (1l) p
y
𝑟𝑠𝑠= exp (8.206 −4.225𝜃/𝜃𝑠) p
y
𝑟𝑠𝑠= exp (8.206 −4.225𝜃/𝜃𝑠) (1l) (1l) Aerodynamic resistances are evaluated using 𝑟𝑎𝑎= 1
𝑘𝑢∗log (𝑧𝑚−𝑑0
ℎ𝑐−𝑑0
) +
ℎ𝑐
𝑘𝑚𝐾ℎ
(𝑒𝑥𝑝 (𝑘𝑚(1 −𝑑0 + 𝑧0
ℎ𝑐
)) −1) (1𝑚)
𝑟𝑎𝑠=
ℎ𝑐exp(𝑘𝑚)
𝑘𝑚𝐾ℎ
(exp (
−𝑘𝑚𝑧0𝑠
ℎ𝑐
) −exp (−𝑘𝑚(
𝑑0+𝑧0
ℎ𝑐))) (1n) (1n) 𝑑0 = 1.1ℎ𝑐log (1 + 𝑋
1
4) (1o) 𝑑0 = 1.1ℎ𝑐log (1 + 𝑋
1
4) (1o) 𝑑0 = 1.1ℎ𝑐log (1 + 𝑋
1
4) (1o)
ss length is calculated using 𝑑0 = 1.1ℎ𝑐log (1 + 𝑋
1
4) (1o)
ess length is calculated using Roughness length is calculated using Roughness length is calculated using 𝑧0 = {
𝑧0𝑠+ 0.3ℎ𝑐𝑋
1
2 0 < 𝑋< 0.2
0.3ℎ𝑐(1 −
𝑑0
ℎ𝑐) 0.2 < 𝑋< 1.5
(1p)
𝑋= 𝐶𝑑𝐿𝐴𝐼 (1q)
𝐾ℎ= 𝑘𝑢∗(ℎ𝑐−𝑑0); 𝑒𝑑𝑑𝑦 𝑑𝑖𝑓𝑓𝑢𝑠𝑖𝑜𝑛 𝑐𝑜𝑒𝑓𝑓. 𝑎𝑡 𝑡ℎ𝑒 𝑡𝑜𝑝 𝑜𝑓 𝑡ℎ𝑒 𝑐𝑎𝑛𝑜𝑝𝑦
𝑟𝑎𝑐=
𝑟𝑏
2𝐿𝐴𝐼 (1r)
𝑟𝑏=
100
𝑘𝑚(
𝑑𝑙
𝑢ℎ)
1−exp(−𝑘𝑚
2 ) (1s) 𝑧0 = {
𝑧0𝑠+ 0.3ℎ𝑐𝑋
1
2 0 < 𝑋< 0.2
0.3ℎ𝑐(1 −
𝑑0
ℎ𝑐) 0.2 < 𝑋< 1.5
(1p) (1p) 𝑘𝑚∈[2.5 4.5]; 𝑒𝑥𝑡𝑖𝑛𝑐𝑡𝑖𝑜𝑛 𝑐𝑜𝑒𝑓𝑓. 𝑜𝑓 𝑒𝑑𝑑𝑦 𝑑𝑖𝑓𝑓𝑢𝑠𝑖𝑜𝑛 𝑧0𝑠= 0.01; 𝑒𝑓𝑓𝑒𝑐𝑡𝑖𝑣𝑒 𝑟𝑜𝑢𝑔ℎ𝑛𝑒𝑠𝑠 𝑙𝑒𝑛𝑔𝑡ℎ
𝑐𝑑= 0.001; 𝑑𝑟𝑎𝑔 𝑐𝑜𝑒𝑓𝑓. 𝑧0𝑠= 0.01; 𝑒𝑓𝑓𝑒𝑐𝑡𝑖𝑣𝑒 𝑟𝑜𝑢𝑔ℎ𝑛𝑒𝑠𝑠 𝑙𝑒𝑛𝑔𝑡ℎ
𝑐𝑑= 0.001; 𝑑𝑟𝑎𝑔 𝑐𝑜𝑒𝑓𝑓. 𝑑𝑙= 0.068; 𝑐ℎ𝑎𝑟𝑎𝑐𝑡𝑒𝑟𝑖𝑠𝑡𝑖𝑐 𝑙𝑒𝑎𝑓 𝑑𝑖𝑚𝑒𝑛𝑠𝑖𝑜𝑛 𝑑𝑙= 0.068; 𝑐ℎ𝑎𝑟𝑎𝑐𝑡𝑒𝑟𝑖𝑠𝑡𝑖𝑐 𝑙𝑒𝑎𝑓 𝑑𝑖𝑚𝑒𝑛𝑠𝑖𝑜𝑛 As we can see in the equations above, one critical variable that still needs to be computed is
stomatal resistance (𝑟𝑠𝑐) or stomatal conductance (𝑔𝑠𝑐) (Note: 𝑟𝑠𝑐= 1/𝑔𝑠𝑐). It is to be noted that
we removed rainy hours (and following four hours) from the analysis to avoid any effect of canopy
interception. In other words, in our study, we assume that only soil evaporation and plant
transpiration contribute to total evapotranspiration. 1. Modeling stomatal conductance, ET, and T To compute 𝑔𝑠𝑐, we tested three different
formulations that are explained below: 1a. Empirical representation
In the empirical representation, the stomatal conductance is modeled as: In the empirical representation, the stomatal conductance is modeled as: 𝑔𝑠𝑐= 𝑔𝑠
∗(1 −𝑚∙𝑙𝑛(𝑉𝑃𝐷)) (2𝑎) 𝑔𝑠𝑐= 𝑔𝑠
∗(1 −𝑚∙𝑙𝑛(𝑉𝑃𝐷)) (2𝑎) (2𝑎) where m is the VPD sensitivity parameter and 𝑔𝑠
∗ is reference stomatal conductance that is
restricted by the soil moisture in plant’s root-zone 62 as: where m is the VPD sensitivity parameter and 𝑔𝑠
∗ is reference stomatal conductance that is
restricted by the soil moisture in plant’s root-zone 62 as: 𝑔𝑠
∗= 𝑔𝑠
𝑟𝑒𝑓(max (0, 𝑚𝑖𝑛( 𝜃−𝜃𝑤
𝜃𝑠−𝜃𝑤
, 1))) (2𝑏)
𝑓 (2𝑏) 𝑔𝑠= 𝑔𝑠
(max (0, 𝑚𝑖𝑛(𝜃𝑠−𝜃𝑤
, 1))) (2𝑏)
where 𝑔𝑠
𝑟𝑒𝑓is the maximum 𝑔𝑠
∗. Here, the two parameters (m and 𝑔𝑠
𝑟𝑒𝑓) are obtained using
MCMC (see section “Model parameter estimation using data assimilation” below). where 𝑔𝑠
𝑟𝑒𝑓is the maximum 𝑔𝑠
∗. Here, the two parameters (m and 𝑔𝑠
𝑟𝑒𝑓) are obtained using
MCMC (see section “Model parameter estimation using data assimilation” below). 1b. Implicit and explicit plant hydraulic models 1b. Implicit and explicit plant hydraulic models 12 In the plant hydraulic model, 𝑔𝑠𝑐 is often modeled based on leaf-gas exchange optimality theory37
where the idea is that plants maximize carbon gain for a unit loss of water. Algebraically, 𝑔𝑠𝑐= 𝑎𝑟𝑔𝑚𝑎𝑥(𝑓𝑐(𝑔𝑠𝑐) −𝜆𝑓𝑒(𝑔𝑠𝑐)) (3𝑎) (3𝑎) where 𝑓𝑐(𝑔𝑠𝑐) is the carbon gain, 𝑓𝑒(𝑔𝑠𝑐) is the water loss, and 𝜆 is the marginal water use
efficiency (MWUE) that describes the cost of water loss in carbon units. Under given 𝜆, 𝑔𝑠𝑐 can
be solved by combining equation (3a) with a biochemical demand model of 𝐶𝑂2 (ref.54), as
described elsewhere15. Also, as described elsewhere25, 𝜆 responds to leaf water potential (Ψ𝑙) as:
𝑐 𝜆(Ψ𝑙) = 𝜆𝑤𝑤
∗
𝑐𝑎
𝑐𝑎∗exp(𝛽0𝛹𝑙) (3𝑏) (3𝑏) where 𝜆𝑤𝑤
∗
is the MWUE in well-watered conditions at atmospheric 𝐶𝑂2 concentration (𝑐𝑎) of 𝑐𝑎
∗
(= 400 𝑢𝑚𝑜𝑙 𝑚𝑜𝑙−1), and 𝛽0 is the sensitivity parameter. 1. Modeling stomatal conductance, ET, and T 𝑔𝑠𝑐= 𝑔𝑠
∗(1 −𝑚∙𝑙𝑛(𝑉𝑃𝐷)) (3𝑔) 𝑔𝑠𝑐= 𝑔𝑠
∗(1 −𝑚∙𝑙𝑛(𝑉𝑃𝐷)) (3𝑔) where 𝑔𝑠
∗ is the reference stomatal conductance at VPD of 1 kPa and m is the VPD sensitivity. When omitting plant hydraulic dynamics, 𝑔𝑠
∗ becomes an independent function of soil moisture
only, and m is a constant value. We call it “empirical representation of 𝑔𝑠𝑐” in this study as
described above. This is how 𝑔𝑠
∗ and m are currently represented in most land surface models26,66. However, the restrictions of soil moisture (through 𝑔𝑠
∗) and VPD (through m) on stomatal
conductance are in fact inextricably coupled via leaf water potential. Specifically, 𝑔𝑠
∗= (1 + g1) 𝐴
𝑐𝑎
, 𝑚= 𝑘𝐷𝑔1
1 + 𝑔1
(3ℎ) (3ℎ) where 𝑘𝐷 varies with VPD as 𝑘𝐷= 2(1 −𝑉𝑃𝐷−1
2)/ ln(𝑉𝑃𝐷). Because 𝑔1 is inversely correlated
to the marginal water use efficiency, it nonlinearly decreases as leaf water potential (Ψ𝑙) drops
under soil moisture (SM) and VPD stresses25. Bai, et al. 67 demonstrated in a semi-arid biome
based on in situ observations that 𝑔1 varies with SM and VPD because of plant hydraulics. Thus,
these two water stresses can be used to emulate the dynamics of 𝑔1 using some functional
relationship as: 𝑔1 = 𝑔1(Ψ𝑙) = 𝑓(𝑆𝑀, 𝑉𝑃𝐷) 𝑔1 = 𝑔1(Ψ𝑙) = 𝑓(𝑆𝑀, 𝑉𝑃𝐷) (3𝑖) (3𝑖) Here, we use a feedforward Artificial Neural Network (ANN) machine learning method to estimate
𝑔1 based on SM and VPD. See the ML structure for plant hydraulics model (PHimp) below
(Extended Fig. 1). We first shuffle the entire data set randomly in time at each site and then separate
the data set into training (80%) and validation (20%). All the input variables are normalized to
accelerate the learning pace. The hyperparameters (number of hidden layers and number of
neurons per layer) are determined for each site by repeating the training of multiple ANNs with
varying number of hidden layers (1-5) and neurons (8, 16, 24, 32, 40, 48, 56, 64, 72, 80). Early
stopping is used to avoid overfitting. Furthermore, total training cost during each epoch is obtained
based on training cost of multiple minibatches to avoid overfitting. The activation function in our
ANN structure is a rectified linear unit (ReLU). ANN structure is trained using the Gradient
Descent Optimization algorithm. The mean squared error (MSE) is used as cost function in the
training process. 1. Modeling stomatal conductance, ET, and T Ψ𝑙 controls water supply through the
plant hydraulic system, and the plant transpiration can be computed as: (3𝑐) p
p
p
𝑇= 𝑔𝑠𝑟𝑝(Ψ𝑠−Ψ𝑙)𝐿𝐴𝐼 (3𝑐) (3𝑐) where Ψ𝑠 is the soil water potential, and 𝑔𝑠𝑟𝑝 is the whole-plant xylem conductance that follows
the vulnerability curve63 as: 𝑔𝑠𝑟𝑝= 𝑔𝑝,𝑚𝑎𝑥[1 + ( Ψ𝑙
Ψ50
)
𝑎
]
−1
(3𝑑) (3𝑑) where 𝑔𝑝,𝑚𝑎𝑥 is the maximum whole-plant xylem conductance, Ψ50 is the xylem water potential
at which 50% of 𝑔𝑝,𝑚𝑎𝑥 is lost, and a is the curvature parameter of vulnerability curve. Ψ𝑠 is
obtained using soil texture and soil moisture content64 as: Ψ𝑠= Ψ𝑠𝑎𝑡( 𝜃
𝜃𝑠𝑎𝑡
)
𝑏𝑠
(3𝑒) (3𝑒) By combining Equations (3a-3e) with Shuttleworth-Wallace Model (Equations 1a-1s), the whole
plant hydraulic system can be solved for the desired fluxes (i.e., ET and T). All the parameters in
plant hydraulic model (𝜆𝑤𝑤
∗
, 𝛽0, 𝑔𝑝,𝑚𝑎𝑥, Ψ50, 𝑎, and, 𝑏𝑠) are obtained using a MCMC method to
identify the most likely set of plant traits that lead to model outputs consistent with the observed
ET and/or T at each site (see section `Model parameters estimation using data assimilation` for
more details). Parameterizing the classic plant hydraulic (PHexp) over large scale remains challenging. Plant
hydraulic processes are highly non-linear, and implementing them and calibrating their parameters
is computationally expensive. In addition, the functional relations used to define water potential
gradient along the root-xylem-leaf continuum may have errors. As an alternative, here, we also
developed a simplified data-centric model, PHimp, that incorporates root-xylem-leaf coupling
implicitly enabling it to emulate the impact of plant hydraulics. Emulating plant hydraulics can be achieved through constrained parameter choices of the standard
ti
lit
b
d
t ti
i
th M dl
’
d l65 Emulating plant hydraulics can be achieved through constrained parameter choices of the standard
optimality-based representation, i.e. the Medlyn’s model 65. p
y
𝑔𝑠𝑐= 𝑔0 + (1 +
𝑔1
√𝑉𝑃𝐷
) 𝐴
𝑐𝑎
(3𝑓) (3𝑓) 13 where 𝑔0 and 𝑔1 are intercept and slope parameters in Medlyn’s model, 𝐴 is carbon assimilation,
𝑐𝑎 is atmospheric 𝐶𝑂2 concentration. Omitting 𝑔0 , the residue stomatal conductance that is
usually close to zero and taking the Taylor series of 𝑉𝑃𝐷−1
2, 𝑔𝑠𝑐 can be expressed as a product of
soil moisture and VPD constraints on stomatal conductance as follows. 1. Modeling stomatal conductance, ET, and T The best model for each site is then obtained based on an Akaike Information
Criterion (AIC) as defined below: 𝐴𝐼𝐶= ln(𝑀𝑆𝐸) + 2𝑘/𝑛 (3𝑗) 𝐴𝐼𝐶= ln(𝑀𝑆𝐸) + 2𝑘/𝑛 𝐴𝐼𝐶= ln(𝑀𝑆𝐸) + 2𝑘/𝑛 (3𝑗) where MSE is the Mean Absolute Error, k is the number of estimated parameters in the model,
and n is the number of observations in the training data set. 14 Extended Fig. 1. Flow chart of the plant hydraulics model (PHimp). Extended Fig. 1. Flow chart of the plant hydraulics model (PHimp). Lastly, a pure ML model for 𝑔𝑠𝑐 is developed with different hydrometeorological variables as its
only input. See the structure of pure ML model below (Extended Fig. 2). Training of the model
here is same as described above for PHimp model. Extended Fig. 2. Flow chart of the pure ML model. 2. Estimation of model parameters Extended Fig. 2. Flow chart of the pure ML model. 2. Estimation of model parameters Extended Fig. 2. Flow chart of the pure ML model. 2. Estimation of model parameters
In the empirical representation of 𝑔𝑠𝑐 model, we have two parameters; 𝑔𝑠
𝑟𝑒𝑓 𝑎𝑛𝑑 𝑚 (see Equations
2a and 2b). In the explicit representation of plant hydraulics representation of 𝑔𝑠𝑐 model, we have
six parameters, including four plant hydraulic traits (𝜆𝑤𝑤
∗
, 𝛽0, 𝑔𝑝,𝑚𝑎𝑥, and Ψ50, see Equations 3a-
3d), one scaling parameter relating whole-plant xylem conductance to leaf water potential (a, see
Equation 3d), and one soil parameter (𝑏𝑠, see Equation 3e). In addition to this, two uncertainty Extended Fig. 2. Flow chart of the pure ML model. 2. Estimation of model parameters 2. Estimation of model parameters In the empirical representation of 𝑔𝑠𝑐 model, we have two parameters; 𝑔𝑠
𝑟𝑒𝑓 𝑎𝑛𝑑 𝑚 (see Equations
2a and 2b). In the explicit representation of plant hydraulics representation of 𝑔𝑠𝑐 model, we have
six parameters, including four plant hydraulic traits (𝜆𝑤𝑤
∗
, 𝛽0, 𝑔𝑝,𝑚𝑎𝑥, and Ψ50, see Equations 3a-
3d), one scaling parameter relating whole-plant xylem conductance to leaf water potential (a, see
Equation 3d), and one soil parameter (𝑏𝑠, see Equation 3e). In addition to this, two uncertainty In the empirical representation of 𝑔𝑠𝑐 model, we have two parameters; 𝑔𝑠
𝑟𝑒𝑓 𝑎𝑛𝑑 𝑚 (see Equations
2a and 2b). In the explicit representation of plant hydraulics representation of 𝑔𝑠𝑐 model, we have
six parameters, including four plant hydraulic traits (𝜆𝑤𝑤
∗
, 𝛽0, 𝑔𝑝,𝑚𝑎𝑥, and Ψ50, see Equations 3a-
3d), one scaling parameter relating whole-plant xylem conductance to leaf water potential (a, see
Equation 3d), and one soil parameter (𝑏𝑠, see Equation 3e). 𝑝(𝜃|𝑍) ∝𝑝(𝑍|𝜃) × 𝑝(𝜃) 𝑝(𝜃|𝑍) ∝𝑝(𝑍|𝜃) × 𝑝(𝜃) (4) (4) where 𝑝(𝜃) represents the prior probability density function (PDF) of parameters (𝜃) and 𝑝(𝜃|𝑍)
represents the posterior PDF of 𝜃. 𝑝(𝑍|𝜃) represents the conditional PDF of observation (𝑍) under
the prior parameters. To produce posterior samples, an adaptive metropolized independence sampler (AMIS68) is used. This sampling method has been developed with the aim of improving convergence, particularly
for non-linear models, and has proven successful in retrieving plant hydraulic traits at flux
towers33. In order to simplify the parameter space and promote convergence, the MCMC jointly
sampled all parameters. As mentioned above, MCMC requires prior knowledge on the PDF of
targeted parameters. Uniform priors of (0,1) 𝑚𝑜𝑙 𝑚−2𝑠−1 and (0,1) ln(𝑘𝑃𝑎)−1 are used for
𝑔𝑠
𝑟𝑒𝑓 𝑎𝑛𝑑 𝑚, respectively, based on the range of estimates in previous studies 45,69. A uniform prior
distribution is assumed for the hydraulic representation, covering the entire possible range of each
hydraulic trait as per meta-analyses32,70, i.e., (10-9,10-5) 𝑚 𝑠−1 𝑀𝑃𝑎−1 for 𝑔𝑝,𝑚𝑎𝑥, (0,10,000)
𝜇𝑚𝑜𝑙 𝑚𝑜𝑙−1 for 𝜆𝑤𝑤
∗
, (-10,0) MPa for Ψ50, (-2,0) 𝑀𝑃𝑎−1 for 𝛽0, and (0,8) for a. A uniform(0,10),
uniform(0,2) 𝑚𝑚 𝑑𝑎𝑦−1, and uniform(0,2) 𝑚𝑚 𝑑𝑎𝑦−1priors were assumed for parameters 𝑏𝑠,
𝜎𝐸𝑇, and 𝜎𝑇, respectively, based on 71. p
y
The estimated posterior distribution of parameters is determined by the cost function in the
MCMC, which is the product of the inverse of the likelihood function and the prior. In
Configuration-1, to calculate the likelihood function, the modeled ET is compared with observed
ET. In Configuration-2, the modeled ET and T are compared with observed ET and T to calculate
the likelihood function. The observations are treated as independent and are assumed to follow a
Gaussian distribution with a mean equal to the modeled value and a standard deviation
corresponding to their respective category (i.e., 𝜎𝐸𝑇 or 𝜎𝑇). After weighting each constraint
according to the number of observations, the likelihood of all observations was combined. Mathematically, For Configuration-1: 𝐴𝐼𝐶= ln(𝑀𝑆𝐸) + 2𝑘/𝑛 In addition to this, two uncertainty 15 values, describing the standard deviation of the observational noises of ET (𝜎𝐸𝑇) and T (𝜎𝑇) are
also retrieved. These parameters are estimated using Markov-Chain Monte-Carlo (MCMC)
method at each site. The method assumes parameters as random variables with some prior
distribution and then the parameters are updated using the prior knowledge based on Bayes’
theorem as: For Configuration-1: For Configuration-1:
log (𝐿(𝑦𝐸𝑇
(1:𝑛𝐸𝑇)|𝜃) = ∑
𝑙𝑜𝑔𝐿(𝑦𝐸𝑇
(𝑖)|𝜃)
𝑛𝐸𝑇
𝑖=0
(5)
For Configuration-2:
log (𝐿(𝑦𝐸𝑇
(1:𝑛𝐸𝑇), 𝑦𝑇
(1:𝑛𝑇)|𝜃) = (
1
𝑛𝐸𝑇∑
𝑙𝑜𝑔𝐿(𝑦𝐸𝑇
(𝑖)|𝜃) +
1
𝑛𝑇∑
𝑙𝑜𝑔𝐿(𝑦𝑇
(𝑖)|𝜃)
𝑛𝑇
𝑖=0
𝑛𝐸𝑇
𝑖=0
)
𝑛𝐸𝑇+𝑛𝑇
2
(6) For Configuration 1:
log (𝐿(𝑦𝐸𝑇
(1:𝑛𝐸𝑇)|𝜃) = ∑
𝑙𝑜𝑔𝐿(𝑦𝐸𝑇
(𝑖)|𝜃)
𝑛𝐸𝑇
𝑖=0
(5)
For Configuration-2: g
(𝑦𝐸𝑇
(1:𝑛𝐸𝑇)|𝜃) = ∑
𝑙𝑜𝑔𝐿(𝑦𝐸𝑇
(𝑖)|𝜃)
𝑛𝐸𝑇
𝑖=0
(5)
onfig ration 2 (5) For Configuration-2: For Configuration-2: For Configuration-2: log (𝐿(𝑦𝐸𝑇
(1:𝑛𝐸𝑇), 𝑦𝑇
(1:𝑛𝑇)|𝜃) = (
1
𝑛𝐸𝑇∑
𝑙𝑜𝑔𝐿(𝑦𝐸𝑇
(𝑖)|𝜃) +
1
𝑛𝑇∑
𝑙𝑜𝑔𝐿(𝑦𝑇
(𝑖)|𝜃)
𝑛𝑇
𝑖=0
𝑛𝐸𝑇
𝑖=0
)
𝑛𝐸𝑇+𝑛𝑇
2
(6) log (𝐿(𝑦𝐸𝑇
(1:𝑛𝐸𝑇), 𝑦𝑇
(1:𝑛𝑇)|𝜃) = (
1
𝑛𝐸𝑇∑
𝑙𝑜𝑔𝐿(𝑦𝐸𝑇
(𝑖)|𝜃) +
1
𝑛𝑇∑
𝑙𝑜𝑔𝐿(𝑦𝑇
(𝑖)|𝜃)
𝑛𝑇
𝑖=0
𝑛𝐸𝑇
𝑖=0
)
𝑛𝐸𝑇+𝑛𝑇
2
(6) (6) where L is likelihood of observed ET and T under given parameters 𝜃. 𝑛𝐸𝑇 and 𝑛𝑇 are the number
of observations of ET and T, respectively. 3. Datasets and site properties 16 All the models are applied at 84 flux tower sites in the PLUMBER2 dataset 72 across the globe. These flux sites span over multiple biomes and climate zones globally (Supplementary Table 1). PLUMBER2 dataset has been compiled from the FLUXNET2015, OzFlux, and La Thuile
collections, after quality control and gap-filling to make it suitable for land surface modeling. The
dataset also provides leaf area index (LAI) for each site derived from MODIS (Moderate
Resolution Imaging Spectroradiometer) product (MCD15A2H.006) with a temporal resolution
same as the flux measurements. Sites are chosen to ensure that soil moisture data are available,
and all other required variables are available for at least over one entire year. Only daytime data is
used for parameters estimation and model evaluation. Measurement and canopy heights are
obtained from site-specific metadata information. Soil properties are obtained from the soil map
used in Noah-LSM73. 4. Evapotranspiration partitioning methods Data availability PLUMBER2 fluxnet data is available at http://doi.org/10.25914/5fdb0902607e1. Soil property
data is available at http://globalchange.bnu.edu.cn/research/soil5d.jsp. 6. Estimating observed stomatal conductance Observed 𝑔𝑠𝑐 is obtained by inverting the SW ET model. Combining Equations 1a-1s in
Methods: Observed 𝑔𝑠𝑐 is obtained by inverting the SW ET model. Combining Equations 1a-1s in
Methods: Observed 𝑔𝑠𝑐 is obtained by inverting the SW ET model. Combining Equations 1a-1s in
M th d 𝐸𝑇= 𝐸+ 𝑇= 𝑓(𝑟𝑠𝑐, 𝑟𝑠𝑠, 𝑟𝑎𝑎, 𝑟𝑎𝑐, 𝑟𝑎𝑠, ℎ𝑦𝑑𝑟𝑜𝑚𝑒𝑡𝑒𝑜𝑟𝑜𝑙𝑜𝑔𝑖𝑐𝑎𝑙 𝑣𝑎𝑟𝑖𝑎𝑏𝑙𝑒𝑠, 𝑠𝑜𝑖𝑙 𝑝𝑟𝑜𝑝𝑒𝑟𝑡𝑖𝑒𝑠) (8)
In this study, the calculation of 𝑟𝑠𝑠, 𝑟𝑎𝑎, 𝑟𝑎𝑐, and 𝑟𝑎𝑠 is performed using the standard parameters in
their calculation as explained in Methods: Modeling stomatal conductance, ET, and T. Notably,
these calculations are kept same for all the models for fair model intercomparison. Observed 𝑟𝑠𝑐
(and hence 𝑔𝑠𝑐) for each site can then be obtained by inverting Equation 8 given the observed ET
of the site. 4. Evapotranspiration partitioning methods To partition ET into E and T, we used three different approaches, namely Transpiration Estimation
Algorithm (TEA)42, based on Underlying Water Use Efficiency (uWUE)43, and based on leaf and
ecosystem water use efficiency (Yu22)44. TEA is a nonlinear ML method that predicts the T using a Random Forest (RF) regressor trained
on ecosystem water use efficiency ( 𝑊𝑈𝐸𝑒𝑐𝑜= 𝐺𝑃𝑃/𝐸𝑇; where GPP is Gross Primary
Productivity) during dry periods of growing season (i.e., periods when 𝐸≈0 𝑜𝑟 𝐸𝑇= 𝑇). Selection of dry periods are done by filtering out the wet periods based on the time series of
precipitation and ET, as detailed in 42. Trained model on filtered data is then used to predict
𝐺𝑃𝑃/𝑇 for the full time series. uWUE-based ET partitioning is quite straightforward and uses only data of GPP, VPD, and ET to
do ET partitioning. Ratio T:ET from this method is obtained as: do ET partitioning. Ratio T:ET from this method is obtained as: 𝑇
𝐸𝑇= 𝑢𝑊𝑈𝐸𝑎
𝑢𝑊𝑈𝐸𝑝
(7𝑎) where 𝑢𝑊𝑈𝐸𝑎 (= 𝐺𝑃𝑃∙√𝑉𝑃𝐷/𝐸𝑇) is apparent WUE and can be calculated at a desired temporal
resolution (temporal resolution of flux observations in this study). 𝑢𝑊𝑈𝐸𝑝 is potential WUE that
basically represents the maximum carbon gain to water loss. 𝑢𝑊𝑈𝐸𝑝 is estimated as 95𝑡ℎ quantile
regression slope between 𝐺𝑃𝑃∙√𝑉𝑃𝐷 and ET for a given year. Lastly, Yu22 method does the ET partitioning based on leaf and ecosystem WUE44. Ratio T:ET
from this method is obtained as: hod does the ET partitioning based on leaf and ecosystem WUE . Ratio T:ET
is obtained as: 𝑇
𝐸𝑇= 𝑊𝑈𝐸𝑒𝑐𝑜
𝑊𝑈𝐸𝑙𝑒𝑎𝑓
=
𝐺𝑃𝑃
𝐸𝑇
𝐶𝑎𝑃𝑎
1.6(𝑉𝑃𝐷+ 𝑔1√𝑉𝑃𝐷)
(7𝑏) (7𝑏) where 𝐶𝑎 is the atmospheric 𝐶𝑂2 concentration, 𝑃𝑎 is atmospheric air pressure, 𝑔1 is a theoretical
parameter of Medlyn’s model65. The estimated 𝑔1 values for the sites are obtained from74,75. As
𝑔1 values could be obtained only for 40 out of 84 sites, so results for Yu22 method are evaluated
only for those 40 sites. where 𝐶𝑎 is the atmospheric 𝐶𝑂2 concentration, 𝑃𝑎 is atmospheric air pressure, 𝑔1 is a theoretical
parameter of Medlyn’s model65. The estimated 𝑔1 values for the sites are obtained from74,75. As
𝑔1 values could be obtained only for 40 out of 84 sites, so results for Yu22 method are evaluated
only for those 40 sites. 5. 4. Evapotranspiration partitioning methods Random forest regressor 17 Random forest (RF) regressors76, are used to determine explanatory factors of stomatal
conductance (𝑔𝑠𝑐) based on 𝑔𝑠𝑐 from empirical model (EMP), explicit representation of plant
hydraulic model (PHexp), implicit representation of plant hydraulic model (PHimp), machine
learning model (ML), and observations of 𝑔𝑠𝑐. We use 6 predictors, including vapor pressure
deficit (VPD), soil moisture (SM), atmospheric 𝐶𝑂2 concentration (𝐶𝑂2), incoming solar radiation
(SRad), leaf area index (LAI), and air temperature (𝑇𝑎). Sources of these variables are described
in Supplementary Table 2. Data from all the sites are used to develop the RF regressors. The RF algorithm with the scikit-learn package in Python is implemented. RF is an ensemble
learning method that generates multiple decision trees using a randomly selected subset of samples
through replacement. In this study, we develop RF regressors with 500 decision trees with a leaf
node size no smaller than 5. Around 4,99,500 samples are used in the RF regressor. We use partial
dependence plots (PDP) to visualize the relations between the hydroclimatic variables and 𝑔𝑠𝑐. PDP calculates the marginal effect each feature variable has on the predicted 𝑔𝑠𝑐, while other
covariates were assumed to be held constant49. Random forest (RF) regressors76, are used to determine explanatory factors of stomatal
conductance (𝑔𝑠𝑐) based on 𝑔𝑠𝑐 from empirical model (EMP), explicit representation of plant
hydraulic model (PHexp), implicit representation of plant hydraulic model (PHimp), machine
learning model (ML), and observations of 𝑔𝑠𝑐. We use 6 predictors, including vapor pressure
deficit (VPD), soil moisture (SM), atmospheric 𝐶𝑂2 concentration (𝐶𝑂2), incoming solar radiation
(SRad), leaf area index (LAI), and air temperature (𝑇𝑎). Sources of these variables are described
in Supplementary Table 2. Data from all the sites are used to develop the RF regressors. The RF algorithm with the scikit-learn package in Python is implemented. RF is an ensemble
learning method that generates multiple decision trees using a randomly selected subset of samples
through replacement. In this study, we develop RF regressors with 500 decision trees with a leaf
node size no smaller than 5. Around 4,99,500 samples are used in the RF regressor. We use partial
dependence plots (PDP) to visualize the relations between the hydroclimatic variables and 𝑔𝑠𝑐. PDP calculates the marginal effect each feature variable has on the predicted 𝑔𝑠𝑐, while other
covariates were assumed to be held constant49. Code availability The codes for the analyses will be made available at GitHub and will be archived on Zenodo
after acceptance. [Editor’s note: upon request, the authors made the code available for review
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Breiman, L. Random forests. Machine learning 45, 5-32 (2001). 22 Acknowledgment
This work was supported by NSF grants OIA-2019561, EAR-1856054, and DGE-2152140. Author contribution
PR and MK conceived the study and designed the methodology. PR compiled the data, developed
relevant codes for analyses and visualizations, and generated the results. YL helped with explicit
plant hydraulic model implementation and parameter calibration. PR and MK performed data
analyses and drafted the manuscript. YL provided thoughtful inputs along the way. All authors
edited the manuscript and helped improve it. MK provided project administration, supervision,
and acquired funding to support the project. Acknowledgment
This work was supported by NSF grants OIA-2019561, EAR-1856054, and DGE-2152140.
Author contribution Acknowledgment Acknowledgment
This work was supported by NSF grants OIA-2019561, EAR-1856054, and DGE-2152140. Author contribution PR and MK conceived the study and designed the methodology. PR compiled the data, developed
relevant codes for analyses and visualizations, and generated the results. YL helped with explicit
plant hydraulic model implementation and parameter calibration. PR and MK performed data
analyses and drafted the manuscript. YL provided thoughtful inputs along the way. All authors
edited the manuscript and helped improve it. MK provided project administration, supervision,
and acquired funding to support the project. 23 Supplementary Files This is a list of supplementary ¦les associated with this preprint. Click to download. SupplementaryDoc.docx NCOMMS2311270rs.pdf
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https://openalex.org/W4290694250
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http://www.scielo.br/pdf/pab/v52n3/1678-3921-pab-52-03-00177.pdf
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English
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Control of the vegetative growth of 'Shinseiki' pear trees by prohexadione calcium and root pruning
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Pesquisa Agropecuária Brasileira
| 2,017
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cc-by
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Control of the vegetative growth of 'Shinseiki' pear
trees by prohexadione calcium and root pruning
Bruno Carra(1), José Carlos Fachinello(2), Everton Sozo de Abreu(1), Mateus da Silveira Pasa(3),
Daniel Spagnol(1), Marcos Antônio Giovanaz(1) and Carina Pereira da Silva(4) (1)Universidade
Federal
de
Pelotas,
Faculdade
de
Agronomia
Eliseu
Maciel,
Departamento
de
Fitotecnia,
Campus
Universitário, s/no, CEP 96010-900 Capão do Leão, RS, Brazil. E-mail: brunocarra@hotmail.com, sozodeabreu@hotmail.com,
spagnol.agro@hotmail.com, giovanazmarcos@gmail.com (2)In memoriam. (3)Empresa de Pesquisa Agropecuária e Extensão Rural
de Santa Catarina, Estação Experimental de São Joaquim, Rua João Araújo Lima, no 102, Jardim Caiçara, CEP 88600-000
São Joaquim, SC, Brazil. E-mail: mateuspasa@epagri.sc.gov.br (4)Rua José de Souza Borges, no 102, CEP 88600-000 São Joaquim, SC,
Brazil. E-mail: carpers.rs@gmail.com Abstract – The objective of this work was to evaluate the effect of prohexadione calcium (P-Ca) and root
pruning (RP) on the control of the vegetative growth and on fruit production and quality of 'Shinseiki' pear
trees, under the climatic conditions of Southern Brazil. The experiment was conducted during the 2013/2014
and 2014/2015 crop seasons, in a 15-year-old orchard of 'Shinseiki' pear trees, trained to a central-leader
system, with a 2x5 m spacing between plants, grafted onto Pyrus calleryana rootstock. The experiment was
carried out in a randomized complete block design, with six treatments and four replicate. The treatments
consisted of the control (without P-Ca application or RP), two P-Ca rates (300 and 600 mg L-1), two root
pruning intensities (performed on one or on both sides of the tree), and root pruning on one side + 300 mg L-1
P-Ca. P-Ca reduced vegetative growth more effectively than RP, in both crop seasons, and the combination
of both techniques did not result in additional control of the vegetative growth, compared with P-Ca alone. Vegetative growth was not affected by increasing the rates of P-Ca. The highest P-Ca dose negativelly affected
fruit yield. Regarding fruit quality, RP increases the total soluble solids content at harvest. Controle do crescimento vegetativo de pereiras 'Shinseiki'
por prohexadiona de cálcio e poda de raiz Resumo – O objetivo deste trabalho foi avaliar o efeito da prohexadiona de cálcio (P-Ca) e da poda de raiz
(PR) no controle do crescimento vegetativo e na produção e na qualidade dos frutos de pereiras 'Shinseiki', sob
condições climáticas do Sul do Brasil. O experimento foi realizado durante as safras de 2013/2014 e 2014/2015,
em pomar de pereiras 'Shinseiki' com 15 anos de idade, conduzido em líder central, com espaçamento de
2x5 m entre plantas, enxertadas em porta-enxerto Pyrus calleryana. O experimento foi realizado em blocos
ao acaso, com seis tratamentos e quatro repetições. Os tratamentos consistiram na testemunha (sem aplicação
de P-Ca e sem PR), em duas doses de P-Ca (300 e 600 mg L-1), em duas intensidades de poda de raiz (realizada
em um ou nos dois lados da planta) e na poda de raiz em um lado da planta + 300 mg L-1 de P-Ca. A P-Ca
reduziu o crescimento vegetativo mais efetivamente do que a PR, em ambas as safras, e a combinação de
ambas as técnicas não resultou em controle adicional do crescimento vegetativo, em comparação à P-Ca
utilizada sozinha. O crescimento vegetativo não foi influenciado pelo incremento nas doses de P-Ca. A maior
dose de P-Ca afetou negativamente a produtividade de frutos. Quanto à qualidade de frutos, a PR aumenta o
teor de sólidos solúveis totais na colheita. Termos para indexação: Pyrus pyrifolia, qualidade de fruto, fitorreguladores, pomar adensado, vigor
vegetativo, componentes da produção. Termos para indexação: Pyrus pyrifolia, qualidade de fruto, fitorreguladores, pomar adensado, vigor
vegetativo, componentes da produção. Pesq. agropec. bras., Brasília, v.52, n.3, p.177-185, mar. 2017
DOI: 10.1590/S0100-204X2017000300005 Introduction The experiment
was arranged in a randomized complete block design,
with six treatments – combinations of prohexadione
calcium (P-Ca) doses and root pruning (RP) – and four
replications of three trees each. Only the central tree
was evaluated (experimental unit), while the other two
were used as border. The trees were selected according
to their uniformity and size (canopy volume), and then
grouped into blocks based on trunk circumference
measured 25 cm above the ground. P-Ca is a gibberellin (GA) biosynthesis inhibitor
(Rademacher, 2004) used to control vegetative growth of
different crops. Several studies have shown its efficiency
in different pear cultivars (Sugar et al., 2004; Asín et
al., 2007, Hawerroth et al., 2012; Einhorn et al., 2014;
Pasa & Einhorn, 2014). As to RP, it is a method used to
reduce root area – reducing the flow of nutrients, water
and hormones from the roots to the shoots –, limiting
its growth. According to Rodriguez-Gamir et al. (2010),
RP is an effective tool for controlling vegetative growth. Reduction in shoot number and shoot length of root
pruned trees have been observed (Asín et al., 2007;
Maas, 2008; Vercammen et al., 2005), as well as a higher
number of flower buds and a more regular production
(Vercammen et al., 2005). Maas (2008) observed that
none or only small benefits can be derived from the use
of plant growth regulators when RP is used to control
vegetative growth, but this demands further verification
for Brazilian orchards conditions. The trees were pruned at the end of the winter period
in both seasons (August 12, 2013; and August 18, 2014),
and, after 15 days, Erger (5%, added to 5% calcium
nitrate) was applied for induction and standardization
of budburst and flowering. The treatments were
applied to the same trees in both growing seasons, and
they consisted of: control, without P-Ca applicaiton
and without RP; RP in one side of the tree (1S); RP
in two sides of the tree (2S); P-Ca at 300 mg L-1 of
active ingredient (a.i.); P-Ca 600 mg L-1 a.i.; and RP
1S + P-Ca 300 mg L-1 of a.i. All P-Ca treatments were
sprayed when shoots were 5–10 cm long. The P-Ca
source was the commercial product Viviful (27.5%
a.i.; Iharabras S.A., Chemical Industries). Solutions of
P-Ca were supplemented with 0.05% (v:v) of surfactant
Silwet L-77 Ag (0.5 mL L-1). Introduction growth negatively affects yield (Pasa et al., 2011) and
the control of pests and diseases – due to the reduced
efficiency of sanitary applications (Miller & Tworkoski,
2003) –, and increases pruning costs (Hawerroth et
al., 2012). For achieving economically satisfactory
production and improving fruit quality, management Vegetative growth control of fruit trees is necessary
to avoid excessive shading within the canopy, in order
to promote interception of solar radiation and improve
fruit quality (Rademacher, 2004). Excessive vegetative Pesq. agropec. bras., Brasília, v.52, n.3, p.177-185, mar. 2017
DOI: 10.1590/S0100-204X2017000300005 178 B. Carra et al. municipality of Capão do Leão, state of Rio Grande
do Sul, Brazil (31º52'00"S, 52º21'24"W, and 48-m
altitude), during the 2013/2014 and 2014/2015 growing
seasons. According to Köppen’s classification, the
climate of the studied region is Cfa, i.e., humid
subtropical, with annual rainfall average of 1,367 mm,
temperature of 17.8oC, and humidity of 80.7%. The
average accumulation of chill hours below 7.2oC is 321,
during the winter, according to data provided by the
Pelotas weather station. The soil in the experimental
area is an Argissolo Amarelo distrófico (Ultisol),
according to the Brazilian classification (Santos et al.,
2013).i techniques that promote a proper balance between
vegetative growth and fruiting must be used (Sharma et
al., 2009), especially in vigorous cultivars and rootstocks. The main techniques for controlling vegetative
growth in fruit trees are branch bending, pruning, and
the use of dwarfing rootstocks. Branch bending leads to
reduced shoot growth by decreasing apical dominance. In Brazil, studies about dwarfing rootstocks are
recent, and there are few options available for pears
(Hawerroth et al., 2012). Control of vegetative growth
by summer and winter pruning, therefore, is essential
to ensure the productivity of orchards and to improve
fruit quality (Cline et al., 2008). However, summer and
winter pruning may increase production costs. In this
context, the use of growth regulators and root pruning
(RP) is an alternative to reduce excessive vegetative
growth of pear trees [Pyrus pyrifolia (Burm) Nakai],
in order to provide an adequate balance between
vegetative growth and fruiting. Plant material consisted of fifteen-year-old 'Shinseiki'
pear trees (P. pyrifolia), grafted onto P. calleryana
rootstock. Trees were spaced 2x5 m, totaling 1,000
trees ha-1, and trained to a central-leader system. The
trees were planted in a north-south row orientation,
so that they could intercept the maximum amount of
sunlight during the growing season. Introduction RP was performed at the
end of August, during the dormancy period, in both
seasons, four to five weeks before the flowering start,
which occurred on September 15, 2013, and October The objective of this work was to evaluate the
effect of prohexadione calcium and root pruning
on the control of the vegetative growth and on fruit
production and quality of 'Shinseiki' pear trees, under
climatic conditions of Southern Brazil. Pesq. agropec. bras., Brasília, v.52, n.3, p.177-185, mar. 2017
DOI: 10.1590/S0100-204X2017000300005 Materials and Methods The experiment was carried out at the experimental
field of the Universidade Federal de Pelotas, in the Pesq. agropec. bras., Brasília, v.52, n.3, p.177-185, mar. 2017
DOI: 10.1590/S0100-204X2017000300005 Pesq. agropec. bras., Brasília, v.52, n.3, p.177-185, mar. 2017
DOI: 10.1590/S0100-204X2017000300005 Control of the vegetative growth of 'Shinseiki' pear trees 179 13, 2014. In the case of RP 1S, root pruning was
performed in the east side. P-Ca was sprayed to the
point of product runoff in the plant, using a motorized
backpack sprayer Jacto. For RP, an agricultural tool
with a blade coupled to a tractor was used. The blade
was set to a cutting depth of 50 cm, and it was passed
50 cm apart from the trunk. within the canopy were cut, which left a portion of
the shoot with two buds (≈10 cm). Winter pruning
was performed at the end of dormancy period, in both
seasons, with the cutting of vigorous, upright, and
poorly positioned shoots in the canopy. Fruit were harvested at commercial timing on
January 23, 2014 (106 DAFB), and fruit per tree were
counted and weighted. Estimated yield was calculated
based on the yield per tree and on the number of trees
per ha. The average fruit weight was calculated by
dividing the yield per tree by the number of fruits
per tree. Fruit qualitative attributes were determined
in a sample of 10 fruits per tree, randomly selected at
harvest. Shoot length was measured with a metric tape on 10
one-year-old shoots, with similar diameters, in the four
quadrants of the plant, at similar heights. The shoots
were tagged at the time of the first P-Ca application and,
then, at biweekly intervals until the end of the season. Similarly, fruit diameter in the 2013/2014 season was
measured on 10 fruits with similar diameters, in the
four quadrants of the tree, at a similar height. The
fruits were evenly selected and tagged between 20 and
25 days after full bloom (DAFB) and, then, at biweekly
intervals until harvest. Average growth rate (mm day-1)
was calculated using biweekly shoot length data. Trunk diameter was measured at 25-cm height from
the soil surface with a digital caliper, at the beginning
of the trial and at the end of each season, and expressed
as trunk cross sectional area (TCSA, cm2), calculated
according to Pasa et al. (2012). Materials and Methods In order to calculate
TCSA increase, the values obtained at the end of each
season were subtracted from previous season TCSA.l The qualitative attributes of the fruit analyzed were
fruit firmness (FF), total soluble solids (TSS), and
titratable acidity (TA). Fruit firmness was determined
by removing fruit skin on two opposite sides of each
fruit, using a digital penetrometer with an 8 mm
diameter probe, and expressed in Newton (N). To
measure the total soluble solids (TSS), the sample
was juiced and placed in a digital refractometer model
Atago PAL-1, and expressed as oBrix. Titratable acidity
(TA) was calculated according to Carra et al. (2016),
and expressed as the percentage of malic acid. Statistical analysis was performed using the R
software (R Core Team, 2014), with the package
ExpDes (Ferreira et al., 2013). Data expressed as
percentage or counts were transformed when they
did not meet the assumptions for analysis of variance,
using the arcsin [square root (n + 1)] and square root
(n + 0.5) transformations, respectively. When the F test
was significant, treatment means were compared with
Duncan’s test, at 5% probability. p
The total number of flower clusters per tree was
recorded at full bloom. After natural fruit drop (30–
40 DAFB), the remaining fruits in each tree were
counted and fruit set was calculated according to the
ratio (number of fruits per tree)/(number of flower
clusters per tree), and expressed as the percentage of
fruit per flower cluster. Full bloom occurred on October
09, 2013; October 28, 2014; and September 18, 2015. After fruit set evaluation, thinning was performed,
which left two to three fruits per flower cluster. Yield
components of 2014/2015 growing season were not
recorded due to almost total abortion of flower buds,
probably due to insufficient accumulation of chilling
hours bellow 7.2oC (264 hours), in Pelotas, during
the winter of 2014. In the previous winter (2013), the
accumulation of chilling hours was 464 hours. Pesq. agropec. bras., Brasília, v.52, n.3, p.177-185, mar. 2017
DOI: 10.1590/S0100-204X2017000300005 Results and Discussion The treatments significantly reduced shoot growth
and TCSA increment compared to control, in both
seasons (Figure 1 and Table 1). Shoots of RP trees (1S
and 2S) showed lower reduction in length compared to
trees treated with P-Ca – regardless of the application
rate – and to RP 1S + P-Ca 300 mg L-1, in the 2013/2014
growing season. Similar results were also observed
in the 2014/2015 growing season, but RP 1S did not
differ from P-Ca 300 mg L-1. The differences in shoot
growth rate observed from 36 DAFB in 2013/2014, and
from 28 DAFB in 2014/2015 are shown in Figure 1. Trees were pruned in the summer and the winter. In both occasions, number and weight of pruned
shoots were recorded and, in the winter pruning,
their length was also measured. Summer pruning was
done in the second half of December, in both seasons,
approximately 60 DAFB, when all the upright shoots Pesq. agropec. bras., Brasília, v.52, n.3, p.177-185, mar. 2017
DOI: 10.1590/S0100-204X2017000300005 B. Carra et al. 180 Pesq. agropec. bras., Brasília, v.52, n.3, p.177-185, mar. 2017
DOI: 10.1590/S0100-204X2017000300005 Control of the vegetative growth of 'Shinseiki' pear trees (2012) reported 31–50%
reduction in shoot length, at 146 DAFB, compared
to control trees. Additionally, Einhorn et al. (2014)
observed reduction of up to 70% in the shoot growth of
'd’Anjou' pear trees treated once with 250 mg L-1 P-Ca. As to RP, Asín et al. (2007) reported that 'Blanquilla'
pear trees, root pruned in both sides of the plants (40 cm
from the stem, at 40-cm depth), showed a reduction of
25% in final shoot lengths. Vercammen et al. (2005)
observed that 'Conference' pear trees treated with RP
1S, performed for two consecutive years, and RP 2S,
in a single year, had their vegetative growth reduced
in 27% and 44%, respectively. In a two-year study
developed by Wang et al. (2014b), pear trees treated
with RP 2S (40 cm from the stem, at 30-cm depth)
showed reduction of 72% in total shoot length. In 'Housui' pear trees sprayed with different rates
of P-Ca, Hawerroth et al. (2012) reported 31–50%
reduction in shoot length, at 146 DAFB, compared
to control trees. Additionally, Einhorn et al. (2014)
observed reduction of up to 70% in the shoot growth of
'd’Anjou' pear trees treated once with 250 mg L-1 P-Ca. As to RP, Asín et al. (2007) reported that 'Blanquilla'
pear trees, root pruned in both sides of the plants (40 cm
from the stem, at 40-cm depth), showed a reduction of
25% in final shoot lengths. Vercammen et al. (2005)
observed that 'Conference' pear trees treated with RP
1S, performed for two consecutive years, and RP 2S,
in a single year, had their vegetative growth reduced
in 27% and 44%, respectively. In a two-year study
developed by Wang et al. (2014b), pear trees treated
with RP 2S (40 cm from the stem, at 30-cm depth)
showed reduction of 72% in total shoot length. Regarding summer pruning, trees treated with RP
and P-Ca showed smaller number and lower weight of
pruned shoots, compared to control, in both seasons,
except for RP 1S in the 2014/2015 season (Table 2). This decrease was most evident in trees treated with
P-Ca, either alone or combined with RP, where a
reduction of ~65% in the number of pruned shoots per
tree, and of ~74% in pruning weight was observed in
both seasons. Pesq. agropec. bras., Brasília, v.52, n.3, p.177-185, mar. 2017
DOI: 10.1590/S0100-204X2017000300005 Control of the vegetative growth of 'Shinseiki' pear trees 181 The results with P-Ca application show the
effectiveness of this plant regulator to control vegetative
growth of 'Shinseiki' pears. Several authors reported
vegetative growth reduction in pear trees by using
P-Ca (Costa et al., 2004; Rademacher, 2004; Smit et
al., 2005; Hawerroth et al., 2012; Einhorn et al., 2014;
Pasa & Einhorn, 2014; Carra et al., 2016). Moreover,
the association of RP with P-Ca application did not
result in additional vegetative growth control, when
compared to P-Ca used alone (Table 1). Similar results
were observed by Maas (2008), who also found no
synergistic effect of the combination of RP and P-Ca. The reduction of vegetative growth in root pruned
pear trees may be related to a lower absorption of
water (Janssens et al., 2011) and nutrients (Wang et al.,
2014a) by the tree, due to the reduced volume of soil
occupied by roots. Moreover, the roots are the main site
of cytokinin synthesis, a plant hormone responsible
for cell division and elongation (Fosket & Kieber,
2013) that plays an important role on the promotion
of vegetative growth. Therefore, the reduction in
root volume would reduce the levels of cytokinin
transported to the canopy, thus limiting its growth. In both seasons, the treatments resulted in lower
increase of TCSA compared to control (Table 1). The
lowest values were observed with P-Ca 300 mg L-1
and 600 mg L-1, and with RP 1S + P-Ca 300 mg L-1. Reduction in TCSA increases in response to P-Ca
application was also found in apple trees (Zadravec et
al., 2008). In the first season, RP 1S and RP 2S resulted
in a reduction of 26.62 to 34.7% in TCSA increases,
compared to control. Significant differences were
observed between these two levels of root pruning;
with RP 2S showing reduction in TCSA increase
similar to that observed with the application of P-Ca
300 mg L-1. Ferree & Knee (1997) reported similar
results for apple trees, where in the first 5 years of RP,
'Golden Delicious' apple trees showed a reduction of
14% in TCSA increase, and, in the sixth year, a 22%
reduction. In the second season, the reduction in TCSA
increases for all treatments ranged from 27 to 37%,
compared to control. synergistic effect of the combination of RP and P Ca. In 'Housui' pear trees sprayed with different rates
of P-Ca, Hawerroth et al. B. Carra et al. The vegetative growth control provided by P-Ca and
RP, and by their association, was effective during the whole season, and P-Ca induced greater reduction in
shoot growth than RP (Figure 1). Pesq. agropec. bras., Brasília, v.52, n.3, p.177-185, mar. 2017
DOI 10 1590/S0100 204X2017000300005
The vegetative growth control provided by P-Ca and
RP, and by their association, was effective during the
whole season, and P-Ca induced greater reduction in
shoot growth than RP (Figure 1). Figure 1. Shoot length (A and B), shoot growth rate (C and D), and fruit diameter (E) of 'Shinseiki' pear trees treated with
prohexadione calcium (P-Ca) and root pruning (RP), in the 2013/2014 (A, C and E) and the 2014/2015 (B and D) growing
seasons. Circle on the x-axis denotes the application of P-Ca. Vertical bars represent ±standard error. Figure 1. Shoot length (A and B), shoot growth rate (C and D), and fruit diameter (E) of 'Shinseiki' pear trees treated with
prohexadione calcium (P-Ca) and root pruning (RP), in the 2013/2014 (A, C and E) and the 2014/2015 (B and D) growing
seasons. Circle on the x-axis denotes the application of P-Ca. Vertical bars represent ±standard error. Pesq. agropec. bras., Brasília, v.52, n.3, p.177-185, mar. 2017
DOI: 10.1590/S0100-204X2017000300005 Control of the vegetative growth of 'Shinseiki' pear trees Control of the vegetative growth of 'Shinseiki' pear trees In the second season, RP 1S + P-Ca
300 mg L-1 and P-Ca 600 mg L-1 showed lower pruning
weight than RP 1S and control. That was the single variable where RP 1S + P-Ca 300 mg L-1 showed a
significant difference compared to treatments with
P-Ca applications alone, in 2013/2014 season, proving
the effects of the combination of both techniques to be
minimal in controlling vegetative growth of pear trees,
as observed by Maas (2008). For total shoot length of
pruned shoots, the major reduction was observed with
RP 1S + P-Ca 300 mg L-1 and P-Ca 600 mg L-1. g
g
Similar results were observed for 'Housui' pear trees
treated with P-Ca, where the reduction in pruning
weight per tree (winter pruning) ranged from 28.6 to
63% compared to control trees, while the reduction
of the number of pruned shoots ranged from 39.3 to
66.7% (Hawerroth et al., 2012). According to Wang et
al. (2014b), root pruned pear trees showed a decrease
of 43% in the number of shoots per tree. The decrease
in number, weight and total length of shoots pruned
in 'Shinseiki' pear trees, in response to RP and P-Ca
application, indicates that these two vegetative growth
control methods minimize the formation of strong
vegetative drains. They also reduce the excessive
growth and the need for summer and winter pruning,
which is an important component of the cost for pear
orchards, representing approximately 14% of total
variable cost (Seavert et al., 2005). Table 2. Effects of prohexadione calcium (P-Ca) application
and root pruning (RP) on the number (NPS) and mass
(MPS) of pruned shoots per tree in summer and winter
prunings, and total length of pruned shoots per tree (LPS)
in winter pruning of 'Shinseiki' pear trees(1). Control of the vegetative growth of 'Shinseiki' pear trees Treatment
Summer pruning
Winter pruning
NPS
MPS (g)
NPS
MPS (g) LPS (cm)
2013/2014 growing season
Control
68.3a
1,747.5a
30.0a
599.0a
1,220.8a
RP 1S
47.0b
1,117.0b
24.0a
416.5b
720.3b
RP 2S
41.5b
960.0b
22.8a
424.5b
818.3b
P-Ca 300 mg L-1
24.5c
494.8c
15.0b
328.0c
602.8bc
P-Ca 600 mg L-1
21.3c
434.0c
9.8b
317.0c
472.8c
RP 1S + P-Ca 300 mg L-1
20.3c
417.0c
11.0b
245.5d
409.8c
CV (%)
6.36
12.34
12.79
10.2
19.76
p
0.01
0.01
0.01
0.01
0.01
2014/2015 growing season
Control
71.3a
1,934.5a
32.5a
657.1a
1,082.9a
RP 1S
59.3ab 1,362.5b
23.0b
339.3b
621.0b
RP 2S
45.5b
1,006.0b
17.5c
313.8bc
494.5bc
P-Ca 300 mg L-1
26.0c
476.5c
15.3c
223.8bc
373.7bc
P-Ca 600 mg L-1
25.8c
496.5c
13.8c
194.5c
351.8bc
RP 1S + P-Ca 300 mg L-1
18.3c
379.8c
14.3c
189.3c
297.9c
CV (%)
12.53
27.02
7.97
26.07
24.67
p
0.01
0.01
0.01
0.01
0.01
(1)Means followed by equal letters do not differ by Duncan’s test at 5%
probability. RP 1S, root pruning performed in one side of the pear tree;
and RP 2S, root pruning performed in both sides of the tree. RP and P-Ca applications did not affect fruit set
and the number of fruits before and after thinning,
compared to control trees in the 2013/2014 season
(Table 3). Similar results were reported for P-Ca
applications (Sugar et al., 2004) and RP (Asín et al.,
2007), where fruit set and the number of fruits per tree
were not significantly affected. Einhorn et al. (2014),
however, observed contrasting results, where the Table 3. Effect of prohexadione calcium (P-Ca) and root pruning (RP) on fruit set, number of fruits before thinning (BT), number of fruits after thinning
(AT), fruit yield, average fruit weight, and estimated yield of 'Shinseiki' pear trees, in the 2013/2014 growing season(1). Table 3. Effect of prohexadione calcium (P-Ca) and root pruning (RP) on fruit set, number of fruits before thinning (BT), number of fruits after thinning
(AT), fruit yield, average fruit weight, and estimated yield of 'Shinseiki' pear trees, in the 2013/2014 growing season(1). Control of the vegetative growth of 'Shinseiki' pear trees The reduction in the number and weight
of pruned shoots of RP treated trees was intermediate,
but significantly different from control, except for
RP 1S in the second season. i Table 1. Total shoot length and trunk cross-sectional area
(TCSA) increases of 'Shinseiki' pear trees as affected by
prohexadione calcium (P-Ca) application and root pruning
(RP) in the 2013/2014 and 2014/2015 growing seasons(1). Table 1. Total shoot length and trunk cross-sectional area
(TCSA) increases of 'Shinseiki' pear trees as affected by
prohexadione calcium (P-Ca) application and root pruning
(RP) in the 2013/2014 and 2014/2015 growing seasons(1). Treatment
2013/2014
2014/2015
Shoot
length
(cm)
TCSA
increase
(cm-2)
Shoot
length
(cm)
TCSA
increase
(cm-2)
Control
34.60a
16.08a
36.72a
22.43a
RP 1S
24.00b
11.80b
18.74bc
15.30b
RP 2S
26.27b
10.50c
20.12b
16.35b
P-Ca 300 mg L-1
8.60c
9.91cd
14.37cd
14.37b
P-Ca 600 mg L-1
7.84c
9.15d
13.58d
14.11b
RP 1S + P-Ca 300 mg L-1
9.74c
9.52d
12.00d
14.55b
CV (%)
23.16
5.14
16.77
11.04
p
0.01
0.01
0.01
0.01
(1)Means followed by equal letters do not differ by Duncan’s test at 5%
probability. RP 1S, root pruning performed in one side of the pear tree;
and RP 2S, root pruning performed in both sides of the tree. Treatment
2013/2014
2014/2015
Shoot
length
(cm)
TCSA
increase
(cm-2)
Shoot
length
(cm)
TCSA
increase
(cm-2)
Control
34.60a
16.08a
36.72a
22.43a
RP 1S
24.00b
11.80b
18.74bc
15.30b
RP 2S
26.27b
10.50c
20.12b
16.35b
P-Ca 300 mg L-1
8.60c
9.91cd
14.37cd
14.37b
P-Ca 600 mg L-1
7.84c
9.15d
13.58d
14.11b
RP 1S + P-Ca 300 mg L-1
9.74c
9.52d
12.00d
14.55b
CV (%)
23.16
5.14
16.77
11.04 Significant results were also observed in winter
pruning for the number, weight, and length of pruned
shoots per tree, (Table 2). In the first season, the
number of pruned shoots was lower with P-Ca and
RP 1S + P-Ca 300 mg L-1. In the second season, the
lowest values were observed with P-Ca application, Pesq. agropec. bras., Brasília, v.52, n.3, p.177-185, mar. 2017
DOI: 10.1590/S0100-204X2017000300005 B. Carra et al. 182 RP 1S + P-Ca 300 mg L-1, and RP 2S, followed by RP
1S. The lowest weight of pruned shoots was observed
with RP 1S + P-Ca 300 mg L-1, followed by treatments
with both rates of P-Ca, and by RP 1S and 2S, in the
2013/2014 season. ans followed by equal letters do not differ by Duncan’s test at 5% probability. RP 1S, root pruning performed in one side
oot pruning performed in both sides of the tree. Pesq. agropec. bras., Brasília, v.52, n.3, p.177-185, mar. 2017
DOI: 10.1590/S0100-204X2017000300005 (1)Means followed by equal letters do not differ by Duncan’s test at 5% probability. RP 1S, root pruning performed in one side of the pear tree; and RP
2S, root pruning performed in both sides of the tree. esq. ag opec. b as.,
as
a, v.5 , .3, p. 77
DOI: 10.1590/S0100-204X2017000300005 Control of the vegetative growth of 'Shinseiki' pear trees Treatment
Fruit set (%)
Number of fruits per tree
Yield
(kg tree-1)
Average fruit
weight (g)
Estimated yield
(Mg ha-1)
Before thinning
After thinning
Control
95.28
317.8
220.5
38.14a
172.93a
38.14a
RP 1S
91.89
255.3
178.3
32.54ab
171.42a
32.54ab
RP 2S
90.54
328.8
206.0
37.97a
178.79a
37.97a
P-Ca 300 mg L-1
89.49
287.3
211.3
38.47a
169.67a
38.47a
P-Ca 600 mg L-1
91.91
216.0
137.0
25.69b
168.51a
25.69b
RP 1S + P-Ca 300 mg L-1
93.96
303.3
184.0
29.94ab
152.83b
29.94ab
CV (%)
15.18
12.45
12.55
15.99
5.15
15.99
p
0.92
0.26
0.18
0.01
0.01
0.01
(1)Means followed by equal letters do not differ by Duncan’s test at 5% probability. RP 1S, root pruning performed in one side of the pear tree; and RP
2S, root pruning performed in both sides of the tree. Control of the vegetative growth of 'Shinseiki' pear trees 183 application of P-Ca 250 mg L-1 increased fruit set of
'd’Anjou' pears.i Table 4. Effect of prohexadione calcium (P-Ca) and root
pruning (RP) treatments on fruit firmness, total soluble
solids and titratable acidity (TA) of 'Shinseiki' pears in the
2013/2014 growing season(1). Fruit diameter during the season and final fruit
diameter were not affected by RP or by P-Ca
applications, compared to control trees (Figure 1). Nonetheless, the average fruit weight was significantly
reduced by RP 1S + P-Ca 300 mg L-1 in the 2013/2014
season (Table 3). These results do not corroborate
those by Hawerroth et al. (2012), which observed a
linear increase of fruit diameter of 'Housui' pear trees
treated with P-Ca. In this study, pear trees treated
with RP 1S + P-Ca 300 mg L-1 had lower average fruit
weight compared to the other treatments, showing
that the association of both techniques may negatively
affect fruit size. Smaller average fruit weight was
also observed in other studies with pears treated with
RP (Wang et al., 2014b) or P-Ca (Smit et al., 2005). Additionally, Maas (2008) observed that average fruit
weight of 'Conference' pears was not affected by RP
and RP + P-Ca. Control of the vegetative growth of 'Shinseiki' pear trees Treatment
Fruit firmness
(N)
Total soluble
solids (oBrix)
TA (% of
malic acid)
Control
43.57
10.10b
0.110
RP 1S
41.70
11.23a
0.124
RP 2S
41.71
11.03a
0.110
P-Ca 300 mg L-1
43.26
10.13b
0.106
P-Ca 600 mg L-1
43.23
10.70ab
0.101
RP 1S + P-Ca 300 mg L-1
42.02
10.08b
0.096
CV (%)
2.97
4.57
13.610
p
0.16
0.01
0.189
(1)Means followed by equal letters do not differ by Duncan’s test at 5%
probability. RP 1S, root pruning performed in one side of the pear tree;
and RP 2S, root pruning performed in both sides of the tree. (1)Means followed by equal letters do not differ by Duncan’s test at 5%
probability. RP 1S, root pruning performed in one side of the pear tree;
and RP 2S, root pruning performed in both sides of the tree. vigorous rootstocks induced the formation of fruits
with highest TSS contents. i Fruit firmness (FF) at harvest was not affected by
treatments, similarly as the reports by Smit et al. (2005)
and Einhorn et al. (2014), which showed no effect of
P-Ca application on FF. Neither P-Ca application nor
RP affected titratable acidity of fruit, which was also
observed by Hawerroth et al. (2012), in 'Hosui' pear
trees treated with P-Ca. Yield was negatively affected by P-Ca 600 mg L-1
in the 2013/2014 season, even though it did not
significantly differ from RP 1S and RP 1S + P-Ca
300 mg L-1 (Table 3). Similar results were found by
Hawerroth et al. (2012), where 'Housui' pear trees
sprayed with P-Ca rates greater than 584 mg L-1 showed
reduced yield. However, contrary results were found
by Asín et al. (2007), who observed that 'Blanquilla'
pears sprayed with P-Ca 1,750 mg L-1, split in four
monthly applications, and 'd’Anjou' pears sprayed once
with P-Ca 250 mg L-1 (Einhorn et al., 2014) did not
significantly reduce its yield per tree. Conclusions 1. Root pruning (RP) and prohexadione calcium (P-
Ca), when applied individually, consistently control
vegetative growth of 'Shinseiki' pear trees cultivated in
Southern Region of Brazil, but prohexadione calcium
is more efficient. RP performed alone did not affect yield in the present
study; and Asín et al. (2007) reported that root pruned
'Blanquilla' trees showed increased yield compared to
control trees. Wang et al. (2014b), however, reported
reduced yield of 'Clara Frijs' pear trees treated with RP,
in the first year, but increased yield in the next season,
compared to control trees. 2. The combination of P-Ca and RP does not further
improve vegetative growth control of pear trees. 3. Given the similar effects of the tested P-Ca
rates in reducing vegetative growth, and the negative
of the highest dose on fruit yield, the lowest one
is recommended to control vegetative growth of
'Shinseiki' pear trees. TSS in harvested fruits was significantly affected
by the treatments (Table 4). The highest content of TSS
was observed with RP 1S and 2S, which, however,
did not significantly differ, from P-Ca 600 mg L-1. Vercammen et al. (2005) also observed higher content
of TSS in fruits of root pruned pear trees. These results
may be explained by the decreased translocation of
water to the fruit at ripening stage, increasing the TSS
concentration. Pasa et al. (2012) observed that the least 4. Root pruning increases the total soluble solids
content at harvest. Pesq. agropec. bras., Brasília, v.52, n.3, p.177-185, mar. 2017
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DOI: 10.1590/S0100-204X2017000300005 185 Control of the vegetative growth of 'Shinseiki' pear trees Control of the vegetative growth of 'Shinseiki' pear trees ZADRAVEC, P.; CMELIK, Z.; TOJNKO, S.; UNUK, T.;
SCHLAUER, B. Vegetative growth, yield and fruit quality
of 'Gala' apple treated with Regalis (Prohexadione-Ca). Acta Horticulturae, v.774, p.287-290, 2008. DOI: 10.17660/
ActaHortic.2008.774.38. WANG, Y.; TRAVERS, S.; BERTELSEN, M.G.; THORUP-
KRISTENSEN, K.; PETERSEN, K.K.; LIU, F. Effect of root
pruning and irrigation regimes on pear tree: growth, yield
and yield components. Horticultural Science, v.41, p.34-43,
2014b. ZADRAVEC, P.; CMELIK, Z.; TOJNKO, S.; UNUK, T.;
SCHLAUER, B. Vegetative growth, yield and fruit quality
of 'Gala' apple treated with Regalis (Prohexadione-Ca). Acta Horticulturae, v.774, p.287-290, 2008. DOI: 10.17660/
ActaHortic.2008.774.38. WANG, Y.; TRAVERS, S.; BERTELSEN, M.G.; THORUP-
KRISTENSEN, K.; PETERSEN, K.K.; LIU, F. Effect of root
pruning and irrigation regimes on pear tree: growth, yield
and yield components. Horticultural Science, v.41, p.34-43,
2014b. Received on June 7, 2016 and accepted on January 13, 2017
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English
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Exploring atypical timescales in the brain
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Copyright Gollo. This article is
distributed under the terms of the
Creative Commons Attribution
License, which permits unrestricted
use and redistribution provided that
the original author and source are
credited. Identifying activity imbalances in specific brain regions may help to
diagnose and treat psychiatric disorders. LEONARDO L GOLLO (Kiebel et al., 2008; Murray et al., 2014;
Heeger, 2017). Now, in eLife, Takamitsu Wata-
nabe, Geraint Rees and Naoki Masuda report
that changes in intrinsic timescales are associ-
ated with the symptoms of autism spectrum dis-
order
in
high-functioning
individuals
(Watanabe et al., 2019). Their study raises the
question of whether the intrinsic timescales can
be used as a biomarker for neuropsychiatric dis-
orders and as a target for potential treatment
therapies. Related research article Watanabe T, Rees
G, Masuda N. 2019. Atypical intrinsic neural
timescale in autism. eLife 8:e42256. DOI:
10.7554/eLife.42256 Related research article Watanabe T, Rees
G, Masuda N. 2019. Atypical intrinsic neural
timescale in autism. eLife 8:e42256. DOI:
10.7554/eLife.42256 T
he electrical activity of any region of the
brain changes with time in a complex
way that can be described as combina-
tions of oscillations with different amplitudes,
frequencies and phases. Different areas of the
brain are also characterized by an intrinsic time-
scale that reflects the length of the time window
over which the signals coming into that brain
region
are
integrated
(Mesulam,
1998;
Honey et al., 2012). T The researchers – who are based at the
RIKEN Centre for Brain Science, University Col-
lege London and the University of Bristol –
used
functional
magnetic
resonance
imaging to measure intrinsic timescales in peo-
ple with and without a high-functioning form of
autism. The results revealed that people with
this form of autism have atypically short time-
scales in primary sensory and visual areas, while
a region called the caudate, which is implicated
in sensorimotor coordination, showed a longer
timescale (Grahn et al., 2008). This reinforces
the theory that intrinsic timescales are central to
brain function, and that imbalances in specific
regions substantially affect the severity of symp-
toms in autism spectrum disorders (Figure 1B). Regions with short intrinsic timescales are
usually located at the periphery of the brain net-
work and are implicated in interactions between
the brain and the external world, for example,
perception and movement. Regions with long
timescales are usually strongly connected hubs
located at the core of the brain. They are impor-
tant for regulating interactions between the
brain and the body, such as emotions, mood
and anxiety (Gollo et al., 2015). COMPUTATIONAL PSYCHIATRY
Exploring atypical timescales in
the brain Identifying activity imbalances in specific brain regions may help to
diagnose and treat psychiatric disorders. Identifying activity imbalances in specific brain regions may help to
diagnose and treat psychiatric disorders. This gradient of
timescales forms a hierarchy in brain dynamics
that recapitulates the hierarchy in brain structure
(Kiebel et al., 2008; Murray et al., 2014;
Figure 1A). Intrinsic timescales can be estimated using
simple autocorrelations, which may be used to
identify biomarkers and to improve our under-
standing
of
diseases
and
treatment
plans
(Figure 1B). But further research is needed to
fully comprehend the causes and implications of
atypical
intrinsic
timescales. In
people
with
autism, shorter timescales in regions of sensory
and visual cortices could relate to a heightened
sensory perception, which is consistent with an This hierarchy of timescales also plays an
important role in perception and many other
behaviors, and modifications to these timescales
can
be
detrimental
to
brain
function Gollo. eLife 2019;8:e45089. DOI: https://doi.org/10.7554/eLife.45089 1 of 3 Insight Insight Computational Psychiatry
Exploring atypical timescales in the brain Figure 1. The hierarchy of timescales in the brain. (A) The brain integrates incoming information over different
timescales that are characteristic for different regions. Such a hierarchy of timescales also mirrors a hierarchy in
brain structure. Brain regions located at the top of the hierarchy are represented as large (yellow) circles and have
longer timescales. They are located at the core and have strong connections to other brain regions. Brain regions
located at the periphery are represented by small (blue) circles and have shorter timescales. (B) Watanabe et al. found that individuals with autism spectrum disorders (ASD, black) have different intrinsic timescales (quantified by
the autocorrelation function) compared to typically developing individuals (TD, blue). These differences correlate
with the severity of symptoms of ASD. (C) In the future, non-invasive brain stimulation (black coil) may be used to
selectively modulate atypical brain regions to restore their intrinsic timescales. Brain figure adapted from
Gollo et al. (2018). Figure 1. The hierarchy of timescales in the brain. (A) The brain integrates incoming information over different
timescales that are characteristic for different regions. Such a hierarchy of timescales also mirrors a hierarchy in
brain structure. Brain regions located at the top of the hierarchy are represented as large (yellow) circles and have
longer timescales. They are located at the core and have strong connections to other brain regions. Brain regions
located at the periphery are represented by small (blue) circles and have shorter timescales. (B) Watanabe et al. Identifying activity imbalances in specific brain regions may help to
diagnose and treat psychiatric disorders. found that individuals with autism spectrum disorders (ASD, black) have different intrinsic timescales (quantified by
the autocorrelation function) compared to typically developing individuals (TD, blue). These differences correlate
with the severity of symptoms of ASD. (C) In the future, non-invasive brain stimulation (black coil) may be used to
selectively modulate atypical brain regions to restore their intrinsic timescales. Brain figure adapted from
Gollo et al. (2018). their susceptibility to dysfunction (Fornito et al.,
2015; Gollo et al., 2018). excessive expectation of changes in their envi-
ronment (Lawson et al., 2017). Moreover, lon-
ger timescales in the caudate might also indicate
a compensation strategy to cope with an over-
load of sensory input due to the heightened sen-
sory perception. The second line of research would explore
the possibility of reducing symptoms by manipu-
lating atypical timescales, such as the ones
Watanabe et al. observed in people with autism. Although drugs might not be specific enough to
selectively act upon precise regions, brain stimu-
lation could be a powerful solution (Figure 1C). For example, superficial cortical regions can be
targeted by non-invasive methods such as trans-
cranial magnetic stimulation. Moreover, recent
advances suggest that brain stimulation can
modify the timescale of the target region, which
may be used to modulate intrinsic timescales to
mitigate
symptoms
(Cocchi
et
al.,
2016;
Gollo et al., 2017). The work of Watanabe et al. opens at least
two main lines of research. The first would
involve mapping the timescales of brain regions
across
different
neuropsychiatric
disorders,
including schizophrenia and obsessive-compul-
sive disorder, to determine where and what type
of timescale deviations occur (King and Lord,
2011). This should also be done in healthy indi-
viduals to use their timescales as a benchmark. Depending on the location, disturbances ought
to have different effects. For example, hub
regions play a role in many disorders, and distur-
bances in their timescales may also evidence Overall, the work of Watanabe, Rees and
Masuda reveals how systems-level approaches
hold
the
potential
to
shift
paradigms
in Gollo. eLife 2019;8:e45089. DOI: https://doi.org/10.7554/eLife.45089 2 of 3 Insight Computational Psychiatry
Exploring atypical timescales in the brain psychiatry. Translating these recent results into
clinical practice will involve many practical chal-
lenges, but they may also be highly beneficial. Although many questions certainly remain, these
are crucial advances on the neurobiological basis
of autism. and volatility of structural hubs in the human
connectome. Nature Neuroscience 21:1107–1116. Identifying activity imbalances in specific brain regions may help to
diagnose and treat psychiatric disorders. DOI: https://doi.org/10.1038/s41593-018-0188-z,
PMID: 30038275 connectome. Nature Neuroscience 21:1107–1116. DOI: https://doi.org/10.1038/s41593-018-0188-z,
PMID: 30038275
Grahn JA, Parkinson JA, Owen AM. 2008. The
cognitive functions of the caudate nucleus. Progress in
Neurobiology 86:141–155. DOI: https://doi.org/10. 1016/j.pneurobio.2008.09.004, PMID: 18824075
Heeger DJ. 2017. Theory of cortical function. PNAS
114:1773–1782. DOI: https://doi.org/10.1073/pnas. 1619788114, PMID: 28167793
Honey CJ, Thesen T, Donner TH, Silbert LJ, Carlson
CE, Devinsky O, Doyle WK, Rubin N, Heeger DJ,
Hasson U. 2012. Slow cortical dynamics and the
accumulation of information over long timescales. Neuron 76:423–434. DOI: https://doi.org/10.1016/j. neuron.2012.08.011, PMID: 23083743
Kiebel SJ, Daunizeau J, Friston KJ. 2008. A hierarchy
of time-scales and the brain. PLoS Computational
Biology 4:e1000209. DOI: https://doi.org/10.1371/
journal.pcbi.1000209, PMID: 19008936
King BH, Lord C. 2011. Is schizophrenia on the autism
spectrum? Brain Research 1380:34–41. DOI: https://
doi.org/10.1016/j.brainres.2010.11.031, PMID: 2107
8305 Grahn JA, Parkinson JA, Owen AM. 2008. The
cognitive functions of the caudate nucleus. Progress in
Neurobiology 86:141–155. DOI: https://doi.org/10. 1016/j.pneurobio.2008.09.004, PMID: 18824075 Grahn JA, Parkinson JA, Owen AM. 2008. The
cognitive functions of the caudate nucleus. Progress in
N
bi l
86 141 155 DOI h
//d i
/10 Heeger DJ. 2017. Theory of cortical function. PNAS
114:1773–1782. DOI: https://doi.org/10.1073/pnas. 1619788114, PMID: 28167793 Leonardo L Gollo is in the Systems Neuroscience
Group, QIMR Berghofer Medical Research Institute,
Brisbane, Australia
leonardo.l.gollo@gmail.com
http://orcid.org/0000-0003-3505-9259
Competing interests:
The author declares that no
competing interests exist. Published 05 February 2019 Honey CJ, Thesen T, Donner TH, Silbert LJ, Carlson
CE, Devinsky O, Doyle WK, Rubin N, Heeger DJ,
Hasson U. 2012. Slow cortical dynamics and the
accumulation of information over long timescales. Competing interests:
The author declares that no
competing interests exist. Published 05 February 2019 Gollo. eLife 2019;8:e45089. DOI: https://doi.org/10.7554/eLife.45089 References Cocchi L, Sale MV, L Gollo L, Bell PT, Nguyen VT,
Zalesky A, Breakspear M, Mattingley JB. 2016. A
hierarchy of timescales explains distinct effects of local
inhibition of primary visual cortex and frontal eye
fields. eLife 5:e15252. DOI: https://doi.org/10.7554/
eLife.15252, PMID: 27596931
Fornito A, Zalesky A, Breakspear M. 2015. The
connectomics of brain disorders. Nature Reviews
Neuroscience 16:159–172. DOI: https://doi.org/10. 1038/nrn3901, PMID: 25697159
Gollo LL, Zalesky A, Hutchison RM, van den Heuvel M,
Breakspear M. 2015. Dwelling quietly in the rich club:
brain network determinants of slow cortical
fluctuations. Philosophical Transactions of the Royal
Society B: Biological Sciences 370:20140165. DOI: https://doi.org/10.1098/rstb.2014.0165
Gollo LL, Roberts JA, Cocchi L. 2017. Mapping how
local perturbations influence systems-level brain
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8126550
Gollo LL, Roberts JA, Cropley VL, Di Biase MA,
Pantelis C Zalesky A Breakspear M 2018 Fragility Cocchi L, Sale MV, L Gollo L, Bell PT, Nguyen VT,
Zalesky A, Breakspear M, Mattingley JB. 2016. A
hierarchy of timescales explains distinct effects of local
inhibition of primary visual cortex and frontal eye
fields. eLife 5:e15252. DOI: https://doi.org/10.7554/
eLife.15252, PMID: 27596931
Fornito A, Zalesky A, Breakspear M. 2015. The
connectomics of brain disorders. Nature Reviews
Neuroscience 16:159–172. DOI: https://doi.org/10. 1038/nrn3901, PMID: 25697159
Gollo LL, Zalesky A, Hutchison RM, van den Heuvel M,
Breakspear M. 2015. Dwelling quietly in the rich club:
brain network determinants of slow cortical
fluctuations. Philosophical Transactions of the Royal
Society B: Biological Sciences 370:20140165. DOI: https://doi.org/10.1098/rstb.2014.0165
Gollo LL, Roberts JA, Cocchi L. 2017. Mapping how
local perturbations influence systems-level brain
dynamics. NeuroImage 160:97–112. DOI: https://doi. org/10.1016/j.neuroimage.2017.01.057, PMID: 2
8126550
Gollo LL, Roberts JA, Cropley VL, Di Biase MA,
Pantelis C, Zalesky A, Breakspear M. 2018. Fragility Cocchi L, Sale MV, L Gollo L, Bell PT, Nguyen VT,
Zalesky A, Breakspear M, Mattingley JB. 2016. A
hierarchy of timescales explains distinct effects of local
inhibition of primary visual cortex and frontal eye
fields. eLife 5:e15252. DOI: https://doi.org/10.7554/
eLife.15252, PMID: 27596931
Fornito A, Zalesky A, Breakspear M. 2015. The
connectomics of brain disorders. Nature Reviews
Neuroscience 16:159–172. DOI: https://doi.org/10. 1038/nrn3901 PMID: 25697159 Cocchi L, Sale MV, L Gollo L, Bell PT, Nguyen VT,
Zalesky A, Breakspear M, Mattingley JB. 2016. A
hierarchy of timescales explains distinct effects of local
inhibition of primary visual cortex and frontal eye
fields. eLife 5:e15252. DOI: https://doi.org/10.7554/
eLife.15252, PMID: 27596931 j
p
,
King BH, Lord C. 2011. Gollo LL, Roberts JA, Cropley VL, Di Biase MA,
Pantelis C, Zalesky A, Breakspear M. 2018. Fragility References Is schizophrenia on the autism
spectrum? Brain Research 1380:34–41. DOI: https://
doi.org/10.1016/j.brainres.2010.11.031, PMID: 2107
8305 Lawson RP, Mathys C, Rees G. 2017. Adults with
autism overestimate the volatility of the sensory
environment. Nature Neuroscience 20:1293–1299. DOI: https://doi.org/10.1038/nn.4615, PMID: 2875
8996 Mesulam MM. 1998. From sensation to cognition. Brain 121:1013–1052. DOI: https://doi.org/10.1093/
brain/121.6.1013, PMID: 9648540
Murray JD, Bernacchia A, Freedman DJ, Romo R,
Wallis JD, Cai X, Padoa-Schioppa C, Pasternak T, Seo
H, Lee D, Wang XJ. 2014. A hierarchy of intrinsic
timescales across primate cortex. Nature Neuroscience
17:1661–1663. DOI: https://doi.org/10.1038/nn.3862,
PMID: 25383900 ,
Gollo LL, Zalesky A, Hutchison RM, van den Heuvel M,
Breakspear M. 2015. Dwelling quietly in the rich club:
brain network determinants of slow cortical
fluctuations. Philosophical Transactions of the Royal
Society B: Biological Sciences 370:20140165. DOI: https://doi.org/10.1098/rstb.2014.0165
Gollo LL, Roberts JA, Cocchi L. 2017. Mapping how
local perturbations influence systems-level brain
dynamics. NeuroImage 160:97–112. DOI: https://doi. org/10.1016/j.neuroimage.2017.01.057, PMID: 2
8126550 Watanabe T, Rees G, Masuda N. 2019. Atypical
intrinsic neural timescale in autism. eLife 8:e42256. DOI: https://doi.org/10.7554/eLife.42256 Gollo LL, Roberts JA, Cropley VL, Di Biase MA,
Pantelis C, Zalesky A, Breakspear M. 2018. Fragility 3 of 3
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Prioritization of software development demands with text mining techniques
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Mühendislik bilimleri dergisi/Mühendislik bilimleri dergisi
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cc-by
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Pamukkale Univ Muh Bilim Derg, 25(5), 615-620, 2019
Pamukkale Üniversitesi Mühendislik Bilimleri Dergisi
Pamukkale University Journal of Engineering Sciences
Yazılım geliştirme taleplerinin metin madenciliği yöntemleriyle
önceliklendirilmesi
Prioritization of software development demands with text mining
techniques
Murat Can TEKİN1
1Bilgisayar
, Volkan TUNALI2*
Mühendisliği Bölümü, Fen Bilimleri Enstitüsü, Maltepe Üniversitesi, İstanbul, Türkiye.
muratcantekin@hotmail.com
2Yazılım
Mühendisliği Bölümü, Mühendislik ve Doğa Bilimleri Fakültesi, Maltepe Üniversitesi, İstanbul, Türkiye.
volkan.tunali@gmail.com
Geliş Tarihi/Received: 19.11.2018, Kabul Tarihi/Accepted: 13.02.2019
* Yazışılan yazar/Corresponding author
doi: 10.5505/pajes.2019.47827
Araştırma Makalesi/Research Article
Öz
Kurumsal şirketlerde, yazılımlardaki hatalar ve değişiklik talepleri
genellikle bir talep yönetim sistemi üzerinden Bilgi Teknolojileri (BT)
birimine iletilir. Bu sistemde yer alan öncelik bilgisi BT birimi için kritik
öneme sahiptir. Ancak, talebi giren kişilerin inisiyatifine bırakılan
öncelik kararı her zaman gerçekçi olmamaktadır. Örneğin, kritik
olmayan ve düşük öncelikli bir değişiklik talebi yüksek öncelikli olarak
girilebilmekte, bu da hatalı planlama ve müşteri memnuniyetsizliği ile
sonuçlanabilmektedir. Bu çalışmada, iç müşteri talepleri metin
madenciliği yöntemleriyle sınıflandırılarak taleplerin önem derecesi
tahmin edilmeye çalışılmıştır. Sistemin eğitimi ve testi için kurumsal bir
şirketin talep yönetim sisteminden alınan kayıtlar kullanılmıştır. Ham
metin formundaki talep verisi üzerinde temizlik ve önişleme
işlemlerinin ardından, doküman-terim matrisinin oluşturulmasında TFIDF (Terim Frekansı – Ters Doküman Frekansı) ağırlıklandırma
yönteminden yararlanılmıştır. Oluşturulan veri seti üzerinde çeşitli
sınıflandırma algoritmaları test edilmiş ve en yüksek başarım %54.1 FSkoru ile Sequential Minimal Optimization algoritmasıyla elde
edilmiştir. Ayrıca, aşırı örnekleme yoluyla sınıfların dengeli hale
getirildiği veri seti üzerinde ise en yüksek başarıma %74.5 F-Skoru
değeri ile Random Forest algoritmasıyla ulaşılmıştır.
Abstract
In corporations, software issues and software change demands are
forwarded to the Information Technology (IT) unit via a demand
management system. The priority information in this system has critical
importance to the IT unit. However, the priority decision that is left to
the individuals who create the demand records may not always be
realistic. For instance, a non-critical and low-priority demand may be
created with the highest priority, and this may lead to faulty planning
and eventually to customer dissatisfaction. In this work, internal
customer demands were classified using text mining techniques and
their priorities were predicted. The system was trained and tested with
the records extracted from the demand management system of a
corporation. After cleaning and preprocessing the raw textual demand
data, TF-IDF (Term Frequency – Inverse Document Frequency)
weighting scheme was used when creating the document-term matrix.
Several classification algorithms were tested on the data set generated,
and the highest performance was obtained by Sequential Minimal
Optimization algorithm with 54.1% F-Score. In addition, on the dataset
made balanced with oversampling technique, the highest performance
was achieved by Random Forest algorithm with 74.5% F-Score.
Anahtar kelimeler: Yazılım mühendisliği, Talep önceliklendirme,
Yapay öğrenme, Metin sınıflandırma, Random forest
Keywords: Software engineering, Demand prioritization, Machine
learning, Text classification, Random forest.
1 Giriş
Rekabetin yoğun olduğu iş dünyasında, şirketler, müşterilerine
sundukları hizmetlerin kalitesini en yüksek düzeyde tutmaya
çalışmaktadır. Bu noktada, müşterilere doğrudan ya da dolaylı
olarak sunulan yazılımların kalitesi büyük öneme sahiptir.
Müşterinin dokunduğu noktalarda alınan hatalar ve fark edilen
eksiklikler iç müşteri birimlerine bildirilir. İç müşteri birimleri
de bu hata ve eksiklikleri ilgili Bilgi Teknolojileri (BT) birimine
yazılım geliştirme talebi olarak iletirler. Küçük ölçekli
şirketlerde bu süreç e-posta yoluyla ya da sözlü olarak
yürütülebilmektedir. Ancak, büyük ölçekli şirketlerde bu
taleplerin uygun şekilde kayıt altına alınması ve tüm süreç
boyunca izlenmesi hem şirket iş süreçleri hem de hizmet
kalitesi açısından bir zorunluluktur. Bunun için bu talepler
genellikle Talep Yönetim Sistemi olarak adlandırılan sistemler
üzerinden oluşturularak ilgili birime iletilirler. Bu sistemde yer
alan öncelik bilgisi BT birimi için kritik öneme sahiptir. Mevcut
talepler öncelik sırasına göre işleme alınarak çözümlenmeye
çalışılır. Ancak, iç müşteri birimlerinin, açtıkları taleplere kendi
işlerinin çabuk halledilmesi için genellikle “yüksek” öncelik
verdiği gözlenmektedir. Önem derecesi gerçekçi seçilmediği
için bu talepler, gerçek “yüksek” öncelikli taleplerin önüne
geçebilmektedir. Bu da hatalı süreç ve kaynak planlamasının
yanı sıra müşteri memnuniyetsizliği ile sonuçlanabilmektedir
[1]. Dolayısıyla, yazılım geliştirme taleplerinin önceliklerinin
doğru belirlenmesi büyük önem taşımaktadır.
Özellikle son yıllarda bu konuda çeşitli çalışmaların yapıldığı ve
bu çalışmaların genellikle hata raporlarının (bug report)
önceliklendirilmesi ya da bu hataların önem (severity)
derecelerinin tahmin edilmesi şeklinde olduğu görülmektedir.
Bu çalışmaların genelinde, çeşitli açık-kaynak yazılımların hata
veritabanlarındaki hata kayıtlarının metin bölümleri, metin
madenciliği yöntemleriyle işlenmekte ve öncelik ve/veya önem
dereceleri çeşitli popüler sınıflandırma algoritmalarıyla tahmin
edilmeye çalışılmaktadır [2]-[5]. Hata kayıtlarındaki hataya
dair metinsel açıklamaların yanı sıra hatanın yığın izlemesi
(stack trace), hatanın yaşandığı platform ve işletim sistemi,
hatanın tespit edildiği yazılım bileşeni/modülü gibi çeşitli
kategorik
bilgilerin
de
sınıflandırma
modelinin
oluşturulmasına dâhil edilerek sınıflandırma başarımının
615
Pamukkale Univ Muh Bilim Derg, 25(5), 615-620, 2019
M. C. Tekin, V. Tunalı
arttırılmaya çalışıldığı çeşitli çalışmalar da bulunmaktadır
[6]-[10].
Bu çalışmada ise, yalnızca hatalar değil, diğer çeşitli geliştirme
isteklerini de içeren iç müşteri talepleri, metin madenciliği
yöntemleriyle sınıflandırılarak taleplerin öncelikleri tahmin
edilmeye çalışılmıştır. Bu sayede, BT birimlerine iç müşteri
taleplerinin önceliklerinin değerlendirilmesi ve doğru
planlama yapılması konusunda destekleyici bir sistem
geliştirilmesi amaçlanmıştır. Bilindiği kadarıyla, yazılım
geliştirme taleplerinin metin madenciliği kullanılarak
önceliklendirilmesine yönelik, Türkçe dilinin yapısal
zorluklarını
dikkate
alan
bir
çalışma
literatürde
bulunmamaktadır.
Sistemin eğitimi ve testi için Türkiye’de faaliyet gösteren
kurumsal bir şirketin talep yönetim sisteminden alınan gerçek
talep kayıtları kullanılmıştır ve bu çalışmanın özgün
yönlerinden biri de budur. Bu kayıtlarda, iç müşteri tarafından
belirlenmiş hatalı öncelikler yerine BT birimi tarafından
gerçekçi olarak revize edilmiş öncelik değerleri bulunmaktadır
ve sistemin geliştirilmesi sırasında bu doğru öncelik değerleri
dikkate alınmıştır. Ham metin formundaki talep verisi üzerinde
temizlik ve önişleme işlemleri gerçekleştirilmiş, ardından,
doküman-terim matrisinin oluşturulmasında TF-IDF (Terim
Frekansı-Ters
Doküman
Frekansı)
ağırlıklandırma
yönteminden yararlanılmıştır [11]. Elde edilen veri seti
üzerinde Naive Bayes [12], Naive Bayes Multinomial [13],
Sequential Minimal Optimization [14], Random Forest [15] ve
Rotation Forest [16] gibi çeşitli sınıflandırma algoritmaları test
edilmiş ve en yüksek başarıma %74.5 F-Skoru değeri ile
Random Forest algoritmasıyla ulaşılmıştır.
Makalenin devam eden bölümleri şu şekilde organize
edilmiştir: İkinci bölümde kullanılan veri seti, metin önişleme
teknikleri, sınıflandırma algoritmaları ile değerlendirme
ölçütlerinden bahsedilmiştir. Üçüncü bölümde, yapılan
deneylerin sonuçları sunulmuştur. Dördüncü ve son bölümde
ise elde edilen sonuçlar değerlendirilmiş ve gelecek çalışmalara
dair önerilere yer verilmiştir.
2 Malzeme ve yöntem
2.1
Veri seti
Bu çalışmada, veri seti olarak özel bir şirketin yazılım geliştirme
biriminde, iç müşteri taleplerinin takibi ve çözümü için
kullanılmakta olan bir yazılımın veritabanından alınan talep
kayıtları kullanılmıştır. Bu kayıtlardan ise yalnızca talep başlığı,
açıklama ve öncelik sınıfı alanlarından yararlanılmış, diğer
alanlar tamamen göz ardı edilmiştir. Kayıtlardaki TC Kimlik
Numarası, Vergi Numarası ve çeşitli evrak numaraları gibi
kişisel bilgiler temizlenmiştir. Verilerin veritabanından
çekilmesi ve ön temizlik işleminin gerçekleştirilmesi için Visual
Studio 2013 geliştirme ortamında C# programlama dili ile
yazılan bir program kullanılmıştır. Bu program ile ayrıca
kayıtların başlık ve açıklama alanları birleştirilerek, her bir
kayıt ayrı bir metin dosyası olacak şekilde, öncelik sınıflarına
göre ayrı bir klasöre kaydedilmiş ve metin madenciliğinin ilk
adımı olan metin önişleme için hazır duruma getirilmiştir.
Tablo 1’de veri setinden örnek kayıtlar görülmektedir. Örnek
kayıtlarda görüldüğü gibi metin alanların yazımında herhangi
bir standart bulunmamakta ve kullanıcılar tarafından çokça
yazım hatası yapılmaktadır. Bu durum genellikle tüm metin
işleme ve metin madenciliği uygulamalarında karşılaşılan ve
aşılması gereken önemli bir zorluktur.
Veri setindeki kayıtlar düşük, orta ve yüksek olmak üzere üç
öncelik kategorisine dengesiz olarak dağılmış durumdadır. Veri
setinde 43 düşük, 150 orta ve 151 yüksek öncelikli olmak üzere
toplam 344 kayıt bulunmaktadır. Sınıflandırma çalışmalarında,
modelin eğitimi için kullanılacak veri setindeki kayıtların ilgili
sınıflara dengeli olarak dağılması, oluşturulan modelin
başarımı bakımından önemlidir. Dengesiz sınıflarla oluşturulan
modelin ürettiği tahminler, çoğunluk durumundaki sınıflara
doğru meyilli olmakta ve bu da azınlık durumundaki sınıflardan
örneklerin doğru tahmin oranının düşmesine neden
olmaktadır.
Tablo 1: Veri setinden örnek kayıtlar.
Başlık
Açıklama
Öncelik
TARİH FORMATINDA HATA
BT SİSTEM LİSTE SORGULAR/POLİÇE HASAR LİSTESİ YENİ alanında tarih aralıkları ile rapor
alındığında exel’deki tarih formatlarında hata olmakta ve düzeltilememekte düzeltilmesi için
desteğinizi rica ederiz.
Düşük
SAĞLIK REASÜRANS RAPORU
REASÜRANS KOLONU
YUVARLAMA HK.
SAĞLIK REASÜRANS RAPORU REASÜRANS KOLONUNDAKİ TUTARLAR LE OLUŞLAN
FİŞLERDEKİ TUTARLAR ARASINDA KURUŞ FARKLARI BULUNMAKTADIR. KONTROLÜNÜ VE
RAPORUN FİŞE YANSIYAN ŞEKLİ İLE DÜZENLENMESİNİ RİCA EDERİZ.
Orta
Acente Tecdit Listesi
Merhaba,
Acente tecdit listesinden bölge kodu olarak Bankasürans departmanı görünmemektedir. İlgili
hatanın giderilmesi için desteğinizi rica ederim.
Saygılarımla,
Orta
346 – M******* Kasko baz
update
K03 G04 T 59,535
Binde 59.535 olan bazın 54 olarak acilen düzeltilmesi gerekmektedir.
Teşekkürler
Yüksek
616
Pamukkale Univ Muh Bilim Derg, 25(5), 615-620, 2019
M. C. Tekin, V. Tunalı
Bundan dolayı, bu çalışmada, azınlık durumunda kalan düşük
öncelikli sınıfına ait örnek sayısı, aşırı örnekleme
(oversampling) yapılarak diğer sınıflardaki örnek sayısına
yaklaştırılmış ve dengeli veri seti ile de deneyler yapılarak
sonuca etkisi araştırılmaya çalışılmıştır [17]. Aşırı örneklemede
temel yaklaşım, azınlık durumundaki sınıftaki kayıtların
çoğaltılmasıdır. Bunun için literatürde çeşitli yöntemler
önerilmiş olmakla birlikte, bu çalışmada, azınlık durumundaki
düşük öncelikli sınıfına ait 43 kaydın her birinin ikişer kopyası
oluşturularak, bu sınıfa ait kayıt sayısı 129’a çıkartılmıştır.
Ayrıca, sınıflardaki kayıt sayısının tam olarak eşit olması adına,
orta ve yüksek öncelikli sınıflardaki kayıtlardan bazılarının
rastgele olarak veri setinden çıkartılması ve böylece bu
sınıflardaki kayıt sayısının da 129’a düşürülmesi yoluna
gidilmiştir.
2.2
Metin önişleme
Ham metin formunda yani yapısal olmayan veri üzerinde veri
madenciliği tekniklerinin uygulanabilmesi için metin verisinin
öncelikle yapısal bir forma dönüştürülmesi gereklidir. Bu
dönüşüm işlemi metin önişleme olarak adlandırılır. Bu
çalışmada, önişleme için PRETO aracından yararlanılmıştır
[18]. PRETO, özellikle Türkçe metinler üzerinde yüksek
performansla önişleme yapmak üzere geliştirilmiş, açık
kaynaklı bir yazılımdır. Kök bulma, durdurma sözcükleri
filtreleme, n-gram oluşturma ve terim ağırlıklandırma gibi
doğal dil işleme yeteneklerinin yanı sıra Doküman-Terim (D-T)
Matrisinin çeşitli dosya formatlarında kaydedilebilmesi gibi
kullanışlı özellikler de sunmaktadır.
Bu çalışmada, kök bulma işlevi için PRETO’daki Zemberek ve Ek
Çıkaran (Affix Stripping) kök bulma seçenekleri kullanılarak
ayrı ayrı deneyler yapılmıştır. Zemberek, eklemeli Türk dilleri
için geliştirilmiş, açık kaynak kodlu, platformdan bağımsız bir
doğal dil işleme kütüphanesidir [19]. Zemberek, sözlük tabanlı
bir kök bulma yöntemi kullanmakta olup, tanımlı bir kök ve ek
sözlüğü kullanarak bu işlemi yapmaktadır. Ek Çıkaran kök
bulucu ise Türkçe’nin kural tabanlı yapısını kullanmaktadır ve
eklerin sondan başa doğru çıkarılması yaklaşımıyla kelimenin
kökünü bulacak şekilde geliştirilmiş bir doğal dil işleme
yöntemidir [20]. Çalışmada kullanılan diğer bir önişleme
tekniği ise durdurma sözcükleri filtrelemedir. PRETO ile
birlikte gelen, Türkçe için belirlenmiş 181 durak sözcüğünü
içeren bir liste kullanılmıştır [18]. Terim ağırlıklandırma
yöntemi olarak, metin işleme alanında bilinen en etkin
yöntemlerden biri olan TF-IDF kullanılmıştır. Ayrıca, önişleme
sonucunda uzunluğu üç karakterden az olan sözcükler ile
rakamlar ve noktalama işaretleri tamamen filtrelenmiştir.
Terim oluşturma yöntemi olarak 1-gram ve 2-gram’ların
sonuçlar üzerindeki etkilerini ayrı ayrı gözlemlemek için
deneyler yapılmıştır.
2.3
Sınıflandırma
Bu çalışmada, kullanıcıların metin formunda girdiği taleplerin
önceliklerinin tahmin edilmesi bir metin sınıflandırma
problemi olarak ele alınmıştır.
Sınıflandırma, sınıfı bilinmeyen bir veri örneğinin, daha önce
sınıfları bilinen veri örnekleriyle eğitilmiş bir model üzerinden
sınıfının tahmin edilmesi olarak tanımlanır [12].
Literatürde
çok
sayıda
sınıflandırma
algoritması
bulunmaktadır. Bu çalışmada ise çeşitli sınıflandırma
algoritmaları denenmiş ve genel olarak etkinliği yüksek olanlar
seçilerek deneyler çeşitlendirilmiş ve sonuçları sunulmuştur.
Seçilen bu algoritmalar Naive Bayes, Naive Bayes Multinomial,
Sequential Minimal Optimization, Random Forest ve Rotation
Forest algoritmalarıdır.
2.3.1 Naive bayes (NB)
Naive Bayes algoritması, veri kümesindeki değerlerin
kombinasyonunu ve frekansını dikkate alarak bir olasılık
kümesi oluşturan temel bir istatistiksel olasılık sınıflandırıcıdır.
Yapısının basitliği, hızlı çalışması ve karmaşık sınıflandırma
algoritmalarıyla kıyaslanabilir sonuçlar üretmesi sayesinde
sıklıkla tercih edilen bir algoritmadır [12].
2.3.2 Naive bayes multinomial (NBM)
Naive Bayes Multinomial algoritması, Naive Bayes
algoritmasının metin belgeleri için özelleştirilmiş bir halidir.
NB’den farklı olarak sözcükler için çok terimli dağılım kullanan
NBM, metin sınıflandırmada NB’ye göre daha yüksek başarım
sergilemektedir [13].
2.3.3 Sequential minimal optimization (SMO)
Destek Vektör Makineleri’nin (SVM - Support Vector Machines)
eğitilmesi için çok büyük bir kuadratik programlama eniyileme
probleminin çözülmesi gerekmektedir. SMO, bunu etkili bir
şekilde gerçekleştiren, hızlı bir SVM eğitim algoritmasıdır [14].
2.3.4 Random forest (RaF)
Karar ağaçları, sınıflandırma görevlerinde yaygın olarak
kullanılan bir öğrenme yöntemidir. Adından da anlaşılacağı gibi
eğitilen model ağaç görüntüsüne benzer bir yapıda olup dal ve
yapraklardan oluşur. Ağaçta bulunan her bir düğüm bir test
koşulunu temsil edip, test sonucu oluşan daldaki yaprak, test
edilen verinin sınıfını belirtir. Random Forest algoritmasında
ise eğitim kümesindeki verilerden rastgele seçimler yapılarak
çok sayıda karar ağacı oluşturulur [15]. Test verisi tüm
ağaçlarda test edildikten sonra çoğunluğun kararı, ilgili verinin
sınıfı olarak belirlenir.
2.3.5 Rotation forest (RoF)
Rotation Forest algoritması, eğitim kümesinden farklı öznitelik
kombinasyonları kullanarak çok sayıda karar ağacı oluşturur
[16]. Test verisi yine tüm ağaçlarda test edildikten sonra
çoğunluğun kararı, ilgili verinin sınıfı olarak belirlenir.
2.4
Deney ortamı
Deney ortamı olarak, çeşitli sınıflandırma algoritmalarını
bünyesinde barındıran ve deney sonuçlarının kolayca elde
edildiği WEKA kullanılmıştır. WEKA, veri madenciliği
çalışmaları için geliştirilmiş, Java tabanlı, açık kaynak kodlu bir
araçtır [21]. PRETO aracından elde edilen doküman-terim
matrisi dosyaları WEKA’da doğrudan kullanılamamaktadır. Bu
nedenle, C# programlama dili kullanılarak geliştirilen bir
program aracılığıyla, doküman-terim matrisi dosyaları,
WEKA’nın ARFF dosya formatına dönüştürülmüştür.
Deneylerde kullanılan sınıflandırıcılar, WEKA’daki varsayılan
parametreleriyle çalıştırılmıştır. Veri setindeki örnek sayısının
azlığı nedeniyle tüm deneylerde 10-kat çapraz geçerleme
uygulanmıştır.
2.5
Değerlendirme ölçütleri
Yapılan sınıflandırma deneylerinde, sınıflandırıcı başarımı için
Kesinlik (Precision), Anımsama (Recall), F-Skoru (F-Measure)
617
Pamukkale Univ Muh Bilim Derg, 25(5), 615-620, 2019
M. C. Tekin, V. Tunalı
ve ROC Alanı (Receiver Operating Characteristic Area) ölçütleri
kullanılmıştır. Bu ölçütlerin tümünde ölçüt değeri ne kadar
yüksek ise sınıflandırma başarımı o kadar yüksek demektir.
Sınıflandırma modelinin test edilmesi sonucunda Doğru Pozitif
(DP-True Positive), Yanlış Pozitif (YP-False Positive), Doğru
Negatif (DN-True Negative) ve Yanlış Negatif (YN-False
Negative) olmak üzere dört farklı çıktı elde edilir. Bu çıktılara
bağlı olarak çeşitli başarım ölçütleri hesaplamak olasıdır.
Kesinlik ve Anımsama değerleri Denklem (1) ve (2)’deki gibi
hesaplanır.
𝐾𝑒𝑠𝑖𝑛𝑙𝑖𝑘 =
𝐷𝑃
𝐷𝑃 + 𝑌𝑃
𝐴𝑛ı𝑚𝑠𝑎𝑚𝑎 =
𝐷𝑃
𝐷𝑃 + 𝑌𝑁
(1)
(2)
F-Skoru, kesinlik ve anımsamanın harmonik ortalamasıdır ve
Denklem (3)’teki gibi hesaplanır.
𝐹-𝑆𝑘𝑜𝑟𝑢 =
2 × 𝐾𝑒𝑠𝑖𝑛𝑙𝑖𝑘 × 𝐴𝑛ı𝑚𝑠𝑎𝑚𝑎
𝐾𝑒𝑠𝑖𝑛𝑙𝑖𝑘 + 𝐴𝑛ı𝑚𝑠𝑎𝑚𝑎
(3)
ROC Alanı ölçütü, sınıflandırıcı başarımı değerlendirmede
kullanılan etkili bir ölçüttür ve Denklem (4)’teki gibi hesaplanır.
1
𝐷𝑃
𝐷𝑁
𝑅𝑂𝐶 𝐴𝑙𝑎𝑛ı = (
+
)
2 𝐷𝑃 + 𝑌𝑁 𝐷𝑁 + 𝑌𝑃
(4)
3 Bulgular
Farklı önişleme seçenekleri uygulanarak elde edilen sekiz farklı
doküman-terim matrisi üzerinde Bölüm 2.3’te bahsedilen
sınıflandırma algoritmaları çalıştırılarak sınıflandırma
başarımları gözlenmiştir. Tablo 2’de bu doküman-terim
matrislerinin özellikleri özetlenmiştir.
Tablo 2: Doküman terim matrislerinin özellikleri.
No
1
2
3
4
5
6
7
8
Sınıftaki Doküman Sayısı
Düşük
Orta
Yüksek
43
150
151
43
150
151
43
150
151
43
150
151
129
129
129
129
129
129
129
129
129
129
129
129
Önişleme Seçenekleri
Kök Bulucu
n-gram
Ek Çıkaran
1-gram
Ek Çıkaran
2-gram
Zemberek
1-gram
Zemberek
2-gram
Ek Çıkaran
1-gram
Ek Çıkaran
2-gram
Zemberek
1-gram
Zemberek
2-gram
Sekiz doküman-terim matrisi üzerinde beş sınıflandırma
algoritması işletilerek toplam 40 deney sonucu elde edilmiş
olup, bu sonuçlar Tablo 3 ve 4’te toplu halde gösterilmektedir.
Tablolarda her bir doküman-terim matrisi üzerinde elde edilen
en yüksek başarım değerleri koyu olarak gösterilmiştir.
Dengesiz sınıflara sahip veri seti ile elde edilen sonuçlara göre
en yüksek F-Skoru ve ROC Alanı değerleri 1 No.lu dokümanterim matrisinde sırasıyla 0.541 ve 0.690 olarak bulunmuştur.
Ek Çıkaran kök bulucu ve 1-gram önişleme seçeneklerinin
genellikle daha iyi sonuç verdiği görülmektedir. Diğer önişleme
seçeneklerinde de karar ağacı tabanlı sınıflandırıcılar olan
Random Forest ve Rotation Forest algoritmalarının yüksek
başarım gösterdikleri görülmektedir.
Dengeli sınıflara sahip veri seti ile elde edilen sonuçlara göre en
yüksek F-Skoru ve ROC Alanı değerleri 5 No.lu doküman-terim
matrisinde sırasıyla 0.745 ve 0.898 olarak bulunmuştur. ROC
Alanı ölçütüne göre tüm önişleme seçeneklerinde en yüksek
başarım yine karar ağaçları tabanlı algoritmalarla elde edilmiş
olup Random Forest algoritması en yüksek başarıma sahiptir.
Diğer ölçütler bakımından ise SMO algoritmasıyla elde edilen
yüksek sonuçlar da dikkat çekicidir.
Tablo 3: Dengesiz sınıflardan oluşan doküman-terim
matrislerinde deney sonuçları.
D-T
FROC
Sınıflandırıcı Kesinlik Anımsama
Matrisi
Skoru Alanı
NB
0.485
0.488
0.486 0.570
NBM
0.533
0.535
0.532 0.665
1
SMO
0.542
0.541
0.541 0.630
RaF
0.496
0.561
0.526 0.690
RoF
0.490
0.523
0.501 0.653
NB
0.573
0.520
0.527 0.660
NBM
0.486
0.462
0.467 0.643
2
SMO
0.506
0.517
0.507 0.597
RaF
0.528
0.515
0.519 0.677
RoF
0.497
0.520
0.496 0.650
NB
0.456
0.456
0.455 0.577
NBM
0.465
0.465
0.465 0.580
3
SMO
0.489
0.491
0.490 0.580
RaF
0.556
0.541
0.512 0.670
RoF
0.494
0.523
0.498 0.628
NB
0.494
0.468
0.474 0.627
NBM
0.449
0.422
0.429 0.606
4
SMO
0.467
0.480
0.469 0.550
RaF
0.481
0.488
0.484 0.625
RoF
0.485
0.532
0.496 0.645
Tablo 4: Dengelenmiş sınıflardan oluşan doküman-terim
matrislerinde deney sonuçları.
D-T
FROC
Sınıflandırıcı Kesinlik Anımsama
Matrisi
Skoru Alanı
NB
0.611
0.612
0.610 0.767
NBM
0.718
0.718
0.717 0.849
5
SMO
0.712
0.724
0.716 0.817
RaF
0.747
0.749
0.745 0.898
RoF
0.687
0.700
0.690 0.878
NB
0.601
0.592
0.560 0.825
NBM
0.681
0.695
0.686 0.872
6
SMO
0.730
0.729
0.727 0.833
RaF
0.724
0.724
0.723 0.880
RoF
0.696
0.700
0.698 0.866
NB
0.569
0.563
0.565 0.745
NBM
0.624
0.612
0.617 0.791
7
SMO
0.631
0.651
0.637 0.766
RaF
0.721
0.726
0.721 0.886
RoF
0.656
0.672
0.660 0.858
NB
0.573
0.592
0.570 0.800
NBM
0.657
0.669
0.662 0.851
8
SMO
0.692
0.693
0.692 0.810
RaF
0.678
0.680
0.679 0.853
RoF
0.667
0.667
0.664 0.829
5 No.lu doküman-terim matrisinde Random Forest
algoritmasıyla 10-kat çapraz geçerleme sonucunda elde edilen
başarım değerlerinin üretildiği karmaşıklık matrisi (confusion
matrix) Tablo 5’te görülmektedir. Karmaşıklık matrisine göre,
düşük öncelikli 129 talebin tamamı yine düşük öncelikli olarak
eksiksiz bir biçimde tahmin edilebilmiştir. Ancak, gerçekte orta
öncelikli olan taleplerin 91 tanesi orta, 34 tanesi yüksek ve 4
tanesi ise düşük öncelikli olarak tahmin edilmiştir. Benzer
şekilde, gerçekte yüksek öncelikli olan taleplerin 70 tanesi
618
Pamukkale Univ Muh Bilim Derg, 25(5), 615-620, 2019
M. C. Tekin, V. Tunalı
yüksek, 56 tanesi orta ve 3 tanesi düşük öncelikli olarak tahmin
edilmiştir.
Tablo 5: 5 No.lu D-T matrisinde Random Forest algoritmasının
çalıştırılmasıyla elde edilen karmaşıklık matrisi.
Gerçek
Sınıf
Düşük
Orta
Yüksek
Toplam
Tahmin Edilen Sınıf
Düşük
Orta
129
0
4
91
3
56
136
147
Yüksek
0
34
70
104
Toplam
129
129
129
387
Görüldüğü gibi düşük öncelikli talepler açıkça diğerlerinden
ayrılabilirken, orta ve yüksek öncelikli taleplerin
ayrıştırılmasında başarı oranı düşmektedir. Bu durumun
oluşmasındaki temel sebebin, farklı öncelik sınıflarına ait talep
kayıtlarında geçen sözcüklerin sıklığı ve dağılımı olduğu
düşünülmektedir. Tablo 6’da her bir öncelik sınıfındaki
taleplerde en sık geçen ilk 20 terim (ön işlemeden sonra
doküman-terim matrisinde yer aldığı ve sınıflandırıcıların
eğitiminde kullanıldığı haliyle) ve bunların sıklıkları
verilmiştir. Tabloda koyu yazı tipi ile yazılan terimler, yalnızca
ilgili öncelik sınıfında bulunan ve diğer sınıflarda yer almayan
(ilk 20 içerisinde) terimlerdir. Tablo 6’daki değerler
incelendiğinde, orta ve yüksek öncelikli taleplerde sıklıkla aynı
sözcüklerin kullanıldığı, düşük öncelikli taleplerde ise sözcük
kullanımının ve sözcüklerin sıklık sıralarının farklılaştığı
görülmektedir.
Tablo 6: Sınıflardaki en sık ilk 20 terim.
Düşük
Sıra
Terim
Sıklık
1
zeyil
60
2
dosya
42
3
poliçe
39
4
rapor
39
5
rica
36
6
siste
36
7
poliç
36
8
ace
36
9
tarih
36
10
eder
33
11
uyar
27
12
kontrol
24
13
hatas
24
14
işle
24
15
nol
21
16
not
21
17 provizyo
21
18
limit
21
19
merhap
18
20
acil
18
Orta
Terim
Sıklık
rica
73
eder
67
merhap
55
poliçe
43
poliç
43
nol
34
iptal
34
dosya
32
transfer
29
siste
27
acil
25
kayıt
25
zeyil
25
kontrol
24
imagı
24
png
24
thumbnail
24
ace
23
hatas
22
sistem
18
Yüksek
Terim
Sıklık
poliç
101
eder
79
rica
76
merhap
67
nol
56
acil
47
poliçe
46
kayıt
46
iptal
40
siste
36
kontrol
31
zeyil
31
hatas
30
teklif
30
prim
28
aşağı
26
hata
25
imagı
25
png
25
thumbnail
25
Orta
ve
yüksek
öncelik
sınıflarındaki
20 terimden 16’sı ortak olmakla birlikte bunların sıklık sıraları
da çoğunlukla birbirine yakındır. Bu durum, bu iki sınıftan talep
kayıtlarının ayrıştırılmasında başarı oranının düşük olmasına
neden olmaktadır. Buna karşın, düşük öncelik sınıfındaki 20
terimden 13’ü orta ve 11’i yüksek öncelik sınıfındaki terimlerle
ortak olmakla birlikte terim sıklık sıraları da önemli ölçüde
farklılık göstermektedir. Örneğin, düşük öncelik sınıfındaki en
sık zeyil teriminin orta ve yüksek öncelik sınıflarındaki sıklık
sıraları 13 ve 12’dir. Benzer şekilde, düşük öncelik sınıfında en
alt sıradaki merhap ve acil terimleri ise diğer sınıfların sıklık
sıralamasında üst sıralarda yer almaktadır. Bu belirgin farklılık,
düşük öncelikli taleplerin orta ve yüksek öncelikli taleplerden
ayrıştırılmasındaki yüksek başarı oranını açıklamaktadır.
Sınıflandırıcı başarımı bakımından elde edilen sonuçlar genel
olarak çok şaşırtıcı değildir çünkü Random Forest gibi topluluk
öğrenmesi yöntemlerinin bireysel yöntemlere göre daha
yüksek sınıflandırma başarımı sergiledikleri bilinmektedir.
Yine bir topluluk öğrenmesi yöntemi olan Rotation Forest
algoritmasının da Random Forest algoritmasına yakın sonuçlar
ürettiği deney sonuçlarında görülmektedir.
4 Tartışma ve sonuç
Bu çalışmada, kullanıcıların metin formunda girdiği yazılım
geliştirme taleplerinin önceliklerinin tahmin edilmesi bir metin
sınıflandırma problemi olarak ele alınmıştır. Yüksek
başarımları nedeniyle metin sınıflandırma uygulamalarında
sıkça kullanılan çeşitli algoritmalar, farklı önişleme
seçenekleriyle elde edilmiş doküman-terim matrisleri üzerinde
çalıştırılarak elde edilen sonuçlar karşılaştırılmıştır. Mevcut
veri setindeki kayıtların öncelik sınıfları üzerindeki dağılımının
dengesiz olması nedeniyle sınıf dengeleme yoluna gidilmiş,
dengesiz ve dengeli veri setleriyle deneyler yapılarak elde
edilen sonuçlar sunulmuştur.
Deney sonuçlarına göre Random Forest algoritmasının
genellikle tüm koşullarda iyi sonuçlar verdiği görülmektedir.
Dengeli veri seti üzerinde Random Forest algoritmasıyla 0.745
F-Skoru ve 0.898 ROC Alanı sonuçlarına ulaşılmıştır. Yine karar
ağacı tabanlı bir algoritma olan Rotation Forest ile de yakın
sonuçlar elde edilmiştir.
Bu çalışmada ele alınan problemin yaşandığı şirketin talep
yönetim sistemine yapılacak bir eklemeyle, talep
önceliklendirme konusunda BT birimine yardımcı olacak bir
karar destek sisteminin oluşturulması ve işlerliğinin
denenmesi planlanmaktadır. Bu iyileştirme çalışması
kapsamında, iç müşterinin belirlediği ilk öncelik değerinin ve
bu sistemin tahmin ettiği öncelik değerinin de kayıtlar üzerinde
saklanması ve böylece sistem başarımının izlenebilir olması
hedeflenmektedir. Ayrıca, saklanacak bu bilgilerin, sistemin
yeniden eğitimi ve hassas ayarlamalar yapılabilmesi
bakımından yararlı olacağı değerlendirilmektedir.
Bu çalışmada, kullanılan veri setinin küçük olmasının
sınıflandırma başarımını düşüren bir etken olduğu
düşünülmektedir. Veri setinin elde edildiği talep yönetim
sisteminden ileride elde edilecek daha geniş bir veri seti ile
benzer deneyler yapılarak başarımın arttırılması sağlanabilir.
Ek olarak, çeşitli önişleme ve ağırlıklandırma teknikleri ile
Yapay Sinir Ağları ve son zamanlarda oldukça etkili sonuçlar
verdiği görülen Derin Öğrenme yöntemleri kullanılarak daha
iyi bir talep önceliklendirme yapılmaya çalışılabilir.
5 Kaynaklar
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English
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Genome-scale identification and characterization of moonlighting proteins
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Biology direct
| 2,014
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cc-by
| 23,878
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Abstract Background: Moonlighting proteins perform two or more cellular functions, which are selected based on various
contexts including the cell type they are expressed, their oligomerization status, and the binding of different
ligands at different sites. To understand overall landscape of their functional diversity, it is important to establish
methods that can identify moonlighting proteins in a systematic fashion. Here, we have developed a computational
framework to find moonlighting proteins on a genome scale and identified multiple proteomic characteristics of
these proteins. Results: First, we analyzed Gene Ontology (GO) annotations of known moonlighting proteins. We found that the
GO annotations of moonlighting proteins can be clustered into multiple groups reflecting their diverse functions. Then, by considering the observed GO term separations, we identified 33 novel moonlighting proteins in Escherichia
coli and confirmed them by literature review. Next, we analyzed moonlighting proteins in terms of protein-protein
interaction, gene expression, phylogenetic profile, and genetic interaction networks. We found that moonlighting
proteins physically interact with a higher number of distinct functional classes of proteins than non-moonlighting
ones and also found that most of the physically interacting partners of moonlighting proteins share the latter’s primary Results: First, we analyzed Gene Ontology (GO) annotations of known moonlighting proteins. We found that the
GO annotations of moonlighting proteins can be clustered into multiple groups reflecting their diverse functions. Results: First, we analyzed Gene Ontology (GO) annotations of known moonlighting proteins. We found that the
GO annotations of moonlighting proteins can be clustered into multiple groups reflecting their diverse functions. Then, by considering the observed GO term separations, we identified 33 novel moonlighting proteins in Escherichia
coli and confirmed them by literature review. Next, we analyzed moonlighting proteins in terms of protein-protein
interaction, gene expression, phylogenetic profile, and genetic interaction networks. We found that moonlighting
proteins physically interact with a higher number of distinct functional classes of proteins than non-moonlighting
ones and also found that most of the physically interacting partners of moonlighting proteins share the latter’s primary
functions. Interestingly, we also found that moonlighting proteins tend to interact with other moonlighting proteins. In
terms of gene expression and phylogenetically related proteins, a weak trend was observed that moonlighting proteins
interact with more functionally diverse proteins. Structural characteristics of moonlighting proteins, i.e. intrinsic
disordered regions and ligand binding sites were also investigated. Open Access * Correspondence: dkihara@purdue.edu
1Department of Computer Science, Purdue University, 305 North University
Street, West Lafayette, IN 47907, USA
2Department of Biological Sciences, Purdue University, 240 Martin Jischke
Drive, West Lafayette, IN 47907, USA
Full list of author information is available at the end of the article Genome-scale identification and characterization
of moonlighting proteins Ishita Khan1, Yuqian Chen2, Tiange Dong2, Xioawei Hong2, Rikiya Takeuchi3, Hirotada Mori3 an Ishita Khan1, Yuqian Chen2, Tiange Dong2, Xioawei Hong2, Rikiya Takeuchi3, Hirotada Mori3 and Daisuke Kihara1,2* Khan et al. Biology Direct (2014) 9:30
DOI 10.1186/s13062-014-0030-9 Khan et al. Biology Direct (2014) 9:30
DOI 10.1186/s13062-014-0030-9 Background and the cell-level benefits given by moonlighting proteins
suggest that the existence of moonlighting proteins in
diverse genomes might be a common phenomenon. With the overwhelming growth of genome sequence
data produced by rapidly advancing sequencing tech-
nologies, the challenge of correctly determining func-
tions of encoded proteins becomes ever more evident. As the number of functionally characterized proteins
increases, it has been observed that there are proteins
involved in more than one function [1-3]. These proteins
were described as “moonlighting” proteins [1]. A moon-
lighting protein demonstrates multiple autonomous and
usually unrelated functions. Diversity of dual functions
of these proteins is in principle not a consequence of gene
fusions, splice variants, multiple proteolytic fragments,
homologous but non-identical proteins, or varying post-
transcriptional modification. Moonlighting proteins also pose a significant challenge
to computational protein function annotation as current
methods do not explicitly consider the possibility of dual
functions for a protein. Conventional sequence-based func-
tional annotation methods that are based on the concept of
homology [21] or conserved motifs/domains [22-24] will
have problems for identifying secondary functions because
there are cases that a homolog of a moonlighting protein
does not possess the secondary function [25] or has a
different secondary function [16,26,27]. There are two
studies that have investigated whether existing sequence-
based function prediction methods can identify distinct
dual functions of moonlighting proteins [28,29]. Gomez
et al. compared eleven methods and reported that
PSI-BLAST [21] performed relatively well in identifying
moonlighting functions [28]. We have compared our
function prediction tools, PFP [30,31] and ESG [32],
with PSI-BLAST and showed that PFP, which mines func-
tion information from weakly similar sequences, had the
best performance in predicting two distinct functions of
moonlighting proteins [29]. These two studies suggest that
secondary functions may be found in distantly related
sequences if not in close homologs; however, further
investigation is needed because the studies are based on
a limited dataset. Gomez et al. have also analyzed
protein-protein interactions (PPIs) of moonlighting pro-
teins and showed that GO terms of secondary function
are enriched in interacting proteins, although they con-
cluded that predicting correct secondary function from
a PPI network is not an easy task [33]. Computational
works on moonlighting proteins were recently summa-
rized in a review article [34]. Background The first and the most widely known example of
moonlighting proteins was identified by Piatigorsky and
Wistow [4] who showed that crystallins, structural pro-
teins in the eye lens, also have enzymatic activity. Crystal-
lins in several mammals, geckos, birds, and some other
species are eye lens proteins that retain their metabolic
functions, including lactate dehydrogenase, arginosucci-
nate lyase, and α-enolase [5-8]. Many known moonlight-
ing proteins were originally recognized as enzymes, but
there are also others that were known as receptors, channel
proteins, chaperone proteins, ribosomal proteins, and scaf-
fold proteins [1,9,10]. The secondary/moonlighting func-
tions of these proteins include transcriptional regulation,
receptor binding, apoptosis-related, and other regulatory
functions. A variety of causes have been found for the
moonlighting activities of these proteins [1], including loca-
tions inside and outside of cell (e.g. thymidine phosphoryl-
ase [11]), different locations within a cell (put A proline
dehydrogenase [12]), ligand binding sites (E. coli aspartate
receptor [13]), oligomerization states (glyceraldehyde-3-
phosphate dehydrogenase [14]), differential expressions
(neuropilin [15]), and ligand concentration (aconitase [16]). Despite the potential abundance of moonlighting
proteins in various genomes and their important roles
in pathways and disease development, systematic studies
of moonlighting proteins are still in their early stage for
obtaining a comprehensive picture of proteins’ moonlight-
ing functions and also for developing computational
methods for predicting moonlighting proteins. The limited
number of known moonlighting proteins is mainly be-
cause secondary functions of proteins are usually found
unexpectedly by experiments. To lay the foundation for
studying moonlighting proteins, the current work is aimed
at establishing a framework for systematically identifying
moonlighting proteins in an organism using currently
available function annotations and omics-scale data. This
work consists of two logical parts. First, we examined
Gene Ontology (GO) annotations [35,36] of known moon-
lighting proteins in the UniProt protein sequence database
[37] to see if functional diversity of moonlighting proteins
is reflected in current GO annotations. Since the systematic As long as the additional functions do not interfere
with the primary function, moonlighting functions can
benefit a cell in several ways. Especially in prokaryotes,
existence of multifunctional proteins aids in saving
energy in cell growth and reproduction and makes their
genomes more compact. Moonlighting proteins can also
help in coordinating cellular activities in signalling path-
ways, transport, biosynthesis, and other functions [17]. It
has been suggested that the presence of moonlighting
proteins is under positive selection [1,10,18]. Abstract Conclusion: Additional functions of moonlighting proteins are difficult to identify by experiments and these proteins
also pose a significant challenge for computational function annotation. Our method enables identification of novel
moonlighting proteins from current functional annotations in public databases. Moreover, we showed that potential
moonlighting proteins without sufficient functional annotations can be identified by analyzing available omics-scale
data. Our findings open up new possibilities for investigating the multi-functional nature of proteins at the systems level
and for exploring the complex functional interplay of proteins in a cell. Reviewers: This article was reviewed by Michael Galperin, Eugine Koonin, and Nick Grishin. Keywords: Moonlighting protein, Multitasking, Function annotation, Genome, Omics data © 2014 Khan et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain
Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article,
unless otherwise stated. Khan et al. Biology Direct (2014) 9:30 Page 2 of 29 Background Recent papers [10,19] indicate that a number of moon-
lighting proteins in mammals play important roles in
cellular activities and biochemical pathways that are
involved in cancer and other diseases. Sriram et al. discussed how moonlighting functions may contribute to
the complexity of metabolic disorders [20]. The positive
selective pressure for developing moonlighting functions Khan et al. Biology Direct (2014) 9:30 Page 3 of 29 Page 3 of 29 study of moonlighting proteins is still in an early stage,
most of the cases they are not explicitly labelled in the data-
base as “moonlighting”, “dual function”, “multitasking”, or
related words, which makes it difficult to collect and reuse
existing knowledge of moonlighting proteins. We analyzed
the GO terms assigned to each known moonlighting pro-
tein and found that the GO term semantic similarity score
can clearly separate the GO terms of the diverse functions
of these proteins. Encouraged by this result, we further ana-
lyzed the GO term annotations of protein genes in the
Escherichia coli K-12 genome and found 33 novel moon-
lighting proteins by identifying genes with clear GO term
separations. We confirmed in literature that the dual func-
tions of the identified proteins had experimental evidence. Among our computationally identified moonlighting
proteins, we later found that DegP was experimentally
identified as a moonlighting protein with both protease
and chaperone activity [38-40], which confirmed that
our procedure was valid. annotations. We used 58 experimentally confirmed moon-
lighting proteins in three datasets (see Methods). We
classified the GO terms of these proteins into four classes:
GO terms that belong to the “primary” function of the
protein (Function 1, F1), terms that belong to the “second-
ary” function (Function 2, F2), terms that belong to both
functions (F3), and terms that do not belong to either of
the functions. For each moonlighting protein, we com-
puted the relevance semantic similarity score (SSRel, Eqn. 1)
for three types of GO term pairs: pairs where both terms
belong to either F1 or F2 and pairs that consist of one GO
term from F1 and the other from F2. SSRel ranges from 0.0
to 1.0 with 0.0 for the least similarity and 1.0 for the high-
est similarity. Figure 1 shows an example of the semantic similarity of
GO pairs for aconitase in yeast (UniProt ID: P19414). Background This
protein was initially identified as an enzyme in the tri-
carboxylic acid (TCA) cycle, which catalyzes the isomeriza-
tion of citrate to iso-citrate via cis-aconitate. The GO terms
for F1 include TCA cycle (GO:0006099), propionate meta-
bolic process (GO:0019541), glutamate biosynthetic process
(GO:0006537), citrate metabolic process (GO:0006101),
cytosol (GO:0005829), cytoplasm (GO:0005737), citrate
hydro-lyase (GO:0052632), lyase activity (GO:0016829),
iso-citrate hydro-lyase (GO:0052633) and aconitate hydra-
tase activity (GO:0003994). The enzyme’s secondary func-
tion (F2) was later found as a “role in mitochondrial DNA
maintenance” [26], which is annotated with GO terms in-
cluding mitochondrial genome maintenance (GO:0000002),
mitochondrial nucleoid (GO:0042645), single-stranded-
DNA binding (GO:0003697), and double-stranded-DNA
binding (GO:0003690). The GO terms that belong to both
the primary and secondary functions (F3) are “mitochon-
drion” and “mitochondrial matrix” (GO:0005759). Figure 1A
shows the SSRel score distribution of GO term pairs, those
within F1 or F2 and pairs across F1 and F2 (F1F2 pairs). It
is apparent that the SSRel scores for all the F1F2 pairs are
very small, below 0.2. All four F2 pairs have large scores
over 0.4. As for F1 pairs, 8 out of 27 have large scores over
0.4. We must note that 12 F1 pairs have a score of 0, which
occurs when the lowest common ancestor for a GO term
pair is at the root of the GO hierarchy. In the case of
aconitase, the majority of the 0 scores for F1 pairs occurred
between terms related to ion-sulfur cluster binding and
aconitase hydrolase (Figure 1B). In the second part of this work, we investigated charac-
teristics of moonlighting proteins in omics-scale data,
namely, protein-protein interaction, gene expression, phy-
logenetic profile [41], and genetic interactions [42]. We
decided to analyze these omics-scale data because moon-
lighting proteins’ distinct functions may display characte-
ristic features in association patterns with other proteins. In analyzing protein-protein interactions, we found that
moonlighting proteins interact with a higher number
of distinct functional classes of proteins than non-
moonlighting ones, which intuitively stems from the func-
tional diversity of these proteins. We found a substantial
number of moonlighting proteins in the PPI network of
moonlighting proteins, suggesting moonlighting proteins
tend to interact with other moonlighting proteins. It is also
notable that moonlighting proteins share their primary
functions with the majority of interacting proteins. Background Simi-
larly, a weak tendency was found that moonlighting pro-
teins interact with proteins from more diverse functional
classes in gene expression and phylogenetic profile net-
works. We have further examined structural features of
proteins, i.e. ligand binding sites and disordered regions. We analysed disordered regions and found that a larger
fraction of moonlighting proteins have intrinsically
disordered regions than non-moonlighting proteins. Finally, although there are only a few moonlighting pro-
teins whose tertiary structures were available, we found
cases where the binding sites that correspond to distinct
functions are located in separate regions of the proteins’
tertiary structures. Figure 1B shows a hierarchical clustering of GO terms
of aconitase based on SSRel. In all three GO categories,
terms in F1 and F2 were clearly separated. In the Biological
Process (BP) ontology, the only GO term in F2 is “mito-
chondrial genome maintenance” (GO:0000002), which is
separated from the other F1 GO terms. In the Molecular
Function (MF) ontology, the GO terms with F2 labels
(ssDNA and dsDNA binding, GO:0003697 and GO:0003690,
respectively) form a cluster that is separate from the Pairwise GO semantic similarity analysis Pairwise GO semantic similarity analysis We investigated whether the distinct dual functions of
moonlighting proteins were reflected in their GO term Page 4 of 29 Khan et al. Biology Direct (2014) 9:30 Figure 1 Semantic similarity distribution. The distribution of the relevance semantic similarity SSRel score of GO term pairs, aconitase, yeast
(Uniprot ID : P19414). (A) SSRel distribution of GO pairs within the primary function (function 1), the secondary function (function 2), and pairs
from function 1 and 2. (B) Hierarchical clustering of GO terms in the three GO categories using pairwise SSRel scores. Figure 1 Semantic similarity distribution. The distribution of the relevance semantic similarity SSRel score of GO term pairs, aconitase, yeast
(Uniprot ID : P19414). (A) SSRel distribution of GO pairs within the primary function (function 1), the secondary function (function 2), and pairs
from function 1 and 2. (B) Hierarchical clustering of GO terms in the three GO categories using pairwise SSRel scores. F1 GO terms. Two separate clusters were formed for F1
terms in MF, “Iron-Sulfer cluster binding” GO terms
(highlighted in yellow) and terms related to aconitase
enzymatic activity. The former F1 cluster lies closer to the
F2 cluster due to a common ancestral term “binding”. In
the Cellular Component (CC) ontology, the F2 term
“mitochondrial nucleoid” (GO:0042645) is separate from
F1 GO terms (related to cytoplasm) but clustered with
two F3 terms. F1 GO terms. Two separate clusters were formed for F1
terms in MF, “Iron-Sulfer cluster binding” GO terms
(highlighted in yellow) and terms related to aconitase
enzymatic activity. The former F1 cluster lies closer to the
F2 cluster due to a common ancestral term “binding”. In
the Cellular Component (CC) ontology, the F2 term
“mitochondrial nucleoid” (GO:0042645) is separate from
F1 GO terms (related to cytoplasm) but clustered with
two F3 terms. Friedman test was performed to evaluate statistical sig-
nificance of score difference between F1, F2, and F1F2
GO term pairs. It was shown that the F1F2 pairs have
significantly smaller scores than F1 and F2 pairs in BP and
CC (p-value < 0.05). As for MF, the score difference of
F1F2 pairs from F1 pairs had a p-value below 0.05 but the
p-value versus F2 pairs was a slightly larger value of 0.097. Moonlighting and non-moonlighting proteins in E. Pairwise GO semantic similarity analysis The Page 5 of 29 Page 5 of 29 Khan et al. Biology Direct (2014) 9:30 Figure 2 Average SSRel of GO term pairs for moonlighting proteins. Average SSRel of GO pairs within function 1, function 2, and pairs from
function 1 and 2 were computed separately. (A) Moonlighting proteins in the MPR1 set. Protein 24 is presenilin in Physcomitrella patens (Uniprot ID:
A9S846). This protein have one GO term each in F1 and F2 (F1 term GO:0004190, “aspartic type endopeptidase activity” and F2 term GO:0016021,
“intergral to membrane”). The two GO terms are in different ontologies, MF and CC respectively, and thus the scores are zero for F1 and F2 (because
there is only one term) as well as F1-F2 (because similarity of GO terms in different categories cannot be considered). (B) the MPR2 set; and (C) the
MPR3 set. Figure 2 Average SSRel of GO term pairs for moonlighting proteins. Average SSRel of GO pairs within function 1, function 2, and pairs from
function 1 and 2 were computed separately. (A) Moonlighting proteins in the MPR1 set. Protein 24 is presenilin in Physcomitrella patens (Uniprot ID:
A9S846). This protein have one GO term each in F1 and F2 (F1 term GO:0004190, “aspartic type endopeptidase activity” and F2 term GO:0016021,
“intergral to membrane”). The two GO terms are in different ontologies, MF and CC respectively, and thus the scores are zero for F1 and F2 (because
there is only one term) as well as F1-F2 (because similarity of GO terms in different categories cannot be considered). (B) the MPR2 set; and (C) the
MPR3 set. proteins that have at least four clusters in the clustering
profile at a 0.5 SSRel. 140 proteins were found to satisfy all
of these three criteria. We have also identified potential
non-moonlighting proteins by applying essentially the
opposite criteria to above: 1) proteins that have at least
eight GO terms in the UniProt annotation; 2) proteins that
have at most one cluster at a SSRel of 0.1; 3) proteins that
have at most one cluster at 0.5 SSRel. There were 150
proteins that satisfied these criteria for non-moonlighting
proteins. proteins that have distinct dual functions. Subsequently,
we used the Pfam database [22] to find domains in the
43 proteins in order to distinguish proteins whose
multi-functionality originates from different domains. Pairwise GO semantic similarity analysis coli
The previous section showed that GO terms of moon-
lighting proteins can be clustered into distinct functions
using the SSRel score. In this section we identified poten-
tial moonlighting proteins in the Escherichia coli K-12
genome by examining clusters of GO term annotations
taken from UniProt. We used GO terms of the BP ontology
because BP GO terms showed a clearer separation between
F1 and F2 functions (Figure 3). Next, we show the mean SSRel score for GO pairs
within F1 or F2 and across F1 and F2 for all moonlight-
ing proteins in the three datasets (Figure 2). The mean
SSRel scores for F1 pairs and F2 pairs are higher than
those for across F1F2 pairs in 51 (87.9%) moonlighting
proteins (MPR1-3 datasets). One exception of this trend is
Protein 17 in MPR1 (Figure 2A). This protein is aconitase
of Mycobacterium tuberculosis (UniProt ID: O53166),
which has “TCA cycle enzyme” as F1 and “iron-responsive
protein” as F2. This protein switches between the two
functions depending on the cellular iron levels, namely,
binding of a 4Fe-4S cluster occurs as a part of the aco-
nitase function whereas binding of a 3Fe-4S cluster trig-
gers the secondary function [16]. Thus, the GO term for
“4 iron, 4 sulfur cluster binding” (GO:0051539) was
classified for F1 and “3 iron, 4 sulfur cluster binding”
(GO:0051538) for F2, which resulted in a relatively high
SSRel score of 0.698 for this F1F2 pair. Figure 4 shows clustering profiles of moonlighting pro-
teins, where GO terms in BP and MF (Figure 4A and B)
were clustered using single linkage clustering at different
SSRel cutoff values. A clustering profile provides a more
thorough picture of GO term similarities than clustering
using a single cutoff value. It can show how the number of
clusters grows at different cutoff values. Using the profiles
for moonlighting proteins in MPR1 (black), MPR2 (red),
and MPR3 (green) as a reference, the following three cri-
teria were used to identify potential moonlighting proteins
in E. coli: 1) proteins that have at least eight GO terms in
the UniProt annotation; 2) proteins that have at least two
clusters in the clustering profile at a SSRel cutoff of 0.1; 3) Figure 3 summarizes the distribution of the average
SSRel score for F1, F2, and F1F2 GO pairs in the BP, MF,
and CC ontologies for the proteins in MPR1-3. Pairwise GO semantic similarity analysis GO terms associated with each Pfam domain in a pro-
tein were compared with the primary and secondary
functions of the protein. Finally, 33 proteins were selected
as moonlighting proteins through this post-processing
(Table 1). The selected moonlighting proteins were further
classified into three categories. The first category is for
moonlighting proteins that have clear experimental evi-
dence for two independent functions. The second category
is proteins for which we found literature evidence of
two diverse functions, but no evidence was found as to
whether those two functions are independent or related. For the 140 identified potential moonlighting proteins,
we manually consulted original literature to determine
the level of experimental support for annotated func-
tions and whether diverse functions are directly related
to each other. This literature check step has selected 43 Page 6 of 29 Khan et al. Biology Direct (2014) 9:30 Figure 3 Average SSRel distribution. Box-and-whisker plots for average
SSRel distribution of BP, MF, and CC GO pairs for the moonlighting
proteins in the MPR1-3 sets excluding proteins with * in Figure 2. The top and the bottom of a box show the first and third quartiles
and the line in the middle of a box is the median. The two ends of
whisker show the minimum and the maximum values. from the final list of moonlighting proteins. These proteins
happen to include five multi-reaction enzymes, which are
enzymes that are generally listed as bi-functional or multi-
functional proteins in UniProt and in literature. They per-
form multiple reactions with similar substrates in the same
or different pathways. A multi-reaction enzyme is not in-
cluded as a moonlighting protein in the original definition
[18]. However, they are kept here along with the five other
multi-domain proteins in Table 2 because they were de-
tected by the GO clustering criteria. The identified 33 moonlighting proteins (Table 1) and
10 multi-domain multi-function proteins (Table 2) do
not have many overlap with the MoonProt database [79]
and MultitaskProtDB [80]. Only two (PepA and DegP)
in Table 1 and one (NadR) in Table 2 were found in the
two databases. Figure 3 Average SSRel distribution. Box-and-whisker plots for average
SSRel distribution of BP, MF, and CC GO pairs for the moonlighting
proteins in the MPR1-3 sets excluding proteins with * in Figure 2. Pairwise GO semantic similarity analysis The top and the bottom of a box show the first and third quartiles
and the line in the middle of a box is the median. The two ends of
whisker show the minimum and the maximum values. Among the 140 proteins that were identified by the GO
clustering criteria, 97 (69.3%) of them were discarded later
by the literature survey. The discarded proteins satisfied
the three GO term clustering criteria but either a) the
sufficient number of GO term clusters was due to a non-
descriptive GO term at a high (general) level of the GO
hierarchy such as “transport” or “biosynthesis”, which
resulted in a small similarity scores with the other GO
terms; or b) experimental evidence of GO terms were
found in literature only for one of its functions but not the
other. Proteins discarded by the latter reason may be
confirmed as moonlighting proteins in the future when
experimental evidence is made available. The third category is for “weak” moonlighting proteins for
which the evidence for the secondary function was found
from a large scale assay or a phenotypic experiment of
mutants and the relationship between the primary and the
newly found secondary function is not known. We would
like to note that some of the moonlighting proteins classi-
fied into the second or the third category are so-called
neomorphic moonlighting proteins [19], which exhibit the
secondary function due to a mutation or conformational
change. Clustering profiles of the identified moonlighting and
non-moonlighting proteins in E. coli are shown in Figure 4
in comparison with the MPR1-3 datasets. Three categories
of moonlighting proteins as well as multi-domain multi-
functional proteins were also separately plotted. Clearly, Table 2 lists ten multi-functional and multi-domain
proteins that were excluded by the Pfam domain search Figure 4 Clustering profiles of sets of moonlighting and non-moonlighting proteins. For each protein in a dataset, GO terms were
clustered using various threshold values of SSRel and average number of GO term clusters were plotted. The datasets plotted were experimentally
known moonlighting proteins (MPR1, 2, and 3) and identified moonlighting and non-moonlighting proteins in E. coli (Ecoli-MP and Ecoli-nonMP). E. coli moonlighting proteins were also plotted separately for each evidence category, 1 to 3 (Ecoli-PosMP-Cat1-3; see Methods) as well as
multi-domain multi-function proteins. (A) BP GO terms were considered. (B) MF GO terms were considered. Pairwise GO semantic similarity analysis Figure 4 Clustering profiles of sets of moonlighting and non-moonlighting proteins. For each protein in a dataset, GO terms were
clustered using various threshold values of SSRel and average number of GO term clusters were plotted. The datasets plotted were experimentally
known moonlighting proteins (MPR1, 2, and 3) and identified moonlighting and non-moonlighting proteins in E. coli (Ecoli-MP and Ecoli-nonMP). E. coli moonlighting proteins were also plotted separately for each evidence category, 1 to 3 (Ecoli-PosMP-Cat1-3; see Methods) as well as
multi-domain multi-function proteins. (A) BP GO terms were considered. (B) MF GO terms were considered. Khan et al. Biology Direct (2014) 9:30 Page 7 of 29 Page 7 of 29 Table 1 Moonlighting proteins identified in E. coli
Proteinname/uniprot
ID/gene ID
First function
Additional functions
Categorya) Ref. b0118/P36683/AcnB
Aconitate hydratase
Post-transcriptional regulation; mRNA binding
I
[27]
b1019/P31545/EfeB
Peroxidase on guaiacol
Iron assimilation from heme; response to DNA
damage stimulas
I
[43]
b1276/P25516/AcnA
Aconitate hydratase
Post-transcriptional regulation; mRNA binding
I
[27]
b1967/P31658/HchA
Molecular chaperone
Glyoxalase activity
I
[44]
b3183/P42641/ObgE
GTPase
Role in ribosome biogenesis
I
[45,46]
b4151/P0A8Q3/FrdD
Membrane bound respiratory protein
(anaerobic condition)
Role in bacterial flagellar switch (aerobic conditions)
I
[47]
b4152/P0A8Q0/FrdC
Membrane bound respiratory protein
(anaerobic condition)
Role in bacterial flagellar switch (aerobic conditions)
I
[47]
b4153/P0AC47/FrdB
Membrane bound respiratory protein
(anaerobic condition)
Role in bacterial flagellar switch (aerobic conditions)
I
[47]
b4154/P00363/FrdA
Membrane bound respiratory protein
(anaerobic condition)
Role in bacterial flagellar switch (aerobic conditions)
I
[47]
b4179/P21499/Rnr
Helicase
RNase
I
[48]
b4260/P68767/
PepA†b)
Plasmid recombination
Peptide catabolic process; DNA binding/transcriptional
control
I
[49]
b0161/P0C0V0/DegP†
Chaperone
Proteolysis
II
[50]
b0509/P77161/GlxR
Glyoxylate metabolism
Allantoin assimilation; DNA damage response
II
[51,52]
b0957/P0A910/OmpA
Transport
1. Viral entry 2.DNA damage response
II
[51,53]
b1317/P77366/YcjU
Carbohydrate metabolism
1. Cell-to-cell plasmid transfer 2. Pairwise GO semantic similarity analysis Reduce the lethal
effects of stress
II
[54,55]
b1710/P06610/BtuE
Glutathione peroxidase
Non-essential role in vitamin-B12 transport
II
[56,57]
b2415/P0AA04/PtsH
Phosphocarrier protein essential in sugar
transport
Positive regulation of glycogen catabolism
II
[58]
b2552/P24232/Hmp
(aerobic condition) Nitric oxide dioxygenase
(NOD)
(anaerobic condition) Amplifier of superoxide stress,
NO and FAD reductase
II
[59,60]
b2949/P0A8I1/YqgF
Putative Holliday junction resolvase
Transcription anti-termination
II
[61,62]
b3414/P63020/NfuA
Fe-S biogenesis
Necessary for the use of extracellular DNA as the sole
source of carbon and energy
II
[63]
b3463/P0A9R7/FtsE
Cell division
Salt transport by ABC-Transporter
II
[64]
b3706/P25522/MnmE
tRNA modification
Regulating glutamate-dependent acid resistance
II
[65]
b0135/P31058/YadC
Cell adhesion
Reduce lethal effects of stress
III
[55]
b0284/P77489/YagR
Putative xanthine dehydrogenase
DNA damage response
III
[51]
b0543/P23895/EmrE
Multidrug transporter
DNA damage response
III
[51]
b1018/P0AB24/EfeO
Involved in Iron uptake
Response to lethal antimicrobial and environmental stress
III
[55]
b2037/P37746/RfbX
Putative O-antigen transporter
DNA damage response
III
[51]
b2147/P25889/PreA
Pyrimidine base degradation
Required for swarming motility
III
[66]
b2290/P0A959/AlaA
Involved in biosynthesis of alanine
Response to lethal antimicrobial and environmental stress
III
[55]
b3191/P64602/MlaB
Phospholipid ABC transporter
Response to lethal antimicrobial and environmental stress
III
[55]
b3233/P0A9Q9/Asd
Aspartate-semialdehyde dehydrogenase
DNA damage response
III
[51]
b4177/P0A7D4/PurA
Adenylosuccinate synthetase
DNA damage response
III
[51] Table 1 Moonlighting proteins identified in E. coli
Proteinname/uniprot
ID/gene ID
First function Table 1 Moonlighting proteins identified in E. coli
Proteinname/uniprot First function Table 1 Moonlighting proteins identified in E. coli Table 1 Moonlighting proteins identified in E. Pairwise GO semantic similarity analysis coli
Proteinname/uniprot
ID/gene ID
First function
b0118/P36683/AcnB
Aconitate hydratase
b1019/P31545/EfeB
Peroxidase on guaiacol
b1276/P25516/AcnA
Aconitate hydratase
b1967/P31658/HchA
Molecular chaperone
b3183/P42641/ObgE
GTPase
b4151/P0A8Q3/FrdD
Membrane bound respiratory protein
(anaerobic condition)
b4152/P0A8Q0/FrdC
Membrane bound respiratory protein
(anaerobic condition)
b4153/P0AC47/FrdB
Membrane bound respiratory protein
(anaerobic condition)
b4154/P00363/FrdA
Membrane bound respiratory protein
(anaerobic condition)
b4179/P21499/Rnr
Helicase
b4260/P68767/
PepA†b)
Plasmid recombination
b0161/P0C0V0/DegP†
Chaperone
b0509/P77161/GlxR
Glyoxylate metabolism
b0957/P0A910/OmpA
Transport
b1317/P77366/YcjU
Carbohydrate metabolism
b1710/P06610/BtuE
Glutathione peroxidase
b2415/P0AA04/PtsH
Phosphocarrier protein essential in sugar
transport
b2552/P24232/Hmp
(aerobic condition) Nitric oxide dioxygenase
(NOD)
b2949/P0A8I1/YqgF
Putative Holliday junction resolvase
b3414/P63020/NfuA
Fe-S biogenesis
b3463/P0A9R7/FtsE
Cell division
b3706/P25522/MnmE
tRNA modification
b0135/P31058/YadC
Cell adhesion
b0284/P77489/YagR
Putative xanthine dehydrogenase
b0543/P23895/EmrE
Multidrug transporter
b1018/P0AB24/EfeO
Involved in Iron uptake
b2037/P37746/RfbX
Putative O-antigen transporter
b2147/P25889/PreA
Pyrimidine base degradation
b2290/P0A959/AlaA
Involved in biosynthesis of alanine
b3191/P64602/MlaB
Phospholipid ABC transporter
b3233/P0A9Q9/Asd
Aspartate-semialdehyde dehydrogenase
b4177/P0A7D4/PurA
Adenylosuccinate synthetase
b4383/P0A6K6/DeoB
Phosphopentomutase
a)Moonlighting proteins are classified into four categories: I, both primary and
independent; II, both primary and the secondary functions have experimenta
moonlighting proteins, evidences for the secondary function is from a large s
primary and the secondary function is not known. Proteins are sorted by the
b)Proteins included in either MoonProt or MultiTaskDB are indicated with†. Pe
and MultiTaskDB Additional functions
Categorya) Ref. Post-transcriptional regulation; mRNA binding
I
[27]
Iron assimilation from heme; response to DNA
damage stimulas
I
[43]
Post-transcriptional regulation; mRNA binding
I
[27]
Glyoxalase activity
I
[44]
Role in ribosome biogenesis
I
[45,46]
Role in bacterial flagellar switch (aerobic conditions)
I
[47]
Role in bacterial flagellar switch (aerobic conditions)
I
[47]
Role in bacterial flagellar switch (aerobic conditions)
I
[47]
Role in bacterial flagellar switch (aerobic conditions)
I
[47]
RNase
I
[48]
Peptide catabolic process; DNA binding/transcriptional
control
I
[49]
Proteolysis
II
[50]
Allantoin assimilation; DNA damage response
II
[51,52]
1. Viral entry 2.DNA damage response
II
[51,53]
1. Cell-to-cell plasmid transfer 2. a)Moonlighting proteins are classified into four categories: I, both primary and the secondary functions have clear experimental evidences that they are
independent; II, both primary and the secondary functions have experimental evidences but it is not clear if the functions are independent; III, “weak”
moonlighting proteins, evidences for the secondary function is from a large scale assay or a phenotypic experiment of mutants and the relationship between the
primary and the secondary function is not known. Proteins are sorted by the b number within each category.
b)Proteins included in either MoonProt or MultiTaskDB are indicated with†. PepA is included in the MoonProt database. DegP is included in both MoonProt
and MultiTaskDB. Pairwise GO semantic similarity analysis Reduce the lethal
effects of stress
II
[54,55]
Non-essential role in vitamin-B12 transport
II
[56,57]
Positive regulation of glycogen catabolism
II
[58]
(anaerobic condition) Amplifier of superoxide stress,
NO and FAD reductase
II
[59,60]
Transcription anti-termination
II
[61,62]
Necessary for the use of extracellular DNA as the sole
source of carbon and energy
II
[63]
Salt transport by ABC-Transporter
II
[64]
Regulating glutamate-dependent acid resistance
II
[65]
Reduce lethal effects of stress
III
[55]
DNA damage response
III
[51]
DNA damage response
III
[51]
Response to lethal antimicrobial and environmental stress
III
[55]
DNA damage response
III
[51]
Required for swarming motility
III
[66]
Response to lethal antimicrobial and environmental stress
III
[55]
Response to lethal antimicrobial and environmental stress
III
[55]
DNA damage response
III
[51]
DNA damage response
III
[51]
DNA damage response
III
[51]
the secondary functions have clear experimental evidences that they are
evidences but it is not clear if the functions are independent; III, “weak”
cale assay or a phenotypic experiment of mutants and the relationship between the
b number within each category. pA is included in the MoonProt database. DegP is included in both MoonProt a)Moonlighting proteins are classified into four categories: I, both primary and the secondary functions have clear experimental evidences that they are
independent; II, both primary and the secondary functions have experimental evidences but it is not clear if the functions are independent; III, “weak”
moonlighting proteins, evidences for the secondary function is from a large scale assay or a phenotypic experiment of mutants and the relationship between the
primary and the secondary function is not known. Proteins are sorted by the b number within each category. b)Proteins included in either MoonProt or MultiTaskDB are indicated with†. PepA is included in the MoonProt database. DegP is included in both MoonProt
and MultiTaskDB. Khan et al. Biology Direct (2014) 9:30 Page 8 of 29 Table 2 Multi-domain proteins with multiple functions identified in E. coli
Gene ID /Protein name/uniprot ID
First function
Additional functions
Ref. Pairwise GO semantic similarity analysis coli non-moonlighting
proteins (Ecoli-NegMP) at the three semantic similarity
thresholds, 0.1, 0.5, and 1.0 for the BP ontology (Figure 4A)
(p-values < 0.05). As for the MF ontology, E. coli moon-
lighting proteins have significantly larger number of clus-
ters than the E. coli non-moonlighting proteins at threshold
1.0, using a p-value cutoff of 0.05. The full results of the KS
tests are provided in (Additional file 1: Table S1). First, we examined the number of interacting pro-
teins of moonlighting and non-moonlighting proteins
(Figure 5A). In addition to the E. coli moonlighting and
non-moonlighting proteins, histograms for the MPR1-3
sets are shown for comparison. Among the E. coli MP set,
11 proteins in the first category (those that have clear
experimental evidence of their dual functions) were also
separately plotted to verify that the observed trend for the
entire E. coli MP set was consistent with its most reliable
subset. Overall MP and nonMP have similar distributions
with the largest peak at 0–5 interacting proteins. A small
peak at 20–25 interacting proteins was observed for E. coli
MP. This peak consists of two proteins, pepA (P68767)
and frdB (P0AC47). It was noticed that known moonlighting proteins in
the MPR1-3 sets have more GO annotations than the
E. coli moonlighting proteins, which is a part of the reason
why the MPR1-3 sets have more GO clusters (Figure 4). The average number of BP GO annotations of the E. coli
moonlighting proteins was 5.76 while the MPR1-3 pro-
teins had 9.65 terms. The clustering profile analysis can
identify new moonlighting proteins from their existing
GO annotations in UniProt. However, a limitation is
that candidate proteins need to be well annotated with a
sufficient number of GO terms. Indeed only 29.1% of
E. coli proteins have eight or more GO terms and were
subject to this analysis. In the subsequent sections, we
will explore different ways to identify potential moon-
lighting proteins that do not require GO annotations. Next, we checked the functional divergence of interacting
proteins. Using the same datasets as Figure 5A, interacting
proteins for each moonlighting or non-moonlighting pro-
teins in the datasets are clustered based on their functional
similarity using the funsim score (Eqn. 4). In Figure 5B, the
average numbers of clusters per interacting protein at dif-
ferent threshold values are plotted. Pairwise GO semantic similarity analysis b0002/P00561/ThrA
Aspartokinase
Homoserine dehydrogenase
[67]
b0529/P24186/FolD
Oxidation of methylenetetrahydrofolate
Hydrolysis of methenyltetrahydrofolate
[68]
b1241/P0A9Q7/AdhE
Alcohol dehydrogenase
Acetaldehyde dehydrogenase; Pyruvate-formate-lyase
deactivase
[69,70]
b1888/P07363/CheA
Chematoxis sensor kinase
Regulation of protein; dephosphorylation
[27,71,72]
b2255/P77398/ArnA
Oxidative decarboxylation of
UDP-glucuronic acid
Formyltransferase
[73]
b3052/P76658/HldE
D-beta-D-heptose 7-phosphate kinase
D-beta-D-heptose 1-phosphate adenosyltransferase
[74]
b3368/P0AEA8/CysG
SAM-dependent methylation
NAD-dependent ring dehydrogenation; Ferrorochelation
[75]
b3650/P0AG24/SpoT
ppGpp synthase
ppGpp hydrolase
[76,77]
b3940/P00562/MetL
Aspartokinase
Homoserine dehydrogenase
[67]
b4390/P27278/NadR†
Transcriptional regulator
Nicotinamide mononucleotide adenylyltransferase;
Ribosylnicotinamide kinase
[78]
†This protein is included in MoonProt. Table 2 Multi-domain proteins with multiple functions identified in E. coli Table 2 Multi-domain proteins with multiple functions identified in E. coli
Gene ID /Protein name/uniprot ID
First function
Additional f †This protein is included in MoonProt. thus a PPI network can be used as a valuable source for
predicting protein function [81]. It was also shown that
PPI networks are helpful in detecting additional novel
function of well-known proteins [82]. We obtained physic-
ally interacting proteins from the STRING database [83]. the number of GO term clusters for moonlighting proteins
is higher than non-moonlighting proteins for both BP and
MF. In the MF ontology, the multi-domain multi-
functional proteins have a larger number of clusters
than the rest for high cutoff values of over 0.4. The
two-sample Kolmogorov-Smirnov (KS) test showed
that the E. coli moonlighting proteins (Ecoli-PosMP in
Figure 4) and the MPR1-3 sets have significantly larger
numbers of clusters than the E. coli non-moonlighting
proteins (Ecoli-NegMP) at the three semantic similarity
thresholds, 0.1, 0.5, and 1.0 for the BP ontology (Figure 4A)
(p-values < 0.05). As for the MF ontology, E. coli moon-
lighting proteins have significantly larger number of clus-
ters than the E. coli non-moonlighting proteins at threshold
1.0, using a p-value cutoff of 0.05. The full results of the KS
tests are provided in (Additional file 1: Table S1). the number of GO term clusters for moonlighting proteins
is higher than non-moonlighting proteins for both BP and
MF. In the MF ontology, the multi-domain multi-
functional proteins have a larger number of clusters
than the rest for high cutoff values of over 0.4. The
two-sample Kolmogorov-Smirnov (KS) test showed
that the E. coli moonlighting proteins (Ecoli-PosMP in
Figure 4) and the MPR1-3 sets have significantly larger
numbers of clusters than the E. Pairwise GO semantic similarity analysis The funsim score of all
three GO categories was used for Figure 5B while the
funsim score with only BP (BP-funsim score) was used for
Figure 5C. In the two clustering profiles (Figures 5B & 5C)
the non-MP set has consistently lower number of clusters
as compared to moonlighting proteins. E. coli MPs and
non-MPs show a clear contrast in the number of clusters
with the former having over twice as many clusters as the
latter. Consistent results were obtained when interacting
proteins were selected from the STRING database using a
score that combines different types of evidence including
physical interactions, comparative genomics approaches, Protein-protein interaction network From this section, we examine characteristic features
of moonlighting proteins in large-scale omics data. We
begin with the protein-protein interaction (PPI) network. Interacting proteins tend to share common function and Khan et al. Biology Direct (2014) 9:30 Page 9 of 29 Page 9 of 29 Figure 5 Interacting proteins of moonlighting and non-moonlighting proteins. Physically interacting proteins were obtained from the STRING
database. (A) Histogram of the number of interacting proteins. Five datasets are shown: known moonlighting proteins in the MPR1-3 sets (MPR-ALL), the
identified moonlighting proteins in E. coli (Ecoli-MP), moonlighting proteins detected in E. coli that have clear experimental evidences for the dual functions
and classified into the category 1 (Ecoli-MP-Cat1), E. coli proteins whose multi-functionality originates from different domains (Ecoli-MultiDomain) and
non-moonlighting proteins in E. coli. Values on the y-axis are the fraction of the proteins among the entire proteins in each dataset. The bin size used was
five. (B), average number of clusters of interacting proteins clustered using the funsim score (Eqn. 4). Seven datasets are plotted: MPR1, MPR2, MPR3,
Ecoli-MP, Ecoli-MP-Cat1, Ecoli-MultiDomain, and Ecoli-nonMP. (C) Clustering was performed using the funsim score of BP terms only (Eqn. 3). Figure 5 Interacting proteins of moonlighting and non-moonlighting proteins. Physically interacting proteins were obtained from the STRING
database. (A) Histogram of the number of interacting proteins. Five datasets are shown: known moonlighting proteins in the MPR1-3 sets (MPR-ALL), the
identified moonlighting proteins in E. coli (Ecoli-MP), moonlighting proteins detected in E. coli that have clear experimental evidences for the dual functions
and classified into the category 1 (Ecoli-MP-Cat1), E. coli proteins whose multi-functionality originates from different domains (Ecoli-MultiDomain) and
non-moonlighting proteins in E. coli. Values on the y-axis are the fraction of the proteins among the entire proteins in each dataset. The bin size used was
five. (B), average number of clusters of interacting proteins clustered using the funsim score (Eqn. 4). Seven datasets are plotted: MPR1, MPR2, MPR3,
Ecoli-MP, Ecoli-MP-Cat1, Ecoli-MultiDomain, and Ecoli-nonMP. (C) Clustering was performed using the funsim score of BP terms only (Eqn. 3). Figure 5 Interacting proteins of moonlighting and non-moonlighting proteins. Physically interacting proteins were obtained from the STRING
database. (A) Histogram of the number of interacting proteins. Five datasets are shown: known moonlighting proteins in the MPR1-3 sets (MPR-ALL), the
identified moonlighting proteins in E. coli (Ecoli-MP), moonlighting proteins detected in E. Protein-protein interaction network coli that have clear experimental evidences for the dual functions
and classified into the category 1 (Ecoli-MP-Cat1), E. coli proteins whose multi-functionality originates from different domains (Ecoli-MultiDomain) and
non-moonlighting proteins in E. coli. Values on the y-axis are the fraction of the proteins among the entire proteins in each dataset. The bin size used was
five. (B), average number of clusters of interacting proteins clustered using the funsim score (Eqn. 4). Seven datasets are plotted: MPR1, MPR2, MPR3,
Ecoli-MP, Ecoli-MP-Cat1, Ecoli-MultiDomain, and Ecoli-nonMP. (C) Clustering was performed using the funsim score of BP terms only (Eqn. 3). Do interacting proteins share moonlighting functions?
l
d h
h h h and gene expression (data not shown). A pairwise two-
sample KS divergence test showed that the average number
of clusters of the E. coli MP and nonMP sets is significantly
different at the funsim-BP threshold values of 0.2, 0.6,
and 0.8 and funsim threshold values 0.6 and 1.0 (Additional
file 1: Table S1). To conclude, the results show that moon-
lighting proteins interact with proteins with more diverse
functions than non-moonlighting ones. Do interacting proteins share moonlighting functions? We also investigated the extent to which the primary
and secondary functions of a moonlighting protein are
shared by its interacting proteins. For this analysis, we
used 27 moonlighting proteins in the MPR1-3 sets that
have interacting proteins because GO terms for their
primary and secondary functions were manually classified. For each moonlighting protein in MPR1-3, we computed Khan et al. Biology Direct (2014) 9:30 Page 10 of 29 is an example of two interacting moonlighting proteins
that have the same primary and secondary functions. the functional similarity of its primary function (F1) and
its secondary function (F2) separately against GO term an-
notation of its interacting proteins. Functional similarity
was quantified by the funsim score (Figure 6A) and the
BP-funsim score (Figure 6B). To determine if an interact-
ing protein was biased to either the F1 or F2 function, the
score difference between F1 and F2 was computed. The second example is mitogen activated protein
kinase 1 (ERK2) (P28482) in human. This protein is
MAP kinase and moonlights as a transcriptional repres-
sor [114]. It has 187 interacting proteins in the PPI
network, among which there are ten proteins with both
F1 and F2 functions. One of the interacting partners is
death-associated protein kinase 3 (DAPK3, UniProt:
O43293), which enhances transcriptional activities of
STAT3/P40763 by phosphorylating them. Besides the
kinase function, DARPK3 is known to have multiple
secondary functions, including involvement in apoptosis
[39], roles in transcription (same as the secondary function
of ERK2), regulation of cell polarity, contractile processes
in non-muscle or smooth muscle cells, and cytokinesis
[40]. Thus, in this example, among interacting moonlight-
ing proteins that share both F1 and F2 functions, one of
them has more secondary functions. It is evident that the F1 function is dominant for the
majority of the interacting proteins. Do interacting proteins share moonlighting functions?
l
d h
h h h When the funsim
score was considered (Figure 6A), 96.3% of the interact-
ing proteins had functions closer to the F1 rather than
the F2 function. The dominance of F1-oriented func-
tions in interacting proteins is consistent in Figure 6B,
where the BP-funsim score was considered. Figure 6C provides results for individual moonlighting
proteins. For a moonlighting protein, GO terms of its F1
and F2 functions were compared separately to the entire
GO annotation of each interacting protein. If GO terms
of an interacting protein have a BP-funsim score that is
larger than the mean SSRel scores of BP terms in F1 or
F2 of the moonlighting protein, the interacting protein
was considered to share common F1 or F2 function, re-
spectively, with the moonlighting protein. In the case
that a moonlighting protein has very diverse F1 or F2
GO terms in itself with the mean SSRel score of 0, we
used a BP-funsim score of 0.4 as a cutoff to determine if
an interacting protein shares F1 or F2 function. Consist-
ent with Figure 6A and 6B, the majority of interacting
proteins have F1 function for 18 out of 27 the moon-
lighting proteins (66.7%) (red bars). On the other hand,
only nine moonlighting proteins (33.3%) have interacting
proteins of F2 functions (blue bars), and among them
interacting proteins with F2 function are dominant for
three (11.1%) moonlighting proteins. Co-expressed proteins Next, we investigated functions of co-expressed genes
with moonlighting proteins in E. coli. The E. coli gene
expression data were taken from the COLOMBOS data-
base [115], which contains expression data of 4295 genes
in 2369 contrasts. We calculated the Pearson correlation
coefficient of expression levels of each pair of genes and
selected pairs as co-expressed if the absolute value of the
correlation coefficient is ranked within the top 2% largest
values among all the pairs. The number of co-expressed
genes of moonlighting and non-moonlighting proteins
do not have large difference, except for a peak observed
at 65 for the moonlighting proteins (Figure 7A), which
consists of four moonlighting proteins (P77489, P0A8Q3,
P0AC47, and P25516). Then, similar to the analysis in
Figure 5B and 5C, we computed functional clustering
profile for co-expressed genes of E. coli moonlighting
proteins to see if co-expressed genes have functional
divergence. The clustering profile using the funsim
score (Figure 7B) and the BP-funsim score (Figure 7C)
showed that the moonlighting proteins have a slightly
larger average number of clusters of functionally similar
proteins per co-expressed genes than that for non-
moonlighting proteins, although this difference is not
statistically significant (Additional file 1: Table S1). The
same conclusion was obtained when we defined co-
expressed genes as those which have over 0.4 of the
correlation coefficient value (data not shown). There are interacting proteins of moonlighting pro-
teins that have functional similarity with both F1 and F2
functions of moonlighting proteins (shown by green bars
in Figure 6C). Fifteen moonlighting proteins have in
total of 30 interacting proteins with both F1 and F2
functions. We analyzed assigned GO terms of these
interacting proteins by referring to literature and found
that 18 out of 30 of these proteins are also moonlighting
proteins while three proteins are multi-domain proteins. This result is summarized in Table 3. This result indi-
cates that moonlighting proteins tend to interact with
moonlighting proteins; thus, novel moonlighting pro-
teins may be identified by analyzing PPIs of moonlight-
ing proteins. We discuss two such cases. The first example is mis-
match repair endonuclease PMS2 (P54279) in mouse,
which also contributes to somatic hypermutation [113]. It has just one interacting protein, which is another
DNA mismatch repair protein Mlh1 (Q9JK91) that is
also involved in somatic hypermutation [105]. Thus, this Phylogenetically related genes (A) The functional similarity score is computed between GO terms of the primary (F1) or the secondary (F2) functions
of a moonlighting protein against the entire GO terms of its interacting protein and the score difference was computed. Interacting proteins
were classified by the range of funsim score difference between F1 and F2 GO terms from their interacting moonlighting proteins. (B) The
same type of chart as panel A, using the BP-funsim score. (C) For each moonlighting protein, percentages (%) of interacting proteins sharing
F1, F2, or both functions of moonlighting proteins are shown. The BP-funsim score was used to determine if proteins share functional similarity. If an interacting protein has a BP-funsim score to both F1 and F2 GO terms of the moonlighting protein, it is classified as both. An interacting
protein is considered to share F1, F2, or both functions if the BP-funsim score is larger than the mean SSRel score of BP GO pairs of F1 or F2 in
the moonlighting protein. In the case that a moonlighting protein has 0 SSRel score, the cutoff was set to 0.4 for an interacting protein to be
considered to share F1, F2, or both functions. P47897 does not have any interacting proteins with F1 or F2 function. Its only interacting protein,
RSBN1, has a BP-funsim score of 0 with F1 and F2 functions of P47897. P36024 also does not have any interacting proteins sharing F1 or F2
function. Out of its four interacting proteins, YKL088W has the highest funsim-BP score with F1/F2 GO terms of P36024 (score 0.25), which is
below the funsim-BP F1/F2 cutoff for P36024 (cutoff 0.4 for both F1 and F2). Figure 6 Function similarity analysis. Functional similarity between interacting proteins and the primary and secondary functions of moonlighting
proteins. 27 moonlighting proteins in the MPR1-3 sets that have physically interacting proteins in STRING database and their 575 interacting
proteins were analyzed. (A) The functional similarity score is computed between GO terms of the primary (F1) or the secondary (F2) functions
of a moonlighting protein against the entire GO terms of its interacting protein and the score difference was computed. Interacting proteins
were classified by the range of funsim score difference between F1 and F2 GO terms from their interacting moonlighting proteins. (B) The
same type of chart as panel A, using the BP-funsim score. Phylogenetically related genes Phylogenetically related genes
We further analyzed genes that have similar comparative
genomic context to the moonlighting proteins [41]. Using the STRING database, for a protein of interest, we
selected proteins as phylogenetically related if they were Page 11 of 29 Page 11 of 29 Khan et al. Biology Direct (2014) 9:30 Figure 6 Function similarity analysis. Functional similarity between interacting proteins and the primary and secondary functions of moonlighting
proteins. 27 moonlighting proteins in the MPR1-3 sets that have physically interacting proteins in STRING database and their 575 interacting
proteins were analyzed. (A) The functional similarity score is computed between GO terms of the primary (F1) or the secondary (F2) functions
of a moonlighting protein against the entire GO terms of its interacting protein and the score difference was computed. Interacting proteins
were classified by the range of funsim score difference between F1 and F2 GO terms from their interacting moonlighting proteins. (B) The
same type of chart as panel A, using the BP-funsim score. (C) For each moonlighting protein, percentages (%) of interacting proteins sharing
F1, F2, or both functions of moonlighting proteins are shown. The BP-funsim score was used to determine if proteins share functional similarity. If an interacting protein has a BP-funsim score to both F1 and F2 GO terms of the moonlighting protein, it is classified as both. An interacting
protein is considered to share F1, F2, or both functions if the BP-funsim score is larger than the mean SSRel score of BP GO pairs of F1 or F2 in
the moonlighting protein. In the case that a moonlighting protein has 0 SSRel score, the cutoff was set to 0.4 for an interacting protein to be
considered to share F1, F2, or both functions. P47897 does not have any interacting proteins with F1 or F2 function. Its only interacting protein,
RSBN1, has a BP-funsim score of 0 with F1 and F2 functions of P47897. P36024 also does not have any interacting proteins sharing F1 or F2
function. Out of its four interacting proteins, YKL088W has the highest funsim-BP score with F1/F2 GO terms of P36024 (score 0.25), which is Figure 6 Function similarity analysis. Functional similarity between interacting proteins and the primary and secondary functions of moonlighting
proteins. 27 moonlighting proteins in the MPR1-3 sets that have physically interacting proteins in STRING database and their 575 interacting
proteins were analyzed. Phylogenetically related genes (C) For each moonlighting protein, percentages (%) of interacting proteins sharing
F1, F2, or both functions of moonlighting proteins are shown. The BP-funsim score was used to determine if proteins share functional similarity. If an interacting protein has a BP-funsim score to both F1 and F2 GO terms of the moonlighting protein, it is classified as both. An interacting
protein is considered to share F1, F2, or both functions if the BP-funsim score is larger than the mean SSRel score of BP GO pairs of F1 or F2 in
the moonlighting protein. In the case that a moonlighting protein has 0 SSRel score, the cutoff was set to 0.4 for an interacting protein to be
considered to share F1, F2, or both functions. P47897 does not have any interacting proteins with F1 or F2 function. Its only interacting protein,
RSBN1, has a BP-funsim score of 0 with F1 and F2 functions of P47897. P36024 also does not have any interacting proteins sharing F1 or F2
function. Out of its four interacting proteins, YKL088W has the highest funsim-BP score with F1/F2 GO terms of P36024 (score 0.25), which is
below the funsim-BP F1/F2 cutoff for P36024 (cutoff 0.4 for both F1 and F2). Table 3 Interacting proteins that have both primary and secondary functions of moonlighting proteins in the
MPR1-3 set
Moonlighting proteins
Interacting proteins
Uniprot ID/Namea)
Primary functionb)
Secondary functionc)
UniProt ID/
Named)
Interacting protein functione)
MP/non-MPf)
Ref. P93834/HXK2
Glucose metabolism
Glucose signalling
Q42525/HXK1
1. Glycolysis
MP, I
[84-86]
2. Sugar mediated signaling programmed cell death
Q99798/ACO2
TCA cycle enzyme
Iron homeostasis
P21399/ACO1
1. Role in TCA cycle
MP, I
[87,88]
mRNA binding and role in iron homeostasis
P00924/ENO1
Galactose catabolism
enzyme
Homotypic vacuole fusion
P00925/ENO2
1. Glycolysis
MP, I
[89]
Vacuole fusion
Q08438/Vhs3
Halotorance determinant
Coenzyme A biosynthesis
P36024/SIS2
1. CoA biosynthesis
MP, I
[90]
Salt tolerance
P28482/ERK2
MAP kinase
Transcriptional
P01100
1. Regulation of transcription
MP, II
[91,92]
Repressor
/FOS
2. Activates phospholipid synthesis in growing cells
(regulated by Mos/MAP kinase pathway)
Q15796/SMAD2-5
1. TGF signaling protein
Multi-domain
[93]
2. Tumor suppressor, dual role in transcriptional activation
P05771/PRKCB
1. Serine/threonine-protein kinase, activates transcription. MP, II
[94]
Inhibition of the insulin gene transcription. O43293
1. Serine/threonine kinase
MP, I
[95,96]
/DAPK3
2. Role in apoptosis, transcription, regulation of cell
polarity, contractile processes in non-muscle or
smooth muscle cells, and cytokinesis
P14921/ETS1
Transcription factor
nonMP
-
P19838/NFKB1
1. O14786/Neuropilin-1 Phylogenetically related genes Transcription factor
MP, I
[97]
2. Cytoplasmic retention of attached NF-kappa-B
proteins by p105, generation of p50 by a
co-translational processing, transcriptional repressor
O43318
1. MAPK
MP, II
[98,99]
/MAP3K7
Regulates TF activator proteins
Q99ML3/STAT3
Transcription factor
Electron transport chain
Q5EG47/Prkaa1-2
1. Protein kinase that phosphorylates TF
MP, II
[100]
Regulation of cellular energy
Q62120/Jak2-3
1. Tyrosine protein kinase
Multi-domain
[101]
Regulation of cellular signaling and cell cycle control
P05480/Src
1. Tyrosine protein kinase
Multi-domain
[102]
Cytokine/cellular receptor
O14786/Neuropilin-1
Vascular endothelial growth
factor
Receptor for semaphorin III
Q14563/SEMA3A-G
1. Development of the olfactory system and in
neuronal control of puberty
MP, II
[103] Q99ML3/STAT3 O14786/Neuropilin-1 Vascular endothelial growth
factor
Receptor for semaphorin III Table 3 Interacting proteins that have both primary and secondary functions of moonlighting proteins in the
MPR1-3 set (Continued)
2. Ensures proper endothelial abundance of soluble
and alternatively spliced form of VEGF receptor(flt1)
P15692/VEGFA
Vascular endothelial growth factor
nonMP
-
P17948
1. VEGF receptor; plays negative role in angiogenesis
in the embryo most likely by trapping VEGF
MP, I
[104]
/FLT1/VEGFR/
2. Plays positive role in adulthood in a tyrosine
kinase-dependent manner
P54279/PMS2
Mismatch repair enzyme
Hypermutation of antibody variable
chains
Q9JK91/Mlh1
1. Mismatch repair protein
MP, I
[105]
Somatic hyper mutation
P19971/PD-ECGF
Thymidine phosphorylase
Platelet-derived endothelial cell
growth factor
P04183/TK1-2
1. Phosphotransferase activity
nonMP
-
Q96B60
Deoxyribonucleotidase, mitochondrial
nonMP
-
/NT5E, NT5M
P06744/Neuroleukin
Phosphoglucose Isomerase
Differentiation, maturation mediator
P52789/HK2
1. Hexokinase-2
MP, III
[106]
HK2 detachment causes apoptosis
P04075/ALDOA-C
1. Glycolysis and gluconeogenesis
MP, III
[107,108]
Regulation of cell shape
P30613/PKLR
1. Pyruvate kinase
MP, III
[109]
Mutation causes hemolytic anemia
P14618/PKM2
1. Pyruvate kinase
MP, III
[110]
Programmed cell death
P06795/P-glycoprotein
P-glycoprotein (transporter)
Regulator of cell-swelling ion
channel (K+/Cl-)
P41233/Abca1
Anion transporter
nonMP
-
Q91XR9/Phospholipid hydroperoxide
glutathione peroxidase
Antioxidant of mature
sperm
Structural protein of the mitochondrial
capsule
Q60928/Ggt1
1. Part of the cell antioxidant defense mechanism
MP, IV
[111,112]
2. Phylogenetically related genes Indirectly regulates multiple aspects of skeletal
biology
Q9P2J5/Leucine-tRNA ligase
tRNA synthetases
Translocation and activation of
mTORC1 to lysosomal membrane
Q9H6Q3/MARS
Methionine-tRNA ligase, cytoplasmic
nonMP
-
Q6P0M4/IARS
tRNA aminoacylation for protein translation
nonMP
-
P11325/Nam2p
Mitochondrial leucyl-tRNA
synthetase
bI4 mitochondrial RNA splicing
activity
P26637/CDC60
Leucine-tRNA ligase, cytoplasmic
nonMP
-
P19414/ACO1
TCA cycle enzyme
Mitochondrial DNA stability
P33421/SDH3
Succinate dehydrogenase involved in mt-electron
transport chain
nonMP
-
Thi t bl
d t
Fi
6C ing proteins that have both primary and secondary functions of moonlighting proteins in the
ntinued) Table 3 Interacting proteins that have both primary and secondary functions of moonlighting proteins in the
MPR1-3 set (Continued) P06744/Neuroleukin P11325/Nam2p P19414/ACO1 Khan et al. Biology Direct (2014) 9:30 Page 14 of 29 Figure 7 Gene expression profile analysis. Average number of clusters of interacting proteins relative to the number of proteins interacting by
gene expression. Proteins considered to be interacting are the top 2% of proteins in the Gene Expression network of E. coli sorted in terms of the
Pearson correlation coefficient. (A) Histogram of number of interacting proteins. (B) Functional clustering using Funsim (BP, MF, CC) score thresholds
between 0.1 and 1.0. (C) Functional clustering using Funsim (BP) score thresholds between 0.1 and 1.0. Figure 7 Gene expression profile analysis. Average number of clusters of interacting proteins relative to the number of proteins interacting by
gene expression. Proteins considered to be interacting are the top 2% of proteins in the Gene Expression network of E. coli sorted in terms of the
Pearson correlation coefficient. (A) Histogram of number of interacting proteins. (B) Functional clustering using Funsim (BP, MF, CC) score thresholds
between 0.1 and 1.0. (C) Functional clustering using Funsim (BP) score thresholds between 0.1 and 1.0. Figure 7 Gene expression profile analysis. Average number of clusters of interacting proteins relative to the number of proteins interacting by
gene expression. Proteins considered to be interacting are the top 2% of proteins in the Gene Expression network of E. coli sorted in terms of the
Pearson correlation coefficient. (A) Histogram of number of interacting proteins. (B) Functional clustering using Funsim (BP, MF, CC) score thresholds
between 0.1 and 1.0. (C) Functional clustering using Funsim (BP) score thresholds between 0.1 and 1.0. A larger fraction of the non-moonlighting proteins
have no phylogenetically related proteins as compared
with the moonlighting ones (0 at the x-axis in Figure 8A). The clustering profiles using the funsim score (Figure 8B)
and the BP-funsim score (Figure 8C) show that the E. coli
moonlighting proteins have slightly more functional
clusters on average, i.e. more functional divergence in
their phylogenetically related proteins, than their non-
moonlighting counterparts. The p-value of this differ-
ence in the number of functional clusters was 0.08 at located in the neighbourhood of the target genes, were
found to co-occur or co-absent, or were fused in multiple
genomes. Concretely, genes that have a sufficient score
(> 0.7 as recommended by STRING) at “neighborhood”,
“co-occurrence”, or “gene-fusion” in the STRING database
[83] were selected. P19414/ACO1 It has been observed that phylogenetic-
ally co-related proteins are functionally related in many
cases [41]. Figure 8 shows the clustering profiles of phylo-
genetically related proteins of the moonlighting and non-
moonlighting proteins. located in the neighbourhood of the target genes, were
found to co-occur or co-absent, or were fused in multiple
genomes. Concretely, genes that have a sufficient score
(> 0.7 as recommended by STRING) at “neighborhood”,
“co-occurrence”, or “gene-fusion” in the STRING database
[83] were selected. It has been observed that phylogenetic-
ally co-related proteins are functionally related in many
cases [41]. Figure 8 shows the clustering profiles of phylo-
genetically related proteins of the moonlighting and non-
moonlighting proteins. Khan et al. Biology Direct (2014) 9:30 Page 15 of 29 Figure 8 Phylogenetic profile analysis. Average number of clusters of phylogenetically related proteins relative to the number of phylogenetically
related proteins. Phylogenetically related proteins are taken from the STRING database. (A) The histogram of number of phylogenetically related
proteins. (B) Functional clustering using Funsim (BP, MF, CC) score with thresholds between 0.1 and 1.0. (C) Functional clustering using Funsim (BP)
score with thresholds from 0.1 to 1.0. Figure 8 Phylogenetic profile analysis. Average number of clusters of phylogenetically related proteins relative to the number of phylogenetically
related proteins. Phylogenetically related proteins are taken from the STRING database. (A) The histogram of number of phylogenetically related
proteins. (B) Functional clustering using Funsim (BP, MF, CC) score with thresholds between 0.1 and 1.0. (C) Functional clustering using Funsim (BP)
score with thresholds from 0.1 to 1.0. the score threshold of 0.8 in the funsim score (Figure 8B)
and larger than 0.05 for the BP-funsim score profile
(Figure 8C). Comparing with the MPR1-3 sets, on average
MPR2 and MPR3 have a higher number of clusters than
the E. coli moonlighting and non-moonlighting proteins,
while the MPR1 set has less functional divergence in their
phylogenetically related proteins. and a double gene knockout mutant. In general, genes in
the same pathway tend to show positive interaction and
those in parallel pathways show negative or synthetic
lethality [116]. Genetic interactions in E. coli were identi-
fied by Takeuchi et al. [117] using conjugation methods
reported as GIANT-coli [118] and eSGA [119] with an
improved quantitative measurement [120]. This dataset
includes genetic interaction data for 215 genes against
3868 genes, which results in total of 813,560 gene combi-
nations. P19414/ACO1 Among them, 2009 pairs were identified as genet-
ically interacting, which were defined as those have a
correlation coefficient of over 0.2 in the maximum growth Genetic interaction network analysis The last omics data we analyzed were genetic interactions. A genetically interacting gene pair was identified by exam-
ining the growth curves of a single gene knockout mutant Khan et al. Biology Direct (2014) 9:30 Page 16 of 29 (Figure 9B & 9C), although the number of proteins avail-
able for the analysis was too small to make a firm conclu-
sion. In terms of the number of genetic interactions
(Figure 9A), there is one moonlighting protein that has 43
genetic interactions. This protein is a subunit of fumarate
reductose flavoprotein in E. coli (P00363), which we classi-
fied as a first category moonlighting protein (Table 1). The
43 interacting proteins belong to 30 different pathways. Panels B & C in Figure 9 show histograms of the number
of functional clusters of genetically interacting proteins for rate in time-series measurements [117]. The interacting
gene pairs overlap with a small portion of the E. coli
moonlighting and non-moonlighting proteins: 5 out of 33
moonlighting proteins, 3 out of 16 first category moon-
lighting proteins, and 5 out of 150 non-moonlighting
proteins. Using these shared proteins, we performed the
clustering profile analysis (Figure 9). Moonlighting and non-moonlighting proteins do not
seem to have difference in the number of genetic interac-
tions (Figure 9A) and the number of functional clusters Figure 9 Genetic interaction network analysis. The number of interacting proteins in the genetic interaction network of E. coli. (A) The number of
interacting proteins selected with a Pearson correlation cutoff of 0.2. E. coli MP and non-MP, multi-domain multi-functional proteins, and the
first category E. coli MPs are plotted. (B) The number of clusters of interacting proteins for individual E. coli moonlighting (blue) and non-moonlighting
(red) proteins at BP-funsim threshold of 0.2. (C) The number of clusters of interacting proteins for individual E. coli moonlighting (blue) and
non-moonlighting (red) proteins at BP-funsim threshold of 0.6. Figure 9 Genetic interaction network analysis. The number of interacting proteins in the genetic interaction network of E. coli. (A) The number of
interacting proteins selected with a Pearson correlation cutoff of 0.2. E. coli MP and non-MP, multi-domain multi-functional proteins, and the
first category E. coli MPs are plotted. (B) The number of clusters of interacting proteins for individual E. coli moonlighting (blue) and non-moonlighting
(red) proteins at BP-funsim threshold of 0.2. (C) The number of clusters of interacting proteins for individual E. Genetic interaction network analysis coli moonlighting (blue) and
non-moonlighting (red) proteins at BP-funsim threshold of 0.6. Khan et al. Biology Direct (2014) 9:30 Page 17 of 29 the E. coli moonlighting and non-moonlighting proteins at
the BP-funsim thresholds of 0.2 and 0.6. There is a moon-
lighting protein that interacts with two proteins with very
different functions (the bar at × = 1.0 in Figure 9B). This
protein is P23895, a third category/weak moonlighting
protein identified to function as a multidrug transporter
and in DNA damage response. It interacts with P77368
(UPF0098 family protein inferred by homology) and
P75719 (endopeptidase that performs host cell lysis). partners through the disordered binding regions [122]. Here, we examined the prevalence of disordered regions
in the proteins in MPR1-3 and the E. coli moonlighting
and non-moonlighting proteins. Disordered regions in the
proteins were obtained from the D2P2 database [123]. The total length of disordered regions and their frac-
tion relative to the full length of a protein are shown in
Figure 10. The distributions for moonlighting proteins
and non-moonlighting proteins were overall similar,
both having the peak at lower end within disordered
region lengths 0 to 5. However, it is noteworthy that
moonlighting proteins had a smaller fraction of proteins
with no disordered regions (Figure 10A) and more
moonlighting proteins had a larger fraction of disordered
regions (Figure 10B). Moonlighting proteins had a small
peak for disordered regions of 47 residues in length and
slightly higher frequency for disordered regions of over
90 residues (Figure 10A). The peak of the moonlighting
proteins at 47 residue-long disordered regions (Figure 10A)
consists of four proteins, fumarate reductase (P00363),
ribonuclease R (P21499) deferrochelatase (P31545), and
GTPase ObgE (P42641). Moonlighting proteins with a
large fraction of disordered region include anion exchange
protein 3 (P48751) and phosphopantothenoylcysteine
decarboxylase subunit VHS3 (Q08438) and subunit S1S2
(P36024). Anion exchange protein 3 does not have known
physical interactions with other proteins while the two
subunits of phosphopantothenoylcysteine decarboxylase
have eight physical interactions in the PPI network. To summarize the omics data analyses, we observed a
clear tendency for moonlighting proteins to have phys-
ical interactions with more diverse classes of proteins
and most of these proteins share the primary function of
the moonlighting protein with which they interact. Moreover, it was found that moonlighting proteins fre-
quently physically interact with other moonlighting pro-
teins. Structural properties of moonlighting proteins p
p
g
g p
Now we turn our attention to structural properties of
moonlighting proteins, namely intrinsically disordered
regions and ligand binding sites. An intrinsically disor-
dered region in a protein lacks a well-defined tertiary
structure in its native condition. Intrinsically disordered
regions have been found to have important roles in
protein function [121], often serving as binding sites for
proteins. There are moonlighting proteins that can both
activate and inhibit their binding partners in the same or
overlapping binding regions which have been found to
be disordered. These proteins can bind the same partner
in different conformations or bind to completely different Genetic interaction network analysis In terms of gene expression and phylogenetically
related proteins, a weak trend was observed that on
average moonlighting proteins interact with more func-
tionally diverse proteins, although not all of the cases
were statistically significant. Ligand binding sites We discuss two cases
below, because the other four are multi-domain pro-
teins (Figure 11). These two proteins to be discussed are
one-domain proteins according to Pfam. The first example is dihydrolipoamide dehydrogenease
(DLD) in human (P09622) (Figure 11A). The primary
function of this protein is as a mitochondrial enzyme in
energy metabolism and its secondary function is protease. To perform the primary function, it utilizes dihydrolipoic
acid and NAD+ to generate lipoic acid. Experiments sug-
gest that mutations that destabilize a DLD homodimer
can simultaneously induce the loss of a primary metabolic
activity and the gain of a moonlighting proteolytic activity
[59]. It was also pointed out that the moonlighting pro-
teolytic activity of DLD could arise under pathological
conditions, including the presence of dimer-destabilizing
mutations or the acidification of the mitochondrial matrix. The latter condition disrupts the quaternary structure of
DLD, leading to a decrease in the dehydrogenase activity
and increase in the diaphorase activity, which is a FAD and
NAD dependent activity. Based on these information we
classified “NAD (nicotinamide adenine dinucleotide) or
NADH binding” (GO:0051287) to both functions and
term “FAD (flavin adenine dicucleotide) or FADH2 bind-
ing” (GO:0050660) to the secondary function. A crystal
structure of DLD (PDB ID: 1ZMC-A) shows that the NAD
and FAD binding sites are located in physically separate
regions in the protein surface. To summarize the structural analyses, about 48% of
moonlighting proteins have disordered regions longer
than five residues and this percentage is larger than that
of non-moonlighting ones (29%). Also examples are
observed in which moonlighting proteins have relatively
longer disordered regions. In terms of the tertiary struc-
tures, examples are found where ligand (including DNA)
binding sites that are related to either the primary or
secondary functions are located in distinct regions on the
protein surface. These structural features may be useful
for predicting the existence of secondary function of
proteins when combined with other evidences. Discussion Moonlighting proteins have more than one independent
function. It is speculated that moonlighting proteins are
not few in number and expected to be found more in
the future. Identification of moonlighting proteins indi-
cates that potential secondary functions need to be con-
sidered when it comes to protein function, which has
significant impact on functional genomics, proteomics,
and computational gene function annotation [10]. In the first part of this work, we examined current GO
annotations of known moonlighting proteins. We found
that the GO term annotations for moonlighting proteins
can be clustered into more than one cluster based on the
semantic similarity between pairs of GO terms. Thus, even
in the case that moonlighting proteins are not labelled as
such in the annotation database, we will be able to identify
them by observing the functional divergence of annotated
GO terms. Based on this intuitive observation, we ana-
lyzed E. coli proteins in the database and identified
novel moonlighting proteins. Figure 11 Moonlighting protein structures. Tertiary structures of
moonlighting proteins. (A) human dihydrolipoamide dehydrogenase
(PDB ID: 1ZMC-A). It binds NAD shown in yellow at residues 208, 243,
279 (“NAD binding” classified as both F1 and F2 function) and FAD
shown in cyan at residues 54, 119, 320 (“FAD binding” classified as F2
term). (B) mitogen activated protein kinase 1 (PDB ID: 4G6N). It binds
ATP (related to F1 function) at residues 31–39 and 54 (shown in yellow),
and DNA (related to F2 function) with residues 259–277 (purple). The second half of this work addressed characteris-
tics of moonlighting proteins in omics data and their
tertiary structures. We found that moonlighting pro-
teins tend to physically interact with proteins of di-
verse functions. The same trend, although weak, was
observed for proteins that are co-expressed with or are
phylogenetically related to moonlighting proteins. The
majority of interacting proteins of a moonlighting pro-
tein shared the primary function of the moonlighting
protein and we found that a substantial fraction of
the interacting proteins were themselves moonlighting
proteins. Figure 11 Moonlighting protein structures. Tertiary structures of
moonlighting proteins. (A) human dihydrolipoamide dehydrogenase
(PDB ID: 1ZMC-A). It binds NAD shown in yellow at residues 208, 243,
279 (“NAD binding” classified as both F1 and F2 function) and FAD
shown in cyan at residues 54, 119, 320 (“FAD binding” classified as F2
term). (B) mitogen activated protein kinase 1 (PDB ID: 4G6N). Ligand binding sites Finally, we discuss ligand binding sites in the tertiary
structures of moonlighting proteins that are related to
either of their primary or secondary functions. Such
examples are limited since the tertiary structures of the Figure 10 Disordered region of moonlighting and non-moonlighting proteins. Histograms of the disordered regions in moonlighting and
non-moonlighting proteins. Five datasets are plotted: MPR1-3 (MPR-All), E. coli moonlighting proteins (Ecoli-MP), E. coli moonlighting proteins in
the first category (Ecoli-MP-Cat1), multi-domain multi-functional proteins, and E. coli non-moonlighting proteins (Ecoli-nonMP). (A) Length of the
disordered regions; (B) Fraction of the length of disordered regions relative to the whole sequence length of the proteins. Figure 10 Disordered region of moonlighting and non-moonlighting proteins. Histograms of the disordered regions in moonlighting and
non-moonlighting proteins. Five datasets are plotted: MPR1-3 (MPR-All), E. coli moonlighting proteins (Ecoli-MP), E. coli moonlighting proteins in
the first category (Ecoli-MP-Cat1), multi-domain multi-functional proteins, and E. coli non-moonlighting proteins (Ecoli-nonMP). (A) Length of the
disordered regions; (B) Fraction of the length of disordered regions relative to the whole sequence length of the proteins. Khan et al. Biology Direct (2014) 9:30 Khan et al. Biology Direct (2014) 9:30 Page 18 of 29 The second example is MAP kinase (ERK2) in human. The secondary function of this protein was identified as a
DNA binding transcriptional repressor that regulates inter-
feron gamma signalling [64]. Naturally, binding ATP is
related to the primary function as a kinase (GO:0005524)
while “DNA binding” (GO:0003677) belongs to the second-
ary function. As shown in Figure 11B, the binding sites for
ATP and DNA are located quite far apart in the protein
structure. proteins must be available for the analysis and multiple
bound ligands need to be involved in the functions. Sixteen proteins in the MPR1-3 sets have their tertiary
structures available in PDB [124,125]. Among them, we
found six structures that have two ligands that bind to
physically different locations. We discuss two cases
below, because the other four are multi-domain pro-
teins (Figure 11). These two proteins to be discussed are
one-domain proteins according to Pfam. proteins must be available for the analysis and multiple
bound ligands need to be involved in the functions. Sixteen proteins in the MPR1-3 sets have their tertiary
structures available in PDB [124,125]. Among them, we
found six structures that have two ligands that bind to
physically different locations. Conclusions The functional diversity of moonlighting proteins poses a
challenge to their experimental identification as well as
computational annotation [10,29]. Our method enables
identification of novel moonlighting proteins from a
current database, even when they are not explicitly
annotated as such. Moreover, we showed that potential
moonlighting proteins without sufficient functional an-
notations could be identified by considering available
omics-scale data and computational structural predic-
tions. Our findings open up a new opportunity to investi-
gate the multi-functional nature of proteins at a systems
level and explore the complex functional interplay of
proteins in a cell. Discussion It binds
ATP (related to F1 function) at residues 31–39 and 54 (shown in yellow),
and DNA (related to F2 function) with residues 259–277 (purple). Figure 11 Moonlighting protein structures. Tertiary structures of
moonlighting proteins. (A) human dihydrolipoamide dehydrogenase
(PDB ID: 1ZMC-A). It binds NAD shown in yellow at residues 208, 243,
279 (“NAD binding” classified as both F1 and F2 function) and FAD
shown in cyan at residues 54, 119, 320 (“FAD binding” classified as F2
term). (B) mitogen activated protein kinase 1 (PDB ID: 4G6N). It binds
ATP (related to F1 function) at residues 31–39 and 54 (shown in yellow),
and DNA (related to F2 function) with residues 259–277 (purple). Khan et al. Biology Direct (2014) 9:30 Page 19 of 29 The characteristics of moonlighting proteins were
investigated by comparing their features with those of
non-moonlighting proteins. In general, finding examples
that do not possess a certain property is not straight-
forward as future research may find that the examples
actually do have the property. So are non-moonlighting
proteins – there is an undeniable possibility that non-
moonlighting proteins used in this study will be found
as moonlighting in the future. Nevertheless we believe
the current research is valuable and has contributed in
progressing our understanding of moonlighting proteins
because the non-moonlighting proteins were selected in
a reasonable way and also because the differences and
similarities of characteristics of moonlighting and non-
moonlighting proteins were clarified that can serve as
hypotheses in the future works. We would also like to
point out that similar approaches of selecting negative
data sets were taken in analyzing protein-protein inter-
actions (by constructing a non-interacting protein dataset,
Negatome [126]) and in analyzing proteins with particular
functions (by constructing the NoGo database [127]),
which contributed in development of computational pre-
diction methods and thereby advance our understanding
and the research field. would be useful in resolving many ambiguities in prote-
omics analysis as well as in unfolding many complex-
ities of protein functions. Improved understanding of
moonlighting functions of proteins can be a touchstone
for our knowledge of molecular biology, because it
requires comprehensive, multilevel data and deep know-
ledge of the cell. Methods
f As for the structural aspects, a larger fraction of
moonlighting proteins than non-moonlighting ones had
intrinsically disordered regions. We have also discussed
examples that ligands related to the primary and second-
ary functions bind at distinct regions in the tertiary struc-
ture. Application of structural analyses is limited because
obviously protein structure information is needed. How-
ever, we would like to point out that disordered regions
can be well predicted from a protein sequence and ligand
binding sites can be also predicted in an experimentally
determined protein structure or in a computational struc-
ture model. Dataset of known moonlighting proteins Dataset of known moonlighting proteins
We constructed three datasets of experimentally confirmed
moonlighting proteins from two review articles [1,18] and
papers we collected from the PubMed database. They are
called the MPR1 (24) [18], MPR2 (18) [1], and MPR3 (16)
set, respectively. In the parentheses is the number of moon-
lighting proteins in the each dataset. The MPR1 dataset
was used in our previous study [29]. The three datasets are
available at http://kiharalab.org/MoonlightingDatasets. The
list of proteins in the MPR3 set is provided in Table 4. In MPR1 and MPR2, we found four proteins (ATF2,
PutA, neuropilin-I, and BirA) are multi-domain proteins. Although these four proteins are also listed as moon-
lighting proteins in MultitaskProtDB and MoonProt, we
excluded them from the dataset in all the results except
for the bar graphs in Figure 2 and Figure 6 where these
proteins are noted with asterisk (*). For each of the moon-
lighting proteins in the three datasets, GO term annota-
tions in UniProt were classified into four classes by
referring to textual description of the protein’s function in
literature: GO annotations that described the “primary”
function of the protein (Function 1, F1), GO annotations
that describe “secondary” function (Function 2, F2), GO
annotations that correspond to both functions of the
protein (usually general GO terms at a higher depth of the
GO hierarchy), and lastly, GO annotations whose func-
tional association to either of the two functions were
unclear. In cases that the description of the secondary
function of a moonlighting protein was absent or We observed significant functional divergence in phys-
ically interacting proteins with moonlighting proteins,
which could be a good feature to use for predicting of
moonlighting proteins. However, the other features of
moonlighting proteins in omics data were weak. Thus,
predicting moonlighting proteins from an individual
feature may not be an easy task. This also reminds us
that moonlighting functions are observed in various
physiological conditions of a cell, which differ for each
moonlighting protein. Therefore, ultimately, prediction of
moonlighting proteins or secondary functions of a protein
needs a holistic understanding of behavior of molecules in
a cell. In practice, this means that integrating various dif-
ferent cell-level data will be effective in prediction,
which includes proteomics, ionomics, phenotypic data
of mutants, bioinformatics predictions, computational
simulations of pathways, and molecular dynamics of
biomolecules. Dataset of known moonlighting proteins Such an automated computational method ble 4 The MPR3 moonlighting protein dataset
iprot ID/Protein name
Organism
Primary function
Secondary function(s)
Ref
9149/Pinin
Canis familiaris
Induce junction formation and enhance
cell aggregation
Component of the RNP structure
[128]
7487/DPP4
Homo sapiens
Serine protease
1. Cell surface glycoprotein receptor for CAV1
[129]
2. Co-stimulatory protein involving in T-cell
receptor-mediated T-cell activation and proliferation. 3. Binding collagen and fibronectin
4. Involvement in apoptosis
1XR9/GPx-4
Mus musculus
Antioxidant of mature sperm
Structural protein of the mitochondrial capsule
[130]
5242/FAN
Mus musculus
Apoptosis
Inflammatory signalling
[131]
D2R2/Fructose-1, 6-bisphosphate
olase
Neisseria meningitidis
Glycolytic enzyme
Host-cell invasion
[132]
L0Y3/MRP1
Homo sapiens
tRNA methyltransferase
Dehydrogenase
[133]
Y7F0/Peroxiredoxin TSA1
Candida albicans
Antioxidant against sulfur-containing
radicals
Involved in morphology
[134]
8237/CCM1
Saccharomyces cerevisiae
Introns removal in mRNA maturation
Maintains the steady-state levels of the mitoribosome
small subunit RNA
[135]
325/Nam2p
Saccharomyces cerevisiae
Mitochondrial leucyl-tRNA synthetase
Mitochondrial RNA splicing activity
[136]
P2J5/LeuRS
Homo sapiens
tRNA synthetase
Translocation and activation of mTORC1 to lysosomal
membrane
[137]
7897/GlnRS
Homo sapiens
tRNA synthetase
Suppresses apoptotic acitivities
[137]
DRC0/SerRS
Danio rerio
tRNA synthetase
Regulates development of closed circulatory system
[137-139]
0883/Fructose-bisphosphate
olase A
Oryctolagus cuniculus
Glycolytic enzyme
Regulation of cell mobility
[140]
A518/Cpn60-1
Mycobacterium tuberculosis
Prototypic molecular chaperone
Osteoclast-inhibitory action
[141]
A518/Cpn60-2
Mycobacterium tuberculosis
Prototypic molecular chaperone
Stimulates macrophage pro-inflammatory cytokine
synthesis
[141] Primary function
Secondary function(s)
Ref
Induce junction formation and enhance
cell aggregation
Component of the RNP structure
[128]
Serine protease
1. Cell surface glycoprotein receptor for CAV1
[129]
2. Co-stimulatory protein involving in T-cell
receptor-mediated T-cell activation and proliferation. 3. Binding collagen and fibronectin
4. Semantic similarity of GO term pair and funsim score General comment: We used the relevance semantic similarity score (SSRel)
[142] for computing functional similarity of a pair of GO
terms, c1 and c2: This manuscript addresses an intriguing problem of
multi-functionality in proteins. "Moonlighting" proteins
that have two or more distinct functions are being dis-
covered at a steady pace which makes this contribution
important and timely. Having said that the current
version of the manuscript has a number of problems
that need to be fixed before it can be considered for
publication. SSRel c1; c2
ð
Þ ¼max
c∈S c1;c2
ð
Þ
2 logp cð Þ
logp c1
ð
Þ þ logp c2
ð
Þ 1−p cð Þ
ð
Þ
ð1Þ ð1Þ Here p(c) is the probability of a GO term c, which is
defined as the fraction of the occurrence of c in the GO
Database [35,36]. The root of the ontology has a prob-
ability of 1.0. s(c1,c2) is the set of common ancestors of
the GO terms c1 and c2. The first term considers the
relative depth of the common ancestor c to the depth of
the two terms c1 and c2 while the second term takes into
account how rare it is to identify the common ancestor c
by chance. 1. This manuscript inexplicably ignores the existence a
publicly available database of moonlighting proteins Mul-
titaskProtDB (http://wallace.uab.es/multitask described in
Hernández et al. Nucleic Acids Res. 42517-D520 2014). There is also MoonProt (http://www.moonlightingpro-
teins.org/ Mani et al. MS thesis University of Illinois at
Chicago 2014). A careful comparison of the results of
this study with the data presented in those two data-
bases is essential to this work. To quantify the functional similarity of two proteins,
both of which are annotated with a set of GO terms, we
used the funsim score [31]. The funsim score of two sets
of terms, GOA and GOB of respective size of N and M, is
calculated from an all-by-all similarity matrix sij. Authors’ response: We searched the 43 moonlighting
(Table 1) and multi-functional multi-domain proteins
(Table 2) we identified from E. coli against both Multi-
taskProtDB and MoonProt. In MultitaskProtDB we
found one protein (b0161/P0C0V0/DegP) in the 43 pro-
teins. The 43 proteins we identified from E. coli include
aconitases (AcnA and AcnB), which are not included in
MultitaskProtDB but their homologs in three species
(aconitase in H. Sapiens, M. Tuberculosis, S. Cerevisiae)
are included. Reviewers’ comments Reviewers’ comments Reviewer 1 (First Round): Dr. Michael Galperin (National
Center for Biotechnology Information, National Library of
Medicine, National Institutes of Health, USA)
General comment: Reviewer 1 (First Round): Dr. Michael Galperin (National
Center for Biotechnology Information, National Library of
Medicine, National Institutes of Health, USA)
General comment: Dataset of known moonlighting proteins Involvement in apoptosis
Antioxidant of mature sperm
Structural protein of the mitochondrial capsule
[130]
Apoptosis
Inflammatory signalling
[131]
Glycolytic enzyme
Host-cell invasion
[132]
tRNA methyltransferase
Dehydrogenase
[133]
Antioxidant against sulfur-containing
radicals
Involved in morphology
[134]
siae
Introns removal in mRNA maturation
Maintains the steady-state levels of the mitoribosome
small subunit RNA
[135]
siae
Mitochondrial leucyl-tRNA synthetase
Mitochondrial RNA splicing activity
[136]
tRNA synthetase
Translocation and activation of mTORC1 to lysosomal
membrane
[137]
tRNA synthetase
Suppresses apoptotic acitivities
[137]
tRNA synthetase
Regulates development of closed circulatory system
[137-139]
Glycolytic enzyme
Regulation of cell mobility
[140]
culosis
Prototypic molecular chaperone
Osteoclast-inhibitory action
[141]
culosis
Prototypic molecular chaperone
Stimulates macrophage pro-inflammatory cytokine
synthesis
[141] Secondary function(s) Q91XR9/GPx-4
Mus musculus
O35242/FAN
Mus musculus
E3D2R2/Fructose-1, 6-bisphosphate
aldolase
Neisseria meningitidis
Q7L0Y3/MRP1
Homo sapiens
Q9Y7F0/Peroxiredoxin TSA1
Candida albicans
P48237/CCM1
Saccharomyces cerevisiae
P11325/Nam2p
Saccharomyces cerevisiae
Q9P2J5/LeuRS
Homo sapiens
P47897/GlnRS
Homo sapiens
Q6DRC0/SerRS
Danio rerio
P00883/Fructose-bisphosphate
aldolase A
Oryctolagus cuniculus
P0A518/Cpn60-1
Mycobacterium tuberculosis
P0A518/Cpn60-2
Mycobacterium tuberculosis Page 21 of 29 Khan et al. Biology Direct (2014) 9:30 incomplete in UniProt, we annotated the protein with
appropriate GO terms selected from the GO database. incomplete in UniProt, we annotated the protein with
appropriate GO terms selected from the GO database. Semantic similarity of GO term pair and funsim score not "not a consequence of gene fusions" (p.3 l.7) but
include in the manuscript numerous examples of pro-
teins that have acquired different functions as a result of
fusion of two or more genes encoding distinct domains. For E. coli examples of two-domain proteins listed in
Table 1 include ThrA (b0002) CysG (b3368) MetL (b3940)
NadR (b4390) HldE (b3052) SpoT (b3650) to name just
a few. In addition ATF2 PutA neuropilin-I and BirA
which are discussed on pp. 23-24 and displayed on
Figure 11 C-G are also multi-domain proteins. As cor-
rectly stated by the authors their distinct functions reside
in distinct domains and therefore none of these proteins is
truly moonlighting. p. 5 l. 14. "poses a challenge to the fundamental con-
cept that genotype can explain phenotype"
Please re-
move or at least reformulate. Genotype cannot "explain"
anything only a human can. I do not see how moonlight-
ing proteins could challenge the fundamental concept
that genotype defines phenotype. Mutations in many
genes have pleiotropic phenotypes even without any
moonlighting. Authors’ response: We deleted the whole sentence. p. 6 l. 14. “the number of currently confirmed moonlight-
ing proteins is too small”. Just how many such proteins are
there? Have you examined the existing databases of such
proteins MultitaskProtDB and MoonProt (see above)? Authors’ response: We appreciate this important com-
ment by the reviewer. In response to this comment, we
have consulted with the Pfam database to find domains
in the 43 E. coli proteins that were originally listed in
Table 1. Then, to determine if the two functions (primary
and secondary functions) of the proteins originate from
different domains, we analyzed GO terms associated with
each Pfam domain. In case the GO terms associated with
a domain are too general or incomplete, we have also
examined the domain’s text description in the Pfam
database. As a result, we identified ten proteins as multi-
domain proteins whose multiple functions are caused by
different domains. These proteins include all the six pro-
teins pointed out by the reviewer, ThrA (b0002), CysG
(b3368), MetL (b3940), NadR (b4390), HldE (b3052), and
SpoT (b3650) and four more proteins. (NadR was found
in the MoonProt database, too). We excluded these ten
multi-domain proteins from Table 1 and separately listed
them in a new table, Table 2. Initially, in Table 1, there
were five proteins that were categorized as category III:
Multi reaction enzymes. Semantic similarity of GO term pair and funsim score In MoonProt, we found 3 proteins (b0161/
P0C0V0/DegP, b4260/P68767/pepA, and b4390/P27278/
nadR). nadR was found in Table 2, which is for multi-
functional multi-domain proteins. This database, too,
contains aconitases of four organisms (H. Sapiens, M. Tuberculosis, S. Cerevisiae, and B. Taurus) but not one
from E. coli. Thus, out of 33 new moonlighting proteins
listed in Table 1, only two are found in the existing two
databases. Sij ¼ sim GOA
i ; GOB
j
∀i∈1 ::N
f
g;∀j∈1 ::M
f
g
ð2Þ ð2Þ sim(GOi
A, GOi
B) is the relevance similarity score for GOi
A
and GOj
B. Since the relevance similarity score is defined
only for GO pairs of the same category, a matrix is com-
puted separately for the three categories, Biological
Process (BP), Molecular Function (MF), and Cellular
Component (CC). Then, the GOscore of the matrix of
each GO category is computed as follows: GOscore ¼ max
1
N
XN
i¼1 1≤i≤M max Sij; 1
M
XM
i¼1 1≤i≤N max Sij
ð3Þ We have indicated the three proteins in Table 1 and
Table 2 that are found in MultitaskProtDB and Moon-
Prot with † (dagger) and mentioned in the text as follows:
“The identified 33 moonlighting proteins (Table 1) and
10 multi-domain multi-function proteins (Table 2) do
not have many overlap with the MoonProt database and
MultitaskProtDB. Only two (PepA and DegP) with Table 1
and one (NadR) in Table 2” (page 13). ð3Þ GOscore will be any of the three category scores
(MFscore, BPscore, CCscore). Finally the funsim score is
computed as funsim ¼ 1
3
MFscore
max MFscore
ð
Þ
2
þ
BPscore
max BPscore
ð
Þ
2
þ
CCscore
max CCscore
ð
Þ
2
"
#
;
ð4Þ 2. This work fails to distinguish between truly moon-
lighting proteins where each part of the polypeptide
chain participates in two different activities and multi-
domain proteins that combine in a single polypeptide
chain two or more different domains each with its own spe-
cific function. The authors correctly define moonlighting as ð4Þ where max(GOscore) = 1 (maximum possible GOscore)
and the range of the funSim score is (0,1). Khan et al. Biology Direct (2014) 9:30 Khan et al. Biology Direct (2014) 9:30 Page 22 of 29 Specific comments. Semantic similarity of GO term pair and funsim score However, since all the five multi-
reaction enzymes turned out to be multi-domain proteins,
now the category III is removed from Table 1. Authors’ response: MultitaskProtDB and MoonProt
have 288 and 289 entries, respectively. We rephrased the
sentence as follows: “systematic studies of moonlighting
proteins are still in their early stage for obtaining a com-
prehensive picture of proteins’ moonlighting functions
and also to develop computational methods for predict-
ing moonlighting proteins.” Ref. 19 cites the 1990 BLAST paper which described
the first ungapped version of BLAST program. Did you
actually use the ungapped version (which is quite diffi-
cult to find these days)? If not you should cite the 1997
BLAST paper (ref. 29) or the later ones. Authors’ response: We cited the 1997 version of the
paper as pointed out. The references to the descriptions of Pfam (ref. 22) Inter-
Pro (ref. 23) GO (ref. 34) STRING (ref. 43) COLOMBOS
(ref. 47) and PDB (ref. 56) are all outdated. If you used
recent versions of these databases you should cite their
most recent descriptions as recommended on the re-
spective web sites.Authors’ response: For Pfam, Inter-
Pro, STRING, and COLOMBOS, we now cited papers that
were published in 2014, 2011, 2014, and 2014, respectively. The paper we originally cited for GO and PDB were those
recommended on their respective websites (GO: http://gen-
eontology.org/page/go-citation-policy, PDB: http://www. rcsb.org/pdb/static.do?p=general_information/about_pdb/
policies_references.html#References). However, as suggested
we now added 2013 papers for these two databases. Moreover, we have removed these ten multi-domain pro-
teins from the E.coli moonlighting proteins datasets, Ecoli-
MP and Ecoli-MP-Cat1 (the First Category moonlighting
proteins in E.coli), and redone all the subsequent analyses
(Figures 3 and 10). The ten multi-domain proteins were
separately plotted in the Figures. Statistical analyses, namely,
p-values in Supplementary Table S1 and the Friedman test
for Figure 3 were also recomputed with the revised datasets. Importantly, removing the 10 proteins from the analyses did
not change the overall trends and conclusions. Reviewer 1 (Second Round): Dr. Michael Galperin Authors’ response: We understand the reviewer’s con-
cern about the validity of the non-moonlighting proteins. We agree that the selection of non-moonlighting proteins
is based on our current knowledge of function of the pro-
teins and it is possible that secondary function may be
found for the proteins in the future. However, to under-
stand characteristics of a certain group of proteins (here
moonlighting proteins) it is effective to select a counter-
part of the protein group (non-moonlighting proteins)
and compare between them as a way of analysing the
data. A similar approach has been taken by Frishman
et al. in analysing protein-protein interactions by con-
structing a database called Negatome, which contains
protein pairs that are unlikely to physically interact [64]
and also in functional analysis of proteins by construct-
ing a database called NoGO, which is a database of pro-
teins that are unlikely to have certain GO terms [65]. Although there is undeniable possibility that some pro-
teins in the non-moonlighting protein dataset may be
found to be moonlighting in future, the analysis using the
non-moonlighting protein dataset gave conclusion at this
point, which can serve as workable hypotheses for future
research as scientific works are desired to do. Moreover,
we believe that the procedure to select non-moonlighting
proteins is quite reasonable: They are proteins which
have a sufficient number of GO term annotations but do
not have as many functionally distinct terms as known
moonlighting proteins. The revised manuscript looks fine to me but several
minor corrections need to be made (these comments do
not need to be included in the printed version): Abstract Abstract Background, 1st sentence: change “more than one cellu-
lar function” to “two or more cellular functions” (functions
need to be plural to correspond to the subsequent “which
are”). Authors’ response: We corrected it to “two or more cellular
functions”. Results, 5th sentence: “most of the physically interacting
proteins share the primary function of the interacting
moonlighting proteins” - could you explain that in simpler
terms? Authors’ response: We rephrased it to “most of the phys-
ically interacting partners of moonlighting proteins share
the latter’s primary functions”. Conclusion, 2nd sentence: change “function annota-
tions in a database” to “functional annotations in public
databases” Authors’ response: We changed the phrase as suggested. Authors’ response: We changed the phrase as suggested. Reviewer 1 (Second Round): Dr. Michael Galperin Main text g
gg
Main text Background, 3d sentence: remove “first” from “first by
Jeffrey [1]”. There have been two earlier papers, PMID:
8543908, 9663383. Authors’ response: Thank you for pointing out the two
papers, which we missed. In addition to modifying the
phrase as suggested we also cited the two papers. Quality of written English: Acceptable Quality of written English: Acceptable Having written our opinion above in response to the
reviewer’s comment, we consider it as an important
point. Therefore, we to clarify our standpoint, added fol-
lowing sentences in Discussion: p. 25: “The characteristics
of moonlighting proteins were investigated by comparing
their features with those of non-moonlighting proteins. In
general, finding examples that do not possess a certain
property is not straightforward as future research may
find that the examples actually do have the property. So
are non-moonlighting proteins – there is an undeniable
possibility that non-moonlighting proteins used in this
study will be found as moonlighting in the future. Reviewer 2 (First Round): Dr. Eugine Koonin (National Table 1 is poorly prepared and must be carefully revised. - While ObgA might indeed have multiple functions
'GTPase' and 'Chromosome segregation' is the same
function. Authors’ response: The second function description
changed to “Role in ribosome biogenesis” Quality of written English: Needs some language cor-
rections before being published Quality of written English: Needs some language cor-
rections before being published Authors’ response: We made language corrections with
help by a native English speaker. Authors’ response: We made language corrections with
help by a native English speaker. Table 1 is poorly prepared and must be carefully revised. Table 1 is poorly prepared and must be carefully revised. Table 1 is poorly prepared and must be carefully revised. - Protein name should start from a capital letter. Authors’ response: We corrected them. - Protein name should start from a capital letter. Authors’ response: We corrected them. Authors’ response: We corrected them. - There must be some order in the list (e.g. by gene
name or b-number) The four proteins in Figure 11 pointed out by reviewer
(ATF2, PutA, neuropilin-I, and BirA) were also confirmed
as multi-domain proteins with multiple functions by
consulting with the Pfam database. Although all of them
are included in both moonlighting protein databases -
MultitaskProtDB and MoonProt, we excluded them from
Figure 11. But we kept them in the bar graphs in Figures 2
and 7 and marked them with asterisk *. Authors’ response: The list was sorted first by the cat-
egory of moonlighting proteins (I to III), then further
sorted according to b-number of proteins. - Why AcnB is (correctly) annotated as? Aconitate hydra-
tase? but AcnA is only annotated as a? TCA cycle enzyme?? - Why AcnB is (correctly) annotated as? Aconitate hydra-
tase? but AcnA is only annotated as a? TCA cycle enzyme?? Authors’ response: Now both annotated as “Aconitate
hydratase”. Authors’ response: Now both annotated as “Aconitate
hydratase”. Page 23 of 29 Khan et al. Biology Direct (2014) 9:30 one of the situations that are rather typical in biology
where the "classic" examples are clear and compelling
(see crystallins) but few and far between whereas away
from the spotlight, matters become fuzzy. I am actually
inclined to think that all proteins perform multiple roles
in organisms and are at some level moonlighting. The
reasons why we think of some but not other proteins as
moonlighting have to do mostly with the level of our
knowledge and the feasibility of defining discrete func-
tional roles for a given protein. Consequently, I am
deeply skeptical about the validity of the control set of
"non-moonlighting" proteins and about any compara-
tive analyses that attempt to contrast properties of
moonlighting and non-moonlighting proteins. - CheA is a Chemotaxis sensor kinase not "Chematoxis"
Authors’ response: We changed it to “Chemotaxis sen-
sor kinase” as pointed out. - While ObgA might indeed have multiple functions
'GTPase' and 'Chromosome segregation' is the same
function. Center for Biotechnology Information, National Library of
Medicine, National Institutes of Health, USA) However, we think knowing the indifference of moon-
lighting and non-moonlighting proteins is also useful for
understanding moonlighting proteins because this is the
first time that moonlighting proteins are analysed in vari-
ous aspects in comparison with non-moonlighting proteins. The activity of CysG as a methyltransferase and syro-
heme synthase are one and the same, this i simply a
confusion about terms. Authors’ response: The description of this protein’s
function in UniProt is “Multifunctional enzyme that
catalyzes the SAM-dependent methylations of uro-
porphyrinogen III at position C-2 and C-7 to form
precorrin-2 via precorrin-1. Then it catalyzes the NAD-
dependent ring dehydrogenation of precorrin-2 to yield
sirohydrochlorin. Finally, it catalyzes the ferrochelation
of sirohydrochlorin to yield siroheme.” Based on this
UniProt description, we changed functions of CysG to
more detailed ones (Function 1: SAM-dependent methy-
lation; Function 2. NAD-dependent ring dehydrogen-
ation; Ferrorochelation). Attempts to mine the GO database in order to identify
new moonlighting proteins are of greater interest but
then, again, what is the status of novelty here if this
cases can be validated through the published literature? Aconitases (aconitate hydratase in the article) seem to
present a good example of moonlighting but this is by
no account new, the dual role of these proteins had been
studied for decades. g
p
Authors’ response: Since this is the first work that
proposes a procedure of identifying moonlighting pro-
teins, we needed to show that the proposed procedure
(clustering GO terms using the semantic similarity
score) can indeed find moonlighting proteins by con-
firming with the literature. Of course one can read
literature of all the genes in an organism to find moon-
lighting proteins. But the proposed automatic procedure
has significantly reduced the amount literature to read. Thus, the proposed computational procedure is an
effective filter to identify potential moonlighting pro-
teins. The proposed computational procedure in this
work would also trigger development of fully-automated or
semi-automated procedure for identifying novel moonlight-
ing proteins that needs none or minimum effort of reading
literature in the future. Authors’ response: Aconitase is listed as moonlighting
proteins for four organisms in in the MoonProt database
(H. Sapiens, M. Tuberculosis, S. Cerevisiae, and B. Taurus)
and from three organisms in MultitaskProtDB (H. Sapiens,
M. Tuberculosis, S. Cerevisiae) but aconitase of E. coli
(AcnA and AcnB) are not included yet. Center for Biotechnology Information, National Library of
Medicine, National Institutes of Health, USA) Khan et al. report an extensive computational analysis
of "moonlighting proteins". They correctly note that
bioinformatic study of such proteins presents a difficult
challenge. Much to my regret, I do not actually believe
that the present manuscript meets the challenge to
enhance the existing understanding of the moonlighting
phenomenon. Moonlighting is not easy to define, it is Khan et al. Biology Direct (2014) 9:30 Page 24 of 29 Nevertheless we believe the current research is valuable
and has contributed in progressing our understanding of
moonlighting proteins since the non-moonlighting pro-
teins were selected in a reasonable way and also because
the differences and similarities of characteristics of moon-
lighting non-moonlighting proteins were clarified that
can serve as hypotheses in the future works. We would
also like to point out that similar approaches of selecting
negative data sets were taken in analyzing protein-protein
interactions (by constructing a non-interacting protein
dataset, Negatome [64]) and also in analyzing proteins
with particular functions (by constructing the NoGo
database [65]).” At best, this analysis can help to systematize the data
on multiple protein functions. And, the authors do not
do a careful job in this systematic survey as one can im-
mediately see from Table 1 that is supposed to present
major results of the study. For instance, aspartokinase/
homoserine dehydrogenase (for some reason, listed twice
in the table) is not a moonlighting protein, it is simply a
two-domain bifunctional proteins. There is a difference
that the authors seem not to recognize. Authors’ response: The two aspartokinase/homoserine
dehydrogenase are aspartokinase/homoserine dehydro-
genase I and II. To clarify, we added gene IDs, ThrA and
MetL. In the revised manuscript, we separated multi-
domain multi-functional proteins to a separate table,
Table 2 and accordingly, ThrA and MetL are now moved
to Table 2. Indeed, the trends in protein-protein interactions re-
ported in this paper are mostly weak and uninformative. Authors’ response: The trends in protein-protein physical
interactions of moonlighting proteins are very clear. The
number of clusters of interacting proteins for moonlighting
proteins is significantly larger than non-moonlighting pro-
teins as the statistical test shows (Supplementary Table S1,
Figures. 5B and 5C). Also, Figure 6C shows that interacting
proteins of a moonlighting protein are clearly dominated
with the moonlighting proteins’ primary function. The
trends of co-expressed genes, phylogenetically related pro-
teins, and genetic interactions of moonlighting proteins are
weak. Center for Biotechnology Information, National Library of
Medicine, National Institutes of Health, USA) Since ortholog of
moonlighting proteins are not necessarily moonlighting, we
thought it was worthwhile to list aconitase of E. coli. For many proteins, e.g. transporters, involvement in
stress response is hardly evidence of moonlighting be-
cause they employ their intrinsic activity. One could
continue through the entire table as more or less every
entry is confusing. One again, bioinformatic study of
moonlighting is not at all easy. Unfortunately, I do not
think the authors of this paper stand up to the challenge. The validation step with published literature also pro-
vided insights about situations when proteins selected by
the proposed procedure are not moonlighting, i.e. when pro-
teins with distinct GO terms are not actually moonlighting. Authors’ response: We agree that levels of experimen-
tal evidence of selected moonlighting proteins differ. That
is the reason why we have classified the identified poten-
tial moonlighting proteins into three categories, I to III Page 25 of 29 Page 25 of 29 Khan et al. Biology Direct (2014) 9:30 Khan et al. Biology Direct (2014) 9:30 (I is the most clear moonlighting proteins, and III is the
weakest), according to the level of certainty based on avail-
able experimental evidence. The cases pointed out by the
reviewer are classified to category III. Thus it is possible that
the pointed out cases are found to be non-moonlighting
once the molecular mechanism of all function of the pro-
teins are revealed. a careful job in this systematic survey as one can imme-
diately see from Table 1 that is supposed to present
major results of the study. For instance, aspartokinase/
homoserine dehydrogenase (for some reason, listed twice
in the table) is not a moonlighting protein, it is simply a
two-domain, bifunctional protein. There is a difference
that the authors seem not to recognize. The activity of CysG as a methyltransferase and syro-
heme synthase are one and the same, this is simply a
confusion about terms. Not for publication but important: the manuscript is
sloppy. There are many non-grammatical sentences,
typos, quite a few references are incomplete etc. Quality of written English: Needs some language cor-
rections before being published Aconitases (aconitate hydratase in the article) seem to
present a good example of moonlighting but this is by
no account new as the dual role of these proteins had
been studied for decades. Center for Biotechnology Information, National Library of
Medicine, National Institutes of Health, USA) Authors’ response: We made language corrections with
help by a native English speaker. For many proteins e.g. transporters involvement in
stress response is hardly evidence of moonlighting be-
cause they employ their intrinsic activity. Reviewer 2 (Second Round): Dr. Eugine Koonin The authors have provided reasonable and informative
responses to the points made in my review, so i refrain
from making further substantive points. One could continue through the entire table as more
or less every entry is confusing. Authors’ response: Thank you. Once again, bioinformatic study of moonlighting is
not at all easy. Unfortunately, I do not think the authors
of this paper stand up to the challenge. However, I have spotted and corrected several typos in
my review, and furthermore, for some reason, all the
punctuation except for periods has disappeared from the
text of the review. The corrected version is below. Authors’ response: As the reviewer pointed out, we
found that the commas were dropped somehow when we
copied the reviewer’s comments to the manuscript. We
put them back to the text of the review. We apologize for
the mistake. Corrected version of the review: Khan et al. report an extensive computational analysis
of "moonlighting proteins". They correctly note that bio-
informatic study of such proteins presents a difficult
challenge. Much to my regret, I do not actually believe
that the present manuscript meets the challenge to en-
hance the existing understanding of the moonlighting
phenomenon. Moonlighting is not easy to define: it is
one of the situations that are rather typical in biology
where the "classic" examples are clear and compelling
(see crystallins) but few and far between, whereas away
from the spotlight matters become fuzzy. I am actually
inclined to think that all proteins perform multiple roles
in organisms and are at some level moonlighting. The
reasons why we think of some but not other proteins as
moonlighting have to do mostly with the level of our
knowledge and the feasibility of defining discrete func-
tional roles for a given protein. Consequently, I am
deeply skeptical about the validity of the control set of
"non-moonlighting" proteins and about any comparative
analyses that attempt to contrast properties of moon-
lighting and non-moonlighting proteins. Quality of written English: Acceptable References
ff Drug Discov Today: TARGETS 2004, 3:71–78. (University of Texas Southwestern Medical Center and
Howard Hughes Medical Institute, Texas, USA) In this well-executed study, the authors investigate the pos-
sibility of a systematic computational approach to find pro-
teins that possess more than one function. Difficulties and
advances along their path are discussed. Several general
conclusions and nice examples are presented. This work is
performed carefully and appears solid. Hopefully, these
computational approaches will mature enough to be used
by biologists in a quest for new functions of old proteins. Authors’ response: Thank you. Authors’ response: Thank you. A general, and more philosophical, comment is about
the definition of "function" and "moonlighting." The
authors already expanded the term to include enzymes
that can perform somewhat different reactions. Should
"moonlighting" further be expanded to cover multido-
main proteins, in which different domains have different
functions, or should the term be applied only to a single
domain that has several functions? I am asking because
if different functions are conveyed by different segments
of the polypeptide chain, it might be difficult to distinguish
these possibilities without a careful case-by-case study. i.e.,
disordered segment that carries a different function may
be thought of as a separate "domain," and such a protein Indeed, the trends in protein-protein interactions re-
ported in this paper are mostly weak and uninformative. Attempts to mine the GO database in order to identify
new moonlighting proteins are of greater interest but
then again, what is the status of novelty here if these
cases can be validated through the published literature? At best this analysis can help to systematize the data
on multiple protein functions.And the authors do not do Khan et al. Biology Direct (2014) 9:30 Page 26 of 29 will not be moonlighting. Basically, if we expand the defin-
ition, almost every protein will be moonlighting. If we
make the definition more stringent: i.e., the same evolu-
tionary domain with at least two very different functions,
maybe moonlighting would be a very rare exception. These comments are not meant as a critique of this excel-
lent study, but just an invitation for thought. General Medical Sciences of the National Institutes of Health (R01GM097528)
and the National Science Foundation (IIS1319551, DBI1262189, IOS1127027),
and National Research Foundation of Korea Grant funded by the Korean
Government (NRF-2011-220-C00004). General Medical Sciences of the National Institutes of Health (R01GM097528)
and the National Science Foundation (IIS1319551, DBI1262189, IOS1127027),
and National Research Foundation of Korea Grant funded by the Korean
Government (NRF-2011-220-C00004). Received: 2 August 2014 Accepted: 2 December 2014 Received: 2 August 2014 Accepted: 2 December 2014 Received: 2 August 2014 Accepted: 2 December 2014 Authors’ contributions IK participated in designing the research, performed computational analyses of
GO terms of moonlighting and non-moonlighting proteins as well as clustering
profile analyses of omics-data, participated in constructing the datasets of
moonlighting proteins, participated in structural analysis of moonlighting
proteins, and wrote the paper. YC and TD constructed the datasets of
moonlighting proteins. X H analyzed structures of moonlighting proteins. R T and HM designed and conducted genetic interaction experiments to
construct the second genetic interaction dataset. DK conceived the study,
participated in its design and coordination and wrote the paper. All authors
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1 1Department of Computer Science, Purdue University, 305 North University
Street, West Lafayette, IN 47907, USA. 2Department of Biological Sciences,
Purdue University, 240 Martin Jischke Drive, West Lafayette, IN 47907, USA. 3Graduate School of Biological Sciences, Nara Institute of Science and
Technology, 8916-5, Takayama, Ikoma, Nara 630-0192, Japan. Authors’ response: Thank you for this very important
comment. As the reviewer pointed out, multi-domain
multi-function proteins add intriguing complexity in in-
vestigating moonlighting proteins. According to the defin-
ition of moonlighting proteins proposed by Jeffrey, one of
the pioneers of studying moonlighting proteins, proteins
with multiple function due to gene fusion are excluded
from moonlighting proteins. However, in her opinion
multi-domain multi-functional proteins that did not gain
multiple domains by gene fusion during evolution (i.e. multi-domain proteins from the beginning) are included
in moonlighting proteins (personal communication). Thus,
defining moonlighting proteins can be complicated. Technology, 8916-5, Takayama, Ikoma, Nara 630-0192, Japan. Acknowledgements
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Challenges in Academic Reading and Overcoming Strategies in Taught Master Programmes: A Case Study of International Graduate Students in Malaysia
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Higher Education Studies; Vol. 4, No. 4; 2014
ISSN 1925-4741 E-ISSN 1925-475X
Published by Canadian Center of Science and Education Higher Education Studies; Vol. 4, No. 4; 2014
ISSN 1925-4741 E-ISSN 1925-475X
Published by Canadian Center of Science and Education Abstract This article focuses on research into academic reading practices of international graduate students in taught
Master programmes in a Malaysian university. The purpose of the study was to examine the challenges faced in
the academic reading practices as well as the strategies employed to overcome the challenges in the academic
reading practices. Quantitative data was collected through the Academic Literacies Questionnaire and analysed. The findings show challenges faced by international graduate students are attributed by the fact that English is
the medium of instruction and English is not their first language. At the same time, the findings also highlight the
strategies employed by the students to overcome the challenges in their academic reading practices. However, it
was found that the students, although exposed to challenges in their academic reading practices were not very
responsive toward overcoming the challenges. This article concludes with administrative policies and reading
related programmes to support the unique academic reading background needs of the students and ensure their
academic success. Keywords: academic reading practice, challenge, international graduate student, Master programme,
overcoming strategy Challenges in Academic Reading and Overcoming Strategies in
Taught Master Programmes: A Case Study of International Graduate
Students in Malaysia Manjet Kaur Mehar Singh1
1 School of Languages, Literacies and Translation, Universiti Sains Malaysia, Malaysia
Correspondence: Manjet Kaur Mehar Singh, School of Languages, Literacies and Translation, Universiti Sains
Malaysia, Minden 11800 Penang, Malaysia. E-mail: manjeet@usm.my Received: June 23, 2014 Accepted: July 22, 2014 Online Published: July 30, 2014
doi:10.5539/hes.v4n4p76 URL: http://dx.doi.org/hes.v4n4p76 1. Introduction English speaking countries have long established popularity among international students from various regions
globally. However, non-English speaking countries, where English is the second language (L2), such as Malaysia,
Singapore and South Africa are only currently increasingly attracting foreign students (Crewe, 2004; Reinties,
Beausaert, Grohnert, Niemantsverdriet, & Kommers, 2012). For example in Malaysia, postgraduate students
from the Middle East countries contribute one of the largest blocks of students (Norhisham, Muhamad, Azizah,
Osman, Abdul, & Norpisah, 2008; Ministry of Higher Education, [MoHE], 2010). The increasing number of international students studying in Malaysia has brought about various issues such as
students coming from different linguistic, educational and cultural backgrounds. Carroll and Ryan (2005) pointed
out that international students bring to an institution a diversity of competencies that have been progressively
built from childhood, primary, secondary and prior tertiary learning experiences. These students have already
experienced a culturally defined approach to academic literacies in learning at first-degree level in their native
country’s higher education system. Furthermore, the students’ prior academic background has been influenced by
the use of their first language (L1) for academic purposes. Second language (L2) academic literacy research results highlight that becoming literate in different discourse
traditions is a challenging, complex and lengthy process (Zhu, 2001; Casanave, 2002; Shi & Beckett, 2002; Leki,
2003; Spack, 2004; Zamel & Spack, 2004). It is also stressful for non-native speakers of English who may try to
reconcile contradictory desires to adjust to and resist new ways of practicing academic literacy in a new
education institution in a different country. Based on past research conducted in English speaking universities,
researchers found non-native speakers of English face hurdles comprehending lectures, taking part in tutorial
discussions, writing essays and theses, reading course books and designated articles and communicating with
lecturers, supervisors and fellow students (Hellstèn, 2002; Hellstèn & Prescott, 2004; Wong, 2004; Sawir, 2005). In the countries mentioned by Crewe (2004) and Rienties et al. (2012), especially Malaysia where English is the 76 Vol. 4, No. 4; 2014 Higher Education Studies www.ccsenet.org/hes second language and is used as the medium of instruction for graduate studies (Mahmud, Amat, Rahman &
Mohd Ishak, 2010; Ministry of Higher Education, 2010) there is a wide gap in research conducted pertaining to
academic literacies practices, especially academic reading practices of the international graduate students (Wahi,
O’Neill, & Chapman, 2012). 1. Introduction This particular research study, therefore, explores the challenges faced by
international graduate students in their academic reading practices in their Master programmes conducted in
English language. 1.1 Statement of the Problem The international graduate students attending higher education institutions in Malaysia who are highly capable
and motivated have successfully met the universities’ academic requirement that are the first-degree CGPA
cut-off point and English language requirement. Subsequently, these students qualify to further their study at
graduate level based on their English language qualifications such as the TOEFL or IELTS results. However,
prior research that has explored academic literacies practices of the international graduate students in Malaysian
local universities found them to be struggling in their reading and writing practices that are the thrust of
academic literacies. The non-native English speaking international graduate students’ exposure to academic literacies from primary
until tertiary level in their native countries has been mainly in their L1. This attribute of the students gives
impact to their academic literacies practices when they come to Malaysia to further their study at graduate level. They are still unable to grasp the new and different academic expectations as well as adapt to the appropriate
academic demands of their Master programmes as mentioned in studies conducted previously in Malaysian
higher education institutions (Rabab’ah, 2003; Hafriza et al., 2004; Kaur & Shakila, 2007; Hisham, 2008; Koo,
2009; Kaur & Sidhu, 2009; Al-Khasawneh, 2010; Al-Zubaidi & Rechards, 2010; Mahmud et al., 2010; Nambiar
& Ibrahim, 2011; Ibrahim & Nambiar, 2011a; Ibrahim & Nambiar, 2011b). Furthermore, to date, most
investigations surrounding the international graduate students conducted locally on the academic literacies issues
have heavily focused on investigating their writing practices. As Braine (2002) puts it, “a fundamental
shortcoming of most studies of socially situated academic literacy is their focus on writing tasks alone” (p. 63)
and also the relationship between writing practices and learning and the production of written assignments
(Wahiza Wahi et al., 2012). Braine (2002) further indicates less research focuses on other academic practices
such as reading and speaking. Prior to the various research conducted in Malaysian context as mentioned above, Green (1987), Stewart and
Spille (1988) and Conrad, Duren, and Haworth (1998) have provided argument that there is extremely limited
literature on how students experience their master programmes, much less the effects of their experience on
students themselves and literature also does not draw on students’ perspectives. Therefore, the findings of this
study add to the existing literature in reading at graduate level and Malaysian international education. 1.3 Related Studies on Academic Reading Practices among International Graduate Students Reading problems and the expected outcomes have been cited as one of the most neglected problems facing the
international graduate students (Smith, 1998). Phakiti and Li (2011) highlighted that their postgraduate
respondents faced difficulty in extracting and synthesizing information from various sources and recognizing and
acquiring academic vocabulary for use. Studies by Burke (1996) with postgraduate Australian students whose
first language is not English, Borland and Pearce (1999) and Cheng, Myles, and Curtis (2004) with international
students whose mother tongue is not English highlighted some of the problems with reading. The findings by
Burke (1996) indicated that new discipline-specific terminology was a major cause of difficulty. Burke (1996)
and Cheng et al. (2004) also found international students consume more time to read as it is done slowly, depend
heavily on dictionaries and re-read a text several times. These challenges mentioned by the researchers above affect the students’ ability to read and engage in critical
discussions of academic texts. Reid, Kirkpatrick, and Mulligan’s (1998) research finding is also consistent with
Burke’s that overseas students take two to three times longer to read an academic text than an English-speaking
background student. In addition, unfamiliarity with English speaking study environment affected students with
linguistic and cultural challenges such as ascertaining the degree of how much critical analysis is required in a
discipline, interpreting unfamiliar texts and lastly, expressing their criticality in the discourse appropriate for that
discipline. In a study conducted by Borland and Pearce (1999), it was found that reading critically is a challenge for most
students and most commonly, they ascribe their difficulty to lack of time. Second language learners tend not to
read as much as an L1 learner as their reading is slower. Second language learners spend a lot of time trying to
understand the material with consequently less time and confidence available to reflect critically on it. Durkin
(2004) also mentioned some issues that can arise in reading and writing critically such as misunderstanding of
concept of scholarly critical evaluation, difficulties with the concept of critical reading, the cultural
inappropriateness of challenging scholarship for some students, difficulties in analysing essay questions and
differences in the way different cultures structure literary texts. In addition, Koda’s (1995) study show that students from four different cultural backgrounds where written texts
used different orthographic structures (Arabic, English, Japanese and Spanish) used different information
processing procedures for reading English text. 1.2 Relevant Scholarship The idea of reading as being concerned primarily with meaning is itself socially constructed. Reading is a
social practice that is often embedded in larger social and institutional contexts. Learning to read and write in
these contexts is, in part, to learn the conventions, norms and standards for compliance if one is to read a
discipline specific text. Furthermore, reading is also related with what is meant by understanding. Understanding consists partly of the inter-textual links the reader makes (Barnett, 1989). According to Olson and
Torrance (2009), different readers in different historical periods or in different cultural contexts might create and
engage with written documents in dramatically distinctive ways. Different types of reading involve special
conventions for use and make different demands on readers. In addition, comprehension is active if the reader is
able to read although peculiar words are missing or not known. In the context of literacy, reading is a set of skills that can be broken into parts, taught and tested. However,
reading is not simply a matter of applying skills to reading. Reading is central to the subject area. The process of
reading involves not only taking the meaning from the text but also interacting with the text. Alderson (2000)
argues that it involves bringing knowledge to a text. There can be no effective analysis, comprehension and
application without reading as a basis. Good reading skills are vital for students to understand the concepts and
in turn transmit their competency in written and oral communication tasks. Moreover, in academic reading, one is expected to distance oneself from the text in an objective way in order to
evaluate its validity, biasness and assumptions. One of the major characteristic of the voice of an academic
proficient reader and proficient writer is authority. Blanton (1994, p. 230) suggests that the student is viewed as
powerless without such an authority. A reader should also become personal to evaluate and speculate on the text
in relation to one’s own reactions, concerns and professional interest. 77 Vol. 4, No. 4; 2014 Higher Education Studies www.ccsenet.org/hes In the context of the present study, there arises necessity for graduates to be able to critically evaluate what is
read and use critical thinking to explain their understanding of the reading. Atkinson and Longman (2003)
stresses that practice in critical thinking helps students to find, sort, evaluate and choose wisely through the
vicarious experience of reading. 1.2 Relevant Scholarship However, lack of competence in reading can negatively affect the written and
spoken output of the student and influence its role as an essential foundation skill in discussing and writing at
graduate level. 1.3 Related Studies on Academic Reading Practices among International Graduate Students 2.2 Sampling Procedures 2.2 Sampling Procedures This quantitative study utilised purposive sampling with the aim of selecting all accessible respondents who
could provide accurate and reliable information regarding the research problem (Teddlie and Yu, 2007; Rubin
and Babbie, 2009; Teddlie and Tashakkori, 2009). 1.4 Objectives of the Study This study looks into a less researched area that is the challenges experienced by the international graduate
students in the academic reading practices and the strategies employed to overcome the challenges in academic
reading practices. The objectives of the study are to identify: - the challenges faced by the international graduate students in their academic reading practices in the taught
Master programmes, - the challenges faced by the international graduate students in their academic reading practices in the taught
Master programmes, - the strategies employed by the international graduate students to overcome the challenges faced in their
academic reading practices in the taught Master programmes. - the strategies employed by the international graduate students to overcome the challenges faced in their
academic reading practices in the taught Master programmes. 1.3 Related Studies on Academic Reading Practices among International Graduate Students It was highlighted that although native speakers of English
experienced difficulty in reading, non-native speakers of English were seen to be at a particular disadvantage in
comprehending what is being said and the cultural implications underpinning much of the material which native
speakers can comprehend with little difficulty. Thus, Koda cautions that while for L1 students is likely that good
readers tend to be good writers and good writers usually display more regular and more sophisticated reading
patterns, the transfer to L2 should not be assumed too readily. Wilson (2003) also concedes to the serious difficulties faced by overseas students in academic reading. Her
research concludes that overseas students who have little experience of academic texts struggle with reading
large volume and time-consuming nature of reading. Another study by Pretorius (2005) showed that students
with poor reading skills seldom achieve academic success. Niven (2005) also postulates that academic reading is
pre-eminent: it precedes academic writing and determines its depth and quality. Her study revealed that students
rarely applied reading strategies that would ensure the sort of comprehension that is aligned with the required
level of learning. When lecturers’ reading frames are explored, there is a marked and significant gap between
lecturer expectations and student realities. Furthermore, Loewy and Vogt (2000) add that international students may be unused to the discourse patterns
used in written text. Vocabulary knowledge may not be broad enough for complete comprehension and requires
dictionary use and re-reading. In one of the latest studies by Johnson (2008), international students found it
difficult to understand assignment requirements, often because they thought the topic was ‘too general’ or they
did not understand the instructional words such as ‘evaluate’ or ‘discuss’. 78 Vol. 4, No. 4; 2014 Higher Education Studies www.ccsenet.org/hes Other studies that have researched on the challenges faced by international students also have some implications
for the present study. Several studies have revealed that students whose first language is not English need to
spend twice and even three times longer than native speakers to finish reading and it takes time for them to form
the habit of critical reading for academic purposes (Goodman, 1976; Reid et al., 1998; Durkin, 2004). Therefore, significant findings in reading challenges faced by the students indicate that academic reading
practices challenges is an area which is worth investigating among international graduate students in Malaysian
higher education context. 2.2.1 Sample Size Data obtained from the Institute of Postgraduate Study (IPS) of the research site university indicated there were
203 international graduate students registered in the 13 coursework and 11 mixed mode Master programmes
offered in the 10 schools comprising three Arts, two Hybrid and five Sciences. One hundred and thirty one
respondents in the age range of 20 to 47 years from the taught Master programmes completed the questionnaire. 2 2 2 2. Method This article reports on part of a larger study (Manjet, 2013) which also examines the academic literacies
challenges faced by international graduate students. The site of the research is one of the higher education
institutions in Malaysia that offers various first-degree and graduate courses with an enrolment of more than
20,000 local and international students. The institution has graduate students from more than 50 countries all
over the world due to the proliferation of its taught Master programmes and to the remarkable record of
accomplishment in sustaining world class education quality with global recognition (Institute of Postgraduate
Studies Website, 2012). Table 1 provides the conceptual framework of the study. The dependent variable is
academic reading practices that are influenced by the challenges faced by the international graduate students and
strategies employed to overcome the challenges in their coursework or mixed mode Master programmes in the
Arts, Hybrid and Sciences schools at the research site university. 2.1 Participant Characteristics The criteria for inclusion of the students in the study are as follow: (a) respondents have to be full-time
international master students in the taught Master programmes; (b) they have to be in their second semester or
later; and (c) they have to be registered students in the schools of Arts, Hybrid or Science at the research site
university. 2.2.2 Measures The Academic Literacies Questionnaire (ALQ) items used to collect data for this study was selected from Chang
(2006) and Evans and Green (2007). Ten items focused on academic reading challenges and five items focused
on overcoming strategies. The ALQ was used to learn about the challenges faced by the students in their
academic reading practices and the strategies taken by them to overcome the challenges. Evans & Green’s
questionnaire is considered suitable to be adapted for this study as it has been pilot tested with 175 subjects and
modified based on the responses obtained. The final version was administered to almost 5000 students with
significant findings (Evans & Green, 2007). Chang’s questionnaire that was used for a thesis research has also
produced reliable and valid data (Chang, 2006). Demographic and background information of respondents were
also collected. The respondents were asked to assess the difficulty level of the challenges in academic reading
practices on a scale from 1 (very difficult) to 4 (very easy). The respondents were also requested to indicate
whether they employed the strategies to overcome the challenges. The data collected were subjected to 79 Higher Education Studies Vol. 4, No. 4; 2014 www.ccsenet.org/hes descriptive statistics, non parametric statistics (Mann-Whitney U Test and Kruskal Wallis Test) and frequency
using the SPSS software. Figure 1 indicates the conceptual framework of the study. Figure 1. Conceptual Framework of the Study
Challenges in academic reading
practices
Strategies employed to overcome the
challenges
in
academic
reading
practices
Schools
Arts
Hybrid
Sciences
Academic reading practices
Taught Master programmes
Coursework
Mixed Mode Strategies employed to overcome the
challenges
in
academic
reading
practices Challenges in academic reading
practices Taught Master programmes
Coursework
Mixed Mode Schools
Arts
Hybrid
Sciences Academic reading practices Figure 1. Conceptual Framework of the Study 3.1 Profile of the Respondents The frequency distribution of each demographic factor was recorded and converted into percentage (%). Majority of the students are from the Middle East countries (64.4%). Iran has the highest number of respondents
(26.5%), followed by Iraq (14.4%), Palestine (6.8%), Libya (6.1%), Yemen (3.8%), Jordan (3.0%), Saudi Arabia
(2.3%) and Egypt (0.8%). The respondents from other Asian and African countries account for 35.6%. Slightly
less than half of the respondents (49.6%) were from the five schools in the Sciences. This was followed by
29.0% of the respondents from two Hybrid schools and 21.4% of the respondents from three Arts schools. Table
1 describes the distribution of students according to their schools. Table 1. Distribution of students according to the schools
Category of Schools
No. of Respondents
%
Sciences (5 schools) 65
49.6%
Hybrid(2 schools) 38
29.0%
Arts (3 schools) 28
21.4%
Total
131
100% 3.3 Challenges in Academic Reading Practices 3.3 Challenges in Academic Reading Practices g
g
Sixty seven respondents from the coursework and 64 respondents from the mixed mode Master programmes
used a Likert scale ranging from one (‘very difficult’) to four (‘very easy’) to assess the degree of difficulty they
experienced in the 10 items on academic reading practices in the questionnaire. Scale one (very difficult) and
scales two (difficult) are combined for reporting purpose. Scale three (easy) and scale four (very easy) are also
combined for reporting purpose. The study has set a mean of 2.5 or above to indicate some degree of ease in the
academic reading practices challenge in the ALQ based on the previous research by Evans and Green (2007). In
cases where the data shows significant result for an individual scale, it is reported individually. Table 3 summarises the respondents’ evaluation on the difficulty level of the academic reading practices. Table 3. Challenges in Academic Reading Practices
Challenges in Academic Reading Practices
1
(%)
2
(%)
3
(%)
4
(%)
Mean
Reading quickly to find information
8.3
44.7
40.2
4.5
2.42
Working out meaning of difficult words
2.3
49.2
38.6
7.6
2.53
Reading quickly to get overall meaning
8.3
42.4
40.9
6.1
2.46
Understanding specialist vocabulary
8.3
41.7
40.9
6.1
2.46
Identifying supporting ideas/examples
2.3
32.6
55.3
6.8
2.69
Taking brief, relevant notes
1.5
30.3
59.1
6.8
2.73
Using own words in note taking
1.5
28.8
57.6
9.8
2.78
Reading carefully to understand a text
1.5
27.3
61.4
7.6
2.77
Identifying key ideas
3.0
25.0
60.6
8.3
2.77
Understanding key ideas
2.3
22.0
65.2
6.1
2.79
Note: Scale: 1 = Very Difficult, 2 = Difficult, 3 = Easy, 4 = Very Easy Reading quickly to find information, reading quickly to get overall meaning and understanding specialist
vocabulary are ranked as the three top challenges in academic reading practices (mean ranging from 2.42 to
2.46). The respondents indicated the other seven items of challenges in academic reading practices range from
difficult to easy. The findings indicate that respondents generally find reading to be a challenging academic
literacies practice. The data in Table 3 indicates that reading quickly to find information is the most difficult (8.3%) and difficult
(44.7%) academic reading practice. The respondents reported working out meaning of difficult words and
reading quickly to get overall meaning as very difficult (51.5%) and difficult (50.7%). 3.2 Results of the Reliability Test A pilot study was administered to a small sample (n=21) similar to the potential respondents. It helped to
determine the consistency between multiple measurements of a variable (Hair et al., 2006). The Cronbach Alpha
reliability coefficient values indicate relatively high internal consistency with higher values than the minimum
accepted value of 0.70 (George & Mallery, 2003; Pallant, 2010). The result of the pilot study indicated high
internal consistency reliability with the alpha coefficient of .822. The statistical analysis indicated that the ALQ
has high internal consistency reliability. Table 2 describes the items in the academic reading practices section of
the ALQ. A pilot study was administered to a small sample (n=21) similar to the potential respondents. It helped to
determine the consistency between multiple measurements of a variable (Hair et al., 2006). The Cronbach Alpha
reliability coefficient values indicate relatively high internal consistency with higher values than the minimum
accepted value of 0.70 (George & Mallery, 2003; Pallant, 2010). The result of the pilot study indicated high
internal consistency reliability with the alpha coefficient of .822. The statistical analysis indicated that the ALQ
has high internal consistency reliability. Table 2 describes the items in the academic reading practices section of
the ALQ. 80 Vol. 4, No. 4; 2014 Higher Education Studies www.ccsenet.org/hes Table 2. Academic Reading Practices Items in the Academic Literacies Questionnaire (ALQ)
Section
Description
Likert Scale
Academic Reading Practices
1. 10 items selected from
Evans and Green (2007)
2. Two items on strategies
taken to overcome the
challenges in academic
reading practices selected
from Chang (2006)
1 = Very difficult
2 = Difficult
3 = Easy
4 = Very easy
Choose
as
many
strategies
employed
to
overcome
academic reading practices
challenges Academic Reading Practices Items in the Academic Literacies Questionnaire (ALQ)
Description
Likert Scale 3.3 Challenges in Academic Reading Practices Half of the respondents
(50%) reported understanding specialist vocabulary as a combination of very difficult (8.3%) and difficult
(41.7%). Identifying supporting ideas/examples, taking brief, relevant notes, using own words in note taking,
reading carefully to understand a text, identifying key ideas, understanding key ideas are reported as easy and
very easy by more than 50% of the respondents. Understanding key ideas is reported by 71.3% of the
respondents to be easy and very easy. Mann-Whitney U test, a non-parametric test for two independent samples carried out to compare the difficulty
level of the academic reading practices between the coursework and mixed mode respondents revealed a 81 Higher Education Studies Vol. 4, No. 4; 2014 www.ccsenet.org/hes significant difference in the difficulty level of identifying key ideas between the coursework and mixed mode
respondents (Z = 2.087, p = .037). The finding indicates that identifying key ideas is more difficult for the
coursework respondents (mean = 69.63) compared to the mixed mode respondents (mean = 58.87). The test also
revealed a significant difference in the difficulty of understanding specialist vocabulary between the coursework
and mixed mode respondents (Z = 2.309, p = .021). The finding indicates that understanding specialist
vocabulary is more difficult for the coursework respondents (mean = 70.75) compared to the mixed mode
respondents (mean = 57.63). significant difference in the difficulty level of identifying key ideas between the coursework and mixed mode
respondents (Z = 2.087, p = .037). The finding indicates that identifying key ideas is more difficult for the
coursework respondents (mean = 69.63) compared to the mixed mode respondents (mean = 58.87). The test also
revealed a significant difference in the difficulty of understanding specialist vocabulary between the coursework
and mixed mode respondents (Z = 2.309, p = .021). The finding indicates that understanding specialist
vocabulary is more difficult for the coursework respondents (mean = 70.75) compared to the mixed mode
respondents (mean = 57.63). Kruskal-Wallis Test, a non-parametric test of four scales carried out to compare the difficulty level of the
academic reading practices among the respondents in the Arts, Hybrid and Sciences schools revealed a
significant difference in the difficulty level of understanding key ideas among the respondents in the Arts,
Hybrid and Sciences (Chi-Square (H) (2) = 7.725, p = .021). 3.3 Challenges in Academic Reading Practices The finding indicates that understanding key ideas
is the most challenging academic reading practice for the respondents in the Arts (mean = 76.05) compared to
those in the Hybrid (mean = 64.36). Understanding key ideas is the easiest academic reading practice for the
respondents in the Sciences (mean = 57.10). 3.4 Overcoming the Challenges in Academic Reading Practices 3.4 Overcoming the Challenges in Academic Reading Practices Table 4 shows the frequency count of the five overcoming strategies employed by the respondents to overcome
the challenges in their academic reading practices. Table 4. Overcoming the Challenges in Academic Reading Practices
Overcoming the Challenges in Academic Reading Practices
YES
(%)
NO
(%)
Read the assigned material more times
72.5%
27.5%
Read extensively in your discipline area
34.4%
65.6%
Refer to your first language text/material for reference
28.2%
71.8%
Use a dictionary
71.8%
28.2%
Read articles with a focus on mastering English speakers' language use
23.7%
76.3% Read the assigned material more times Read extensively in your discipline area Based on frequency count, the findings indicate that the most vital strategies employed by the respondents are
“Read the assigned material more times” (72.5%) and “Use a dictionary” (71.8%). The least popular strategies
among the respondents are “Read articles with a focus on mastering English speakers’ language use” (76.3%)
and “Refer to your first language text/material for reference” (71.8%). Mann-Whitney U test, a non-parametric test for two independent samples carried out to compare the overcoming
strategies employed by the coursework and mixed mode respondents to overcome the challenges in academic
reading practices revealed significant difference in “Refer to your first language text/material for reference” (Z=
2.014, p=.044). The finding indicates that referring to first language text/material for reference is employed more
by the mixed mode respondents (mean = 71.41) compared to the coursework respondents (mean = 60.99). The findings from Likert scale items and open-ended response revealed that the challenges in academic reading
practices were lengthy reading duration and understanding of what is read. More specifically, the respondents
experienced challenges in reading quickly to find information, reading quickly to get overall meaning and
understanding of specialist vocabulary. The less challenging academic reading practices were the understanding
of key ideas and using own words in note taking. 4. Discussion Doing academic reading in a particular situated context at times causes the challenges with academic reading
rather that the international graduate students themselves. Academic reading practices and knowledge the
students bring from their prior academic learning background and culture influence the current notions of
academic reading practices and socialisation in the Master’s community of practice at the research site university. Cultural variations in discourse patterns and previous learning patterns affect how academic reading is done in
the Master’s context. These influences did not help them to grasp and adapt quickly to the new and different
academic expectations in the Master programmes. Previous research has indicated that to become accustomed to
an unfamiliar culture and to a new education system, as well as navigating these differences in a foreign or L2
(Andrade, 2008; Campbell & Li, 2008) is a lengthy process. It was also found that being qualified as competent users of English language based on the language requirement 82 Higher Education Studies Vol. 4, No. 4; 2014 www.ccsenet.org/hes (TOEFL, IELTS, or its equivalent) set by the university does not ensure that the international graduate students
do not struggle with the academic reading practices at Master’s level. The findings of this study strongly
advocate that being non-native speakers of English and attaining required English language qualification, the
students still faced academic literacies challenges in reading. The students need to be assisted with more
trainings and preparation to face the academic literacies challenges. This is necessary as the respondents’ English
language qualifications are not the true indicator of their English language proficiency. Furthermore, their
first-degree academic literacies training did not provide strong foundation to proceed to the graduate level. Hellsten (2002), Hellsten and Prescott (2004), Wong (2004) and Sawir (2005) also confirmed similar results that
students’ language barrier is one of the most known and widely recognised influence on the academic literacies
challenges. Many of the respondents confirmed that low proficiency in English language prevented them from taking part in
academic literacies practices such as reading fluently and comprehending the material read. According to
Ringbom (1987: 113-114) and Odlin (1989; 77-80), a justification for this challenge is the language distance
between their L1 and L2 or third language has effect on the amount of transfer that can take place between
languages. 4. Discussion Ringbom explained that for example, the Arabic speakers take a longer time to acquire English
vocabulary because transfer from third languages seems to depend very much on relative language distance. This
situation makes it difficult for them to learn English language. A research finding by Andrade (2006) is also
consistent with this study’s finding that international students compensate for their low level of English language
proficiency and socio-cultural knowledge through effort, study habits and self-help strategies. The students encountered difficulties in many academic tasks negotiated in their graduate study. The source of
their struggle in their reading was the lack of strong background in their discipline content knowledge. Repeated
re-reading to understand a text was a common trend among the respondents. They highlighted that they put in
more effort and time into their reading compared to the local students. At times, they translated the text from
English into their native language to read and understand. Then, the text was translated into English again to read
and understand. Borland and Pearce (1999) found that reading critically is a challenge mainly due the lack of
time. Students whose L2 is English can take a third to two-times as long to read as L1 students, often reading a
text over and over to gain understanding. Therefore, L2 learners tend not to read as much as L1 learners as their
reading is slower and they spend a lot of time trying to understand the material with consequently less time and
confidence available to reflect critically on it. The respondents had to depend extensively on dictionaries and re-read a text several times as they were
overpowered by the new discipline-specific terminologies. This challenge is cognitively very demanding. Burke
(1996) also identified similar predicaments in his study. Burke stated a new challenge for many international
graduate students in their ability to read and engage in critical discussions of academic texts with the use of new
discipline-specific terminology. Bell (2000) and Nambiar and Ibrahim (2011) indicated that these challenges in
academic reading practices present a heavy burden on the international students who have little experience of
studying in an English speaking environment. In relation to students’ different culture and background
knowledge influencing their learning, there is a dire need for schemata for the international graduate students to
make the necessary connection to the academic context and expectations. 4. Discussion Students’ results in this test will be a good indicator of their reading preparedness
to enroll into the programmes of their choice at the research site university. Furthermore, the in-house
standardised English language placement test is necessary to categorise students based on their language
qualification into linguistically demanding and non-linguistically demanding Master programmes as these
programmes require different levels of English language proficiency. in handling the challenges in their academic reading practices. Firstly, the lack of strict enforcement on the
English language entry requirement by either the individual schools or the Institute of Postgraduate Studies at the
research site university needs to be improved and adhered to. Concurrently, the university should also not only
depend on the standardised English language requirement across the board for all the taught Master programmes. Alco’s (2008) study has found IELTS and TOEFL not to be consistently reliable indicators of language ability in
the academic setting. Therefore, the university should also look into the possibility of designing and
implementing a more effective in-house standardised English language placement test to evaluate students’
English language proficiency. Students’ results in this test will be a good indicator of their reading preparedness
to enroll into the programmes of their choice at the research site university. Furthermore, the in-house
standardised English language placement test is necessary to categorise students based on their language
qualification into linguistically demanding and non-linguistically demanding Master programmes as these
programmes require different levels of English language proficiency. Secondly, in order to ensure the development of effective learning that will enable the international graduate
students to become skilled readers within the graduate education environment, the research site university should
enhance teaching and learning through trans-disciplinary collaboration between content and language specialists’
lecturers. Trans-disciplinary collaboration is a new field that is not yet given the deserved attention in Malaysian
universities. Lecturers from both areas should also be encouraged to cross the boundaries of their discipline,
collaborate and become familiar with a wide range of disciplines. Language specialists should be trained to
become familiar with the effective reading techniques to prepare the students to handle the discipline specific
concepts and the language of the particular discipline. At the same time, the content lecturers should be trained
on the linguistic aspects and the academic reading practices demands of their discipline. 4. Discussion The expertise of both
area lecturers can be employed to create integrative language and content instruction courses that focus on
specific discourses within the discipline to bring about an optimum exposure of the academic reading
expectations to the students. Academic Book Club programmes should also be implemented at the universities to cultivate the habit of
reading and proceeding with Book Club discussions. Activities of this nature will encourage the students to
increase their reading frequency and concurrently improve their academic socialisation with other academic
members of their graduate community through discussions. Lastly, this study also recommends that the lecturers’ role as educators should be enhanced to accommodate the
teaching of international students. The enhancement of the lecturers’ role as educators should focus on educating
lecturers on the intercultural diversity of the international graduate students in the context of how the students
approach reading activities or tasks. Lastly, the implications of the results for improvement of the international
graduate students’ academic reading experience at the research site university are significant. The research site
university’s academic fraternity needs to accept the students with clear teaching and learning plan that addresses
the multifaceted nature of the linguistic and cultural background of the coursework and mixed mode Master
programmes’ international students. 4. Discussion Nambiar and Ibrahim (2011) similarly echoed that a translator is a useful tool to deal with unfamiliar lexicon in
literacies practices. The respondents also depended heavily on lecture notes and the lecture notes became their
only source of reading. This was found to be consistent with other studies carried out by Van Meter, Yokoi and
Pressley (1994) and Harklau (2001) that students depended on the lecture notes and did not take the initiative to
do further reading. Beaufort (2005) also indicated that serious engagement especially with the genre knowledge
is difficult without guidance from within the community that uses the genre especially the lecturers. In addition, it is consistent with research on student engagement in higher education by Christie, Munro and
Fisher (2004). Christie et al. indicated most students tend not to actively seek assistance to overcome the
academic literacies challenges. Trotter and Roberts (2006) added that the international students do not seek
assistance because universities provide poorly articulated orientation programmes, students’ own inability to
self-assess their learning needs, disappointment with performance in courses and lack of awareness of what help
is available or how to access it. When the respondents are confronted by academic challenges such as reading a
research paper, there is high possibility that absence or lack of the ‘correct’ understanding of the academic
culture might lead the respondents to apply their earlier held assumptions, values, beliefs and approaches that
had given them stability, consistency and meaning (Schein, 2004). Therefore, this research presents four vital recommendations that would assist the international graduate students 83 Higher Education Studies Vol. 4, No. 4; 2014 www.ccsenet.org/hes in handling the challenges in their academic reading practices. Firstly, the lack of strict enforcement on the
English language entry requirement by either the individual schools or the Institute of Postgraduate Studies at the
research site university needs to be improved and adhered to. Concurrently, the university should also not only
depend on the standardised English language requirement across the board for all the taught Master programmes. Alco’s (2008) study has found IELTS and TOEFL not to be consistently reliable indicators of language ability in
the academic setting. Therefore, the university should also look into the possibility of designing and
implementing a more effective in-house standardised English language placement test to evaluate students’
English language proficiency. 5. Conclusion In conclusion, this study has revealed how the international graduate students in the taught Master programmes
that bring with them academic reading experiences that differ from the present institutionally accepted codes and
conventions faced the challenges in their academic reading practices and employed strategies to overcome the
challenges. The findings present only a small part of a much larger picture of the academic endeavour especially
the academic reading experience of the students within a broader context of their past and present cultural,
linguistic and educational experiences. It is hoped that this study has shed some light on the academic challenges
such as in reading that international graduate students experience. As such, an understanding of learning process
of international graduate students is pivotal to ensure their academic success. The study also reaffirms the idea
that “an understanding of literacy requires detailed, in-depth accounts of actual practice in different cultural
settings” (Street, 1993) and the situatedness of academic reading are multiple, changing and different from one
academic context to another (Lea & Street, 2000). Finally, the recommendations provided in this research should
be seriously considered by all the involved parties to ensure a positive learning experience for the international
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Copyright for this article is retained by the author(s), with first publication rights granted to the journal. py g
Copyright for this article is retained by the author(s), with first publication rights granted to the journal. Copyright for this article is retained by the author(s), with first publication rights granted to the journal. This is an open-access article distributed under the terms and conditions of the Creative Commons Attribution
license (http://creativecommons.org/licenses/by/3.0/). 88
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Research Priorities for Mental Health and Psychosocial Support in Humanitarian Settings
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Introduction In 2009, more than 119 million people were affected by natural
disasters [1], and 36 armed conflicts were recorded in 26 countries
[2]. Research in such settings has demonstrated the negative
impact of humanitarian crises on mental health and psychosocial
well-being, including increased psychological distress [3], social
problems [4,5], common mental disorders (depression and anxiety,
including post-traumatic stress disorder [PTSD]), and severe
mental disorders (e.g., psychotic disorders) [6]. Citation: TolWA, Patel V, Tomlinson M, BainganaF, Galappatti A,et al. (2011) Research
Priorities for Mental Health and Psychosocial Support in Humanitarian Settings. PLoS
Med 8(9): e1001096. doi:10.1371/journal.pmed.1001096 Published September 20, 2011 Copyright: 2011 Tol 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. Recent international policy [7,8] indicates a growing consensus
in the approaches recommended for mental health and psycho-
social support interventions in humanitarian settings, despite a
weak empirical evidence base to support specific approaches
[9,10]. Amongst both researchers and practitioners, however,
divisions remain on key issues, notably (a) the extent to which
PTSD should be a central research and intervention focus [11], (b)
the distinction between normal psychological distress and mental
disorders in situations of adversity [12], and (c) the extent to which
interventions should aim to target mental disorders or ongoing
structural and situational stressors in the recovery environment
[9,10,13]. In addition, (d) practitioners and researchers have been
divided over the extent to which research has led to tangible Funding: Research for the Mental Health and Psychosocial Support in
Humanitarian Settings – Research Priority Setting (MH-SET) project was funded
by the Department of Mental Health and Substance Abuse, World Health
Organization; Durham University; and HealthNet TPO. VP is supported by a
Wellcome Trust Senior Research Fellowship in Clinical Science. The funders had
no role in study design, data collection and analysis, decision to publish, or
preparation of the manuscript. Competing Interests: Because the authors all work in this research area, it is
conceivable that they could benefit from any increased attention and financing
regarding research on mental health and psychosocial support in humanitarian
settings. The views expressed in this article are those of the authors and not
necessarily those of the institutions that they serve. VP and ES are members of the
PLoS Medicine Editorial Board. The Guidelines and Guidance section contains advice on conducting and
reporting medical research. Summary Points N Addressing this research agenda requires a better alignment between researchers and practitioners,
attention to perspectives of populations affected by humanitarian crises, and sensitivity to sociocultural context. Research Priorities for Mental Health and Psychosocial
Support in Humanitarian Settings Wietse A. Tol1,2*, Vikram Patel3,4, Mark Tomlinson5, Florence Baingana6, Ananda Galappatti7,8,
Catherine Panter-Brick9, Derrick Silove10, Egbert Sondorp4, Michael Wessells11, Mark van Ommeren12 1 Global Health Initiative, MacMillan Center, Yale University, New Haven, Connecticut, United States of America, 2 HealthNet TPO, Amsterdam, The Netherlands, 3 Sangath, Goa,
India, 4London School of Hygiene & Tropical Medicine, London, United Kingdom, 5 Department of Psychology, Stellenbosch University, Stellenbosch, South Africa, 6 Makerere
University School of Public Health, Kampala, Uganda, and Personal Social Services Research Unit, London School of Economics and Political Science, London, United Kingdom,
7 Good Practice Group, Colombo, Sri Lanka, 8 Colombo University, Colombo, Sri Lanka, 9 Jackson Institute for Global Affairs and Department of Anthropology, Yale University,
New Haven, Connecticut, United States of America, 10 School of Psychiatry, University of New South Wales, Sydney, Australia, 11 Program on Forced Migration and Health,
Columbia University, New York, New York, United States of America, 12 Department of Mental Health and Substance Abuse, World Health Organization, Geneva, Switzerland Summary Points N There has been a great need to develop a research agenda to strengthen mental health and psychosocial support in
humanitarian settings; prior research in this area has had limited inputs from practitioners. N There has been a great need to develop a research agenda to strengthen mental health and psychosocial support in
humanitarian settings; prior research in this area has had limited inputs from practitioners. N We developed a consensus-based research agenda for the next ten years through inputs from an in
academics, policy makers, and practitioners (n = 82) representing regions where humanitarian crises We developed a consensus-based research agenda for the next ten years through inputs from an interd
academics, policy makers, and practitioners (n = 82) representing regions where humanitarian crises occ N Participants reached a high level of agreement on the ten most highly prioritized research questions, which consisted of
questions related to: problem analysis (four questions on identifying stressors, problems, and protective factors from the
perspective of affected populations), mental health and psychosocial support interventions (three questions on sociocultural
adaptation and on effectiveness of family- and school-based prevention), research and information management (two
questions on assessment methods and indicators for monitoring and evaluation), and mental health and psychosocial support
context (one question on whether interventions address locally perceived needs). N Participants reached a high level of agreement on the ten most highly prioritized research questions, which consisted of
questions related to: problem analysis (four questions on identifying stressors, problems, and protective factors from the
perspective of affected populations), mental health and psychosocial support interventions (three questions on sociocultural
adaptation and on effectiveness of family- and school-based prevention), research and information management (two
questions on assessment methods and indicators for monitoring and evaluation), and mental health and psychosocial support
context (one question on whether interventions address locally perceived needs). N This research agenda emphasizes the generation of practical knowledge that could translate to immediate tangible benefits
for programming in humanitarian settings, rather than addressing the key debates that have dominated the academic
literature. N This research agenda emphasizes the generation of practical knowledge that could translate to immediate tangible benefits
for programming in humanitarian settings, rather than addressing the key debates that have dominated the academic
literature. N Addressing this research agenda requires a better alignment between researchers and practitioners,
attention to perspectives of populations affected by humanitarian crises, and sensitivity to sociocultural context. Guidelines and Guidance Introduction Abbreviations: CHNRI, Child Health and Nutrition Research Initiative; LMIC, low-
and middle-income country(ies); MH-SET, Mental Health and Psychosocial
Support in Humanitarian Settings – Research Priority Setting [project]; PTSD,
post-traumatic stress disorder The Guidelines and Guidance section contains advice on conducting and
reporting medical research. * E-mail: wietse.tol@yale.edu Provenance: Not commissioned; externally peer reviewed. PLoS Medicine | www.plosmedicine.org September 2011 | Volume 8 | Issue 9 | e1001096 September 2011 | Volume 8 | Issue 9 | e1001096 September 2011 | Volume 8 | Issue 9 | e1001096 1 Figure 1. Research priority setting flow chart. doi:10.1371/journal.pmed.1001096.g001 described in more detail in Text S1). In brief, this method allows
for the systematic generation and scoring of research questions
using predetermined criteria (Figure 1). We selected this method
because it allows for a structured approach to research priority
setting, and because it defines research as an activity that aims to
improve the lives of people rather than focusing on the generation
of new knowledge per se. The CHNRI methodology has been
used to set research priorities in a variety of fields, including child
health, health of people with disabilities, zinc-related health
research, mental health, preterm birth, stillbirth, and birth
asphyxia [16–22]. benefits for implementing programs, and about the universality of
applied constructs of mental disorder [14]. A consensus-based
research agenda for this area of work does not exist. Furthermore,
the fact that the power to set the research agenda typically is vested
in researchers from outside of humanitarian settings has the
potential to marginalize many practitioners. Currently prioritized
research may therefore not improve the knowledge that is needed
by practitioners on the ground [15]. benefits for implementing programs, and about the universality of
applied constructs of mental disorder [14]. A consensus-based
research agenda for this area of work does not exist. Furthermore,
the fact that the power to set the research agenda typically is vested
in researchers from outside of humanitarian settings has the
potential to marginalize many practitioners. Currently prioritized
research may therefore not improve the knowledge that is needed
by practitioners on the ground [15]. The Mental Health and Psychosocial Support in Humanitar-
ian Settings – Research Priority Setting (MH-SET) project was
initiated to establish a consensus-based research agenda aimed at
supporting the prevention and treatment of mental disorders and
the protection and promotion of psychosocial well-being in
humanitarian
settings. Stage 1: Defining the Research Context An international steering committee (authors; 40% from low-
and middle-income countries [LMIC]) decided to address global
research priorities, focus on child and adult populations, and look
broadly at mental disorders and psychosocial issues. In addition,
we focused on setting priorities for questions that could be
answered within the coming ten years. Introduction The
project
aimed
to
set
research
priorities based on the perspectives of a range of key stakeholders,
including academics, practitioners, and policy makers from a
variety of disciplines, ensuring representation from locations
where humanitarian crises occur. This report lays out the results
of the, to our knowledge, first systematic effort to set research
priorities in this field. PLoS Medicine | www.plosmedicine.org September 2011 | Volume 8 | Issue 9 | e1001096 Stage 5: Scoring Research Questions Research questions were scored by (a) 53% of the advisory
group that generated research options (n = 72) and (b) the ten
members of the steering committee (authors) using five predeter-
mined criteria related to significance, answerability, applicability,
equity, and ethics (Box 1). Scoring was conducted through an
online survey format, with follow-up reminders by email. We
assessed whether the 72 respondents in this step were different
from those participating in the generation of research options,
and did not find any differences with regard to gender, region of
work/origin/residence, affiliation, implementation focus, or work
setting
(Chi-square
comparisons;
p-values
0.11–0.79). Final
endorsement scores were calculated by taking the overall average Criterion 5. Ethics Would you say that a study to answer
this question would be perceived as ethical by all of the
key stakeholder groups (e.g. affected population, national
governments, humanitarian agencies, donors)? In other
words, do you think this question may be answered in an
ethical manner in the coming 10 years? Please rate as ‘‘0. No’’ (Not ethical) or ‘‘1. Yes’’ (Ethical). Table 1. Ten most highly endorsed research questions. Table 1. Ten most highly endorsed research questions. Research Option
Category
Average Rating (%)
1. What are the stressors faced by populations in humanitarian settings? Problem Analysis
86.7
2. What are appropriate methods to assess mental health and psychosocial
needs of populations in humanitarian settings? Research and Information Management
85.9
3. How do affected populations themselves describe and perceive mental health
and psychosocial problems in humanitarian settings? Problem Analysis
85.9
4. What are appropriate indicators to use when monitoring and evaluating the
results of mental health and psychosocial support in humanitarian settings? Research and Information Management
85.4
5. How can we best adapt existing mental health and psychosocial interventions
to different sociocultural settings? MHPSS Interventions
85.2
6. What is the effectiveness of family-based interventions to prevent mental
disorders and protect and promote psychosocial well-being and mental health
among children and adolescents in humanitarian settings? MHPSS Interventions
84.7
7. What are the major protective factors (including individual [e.g., coping,
hope] and contextual [e.g., justice mechanisms, religious practices]) for mental
health and psychosocial problems in humanitarian settings? Problem Analysis
84.4
8. What is the effectiveness of school-based psychosocial and mental health
interventions to prevent mental disorders and protect and promote psychosocial
well-being and mental health among children and adolescent in humanitarian settings? MHPSS Interventions
83.2
9. Methods The MH-SET initiative adopted the widely implemented
consensus-building methodology developed by the Child Health
and
Nutrition
Research
Initiative
(CHNRI)
(methods
are Research questions were generated by 136 advisory group
members, and participants of nine focus group discussions in
Peru,
Uganda,
and
Nepal
(n = 114). The
advisory
group September 2011 | Volume 8 | Issue 9 | e1001096 2 generated research questions through an online survey format,
where participants were invited to list up to five research
questions they felt were crucial to support mental health and
psychosocial support in humanitarian settings. Advisory group
members in this step were 43.3% female, and worked in a variety
of settings (academia 13.4%; implementation 38.8%; both
47.8%). Most members focused on both mental disorders and
psychosocial well-being (65.4%; only mental disorders 3.7%; only
psychosocial well-being 29.9%). Members of the advisory group
were broadly representative of locations where humanitarian
settings occur. The focus group discussions are described in more
detail elsewhere [14]. Altogether, 733 research questions were
generated, which were consolidated into a list of 74 research
questions through qualitative data analysis by two independent
analysts. This list contained research questions grouped into four
categories: (1) Problem Analysis (14 research questions), (2)
Mental Health and Psychosocial Support Interventions (25
research questions), (3) Mental Health and Psychosocial Support
Context (25 research questions), (4) Research and Information
Management (ten questions). Box 1. Criteria Used to Score Research
Questions Criterion 1. Significance Would you say the research
question is an important question that needs answering? In other words, do you think this research question is
essential to address in the coming 10 years? Please rate as
‘‘0. No’’ (Not at all important) or ‘‘1. Yes’’ (Essential). Criterion 2. Answerability Would you say that a study
to answer this question is feasible? In other words, do you
think it is possible to actually design a study that addresses
this research question in the coming 10 years? Please rate
as ‘‘0. No’’ (Not at all feasible) or ‘‘1. Yes’’ (Very feasible). Criterion 3. Applicability Would you say that an answer
to this research question would influence humanitarian
policy and practice? In other words, do you think
answering this research question will lead to tangible
practice results in the coming 10 years? Please rate as ‘‘0. No’’ (Not at all applicable) or ‘‘1. Yes’’ (Very applicable). Criterion 4. Equity Would you say that an answer to this
question would help to improve the conditions of
marginalized groups in humanitarian settings? In other
words, do you think answering this research question will
aid underprivileged populations in the coming 10 years? Please rate as ‘‘0. No’’ (Does not improve) or ‘‘1. Yes’’ (Will
very much improve). Stage 5: Scoring Research Questions To what extent do current mental health and psychosocial supports address
locally perceived needs? MHPSS Context
82.5
10. Which are the most common mental health and psychosocial problems in
the general population in humanitarian settings? Problem Analysis
82.2
MHPSS, Mental Health and Psychosocial Support. doi:10.1371/journal.pmed.1001096.t001
PLoS Medicine | www.plosmedicine.org
3
September 2011 | Volume 8 | Issue 9 | e1001096 September 2011 | Volume 8 | Issue 9 | e1001096 PLoS Medicine | www.plosmedicine.org 3 of the average endorsement (Yes = 1, No = 0) for each of the five
research criteria. of the average endorsement (Yes = 1, No = 0) for each of the five
research criteria. Discussion The MH-SET initiative used established methods of research
priority setting and incorporated the perspectives of academics,
practitioners, and policy makers from a variety of disciplines
working in geographically diverse humanitarian settings. 3. Research and Information Management Two questions in this category were prioritized—one question
on assessment methods (#2) and one on the selection of indicators
for monitoring and evaluation (#4). Again, these research
questions appear to support an agenda that is focused on
improving practice and that supports a fresh look at how to
measure the impact and changes over time of mental health and
psychosocial well-being in humanitarian crises. Fourth, in relation to specific interventions, research into the
effectiveness of family- and school-based preventive interventions
scored highly. Research on children and adolescents was similarly
highly rated in two previous research priority setting efforts in the
general field of global mental health [18,25]. Results Table 1 lists the ten most highly prioritized research questions. These ten research questions were rated with high agreement by
participants; all of the research questions scored above 80%
average endorsement as ‘‘essential’’ on each of the five research
criteria. Overall, the top ten appears to emphasize a research
agenda for the next ten years focusing on research questions that
may have immediate benefit for humanitarian programs, rather
than addressing the key debates that have dominated the
academic literature (e.g. the controversy surrounding PTSD,
the distinction between distress and disorder). Specific categories
of research questions that feature prominently in the ten most
highly prioritized research questions are (in order of importance):
(1) Problem Analysis (four research questions), (2) Mental Health
and Psychosocial Support Interventions (three questions), (3)
Research and Information Management (two questions), and (4)
Mental Health and Psychosocial Support Context (one question). The findings offer a number of directions for further research
and practice. First, the most highly prioritized research questions
favored practical initiatives with a strong potential for translation
of knowledge into mental health and psychosocial support
programming. The tendency to emphasize research that informs
practice is further suggested by the fact that the majority of
research questions in the overall compilation were related to
effectiveness and implementation (50 out of 74; 67.6%). To
illustrate, as we note in the Introduction, controversy has
surrounded the psychiatric diagnosis of PTSD. Despite the central
role this debate has played in the literature, only a limited portion
of the generated research questions were trauma-focused (42 out of
the initially generated 733 questions [6%]). Based on this
incongruity, we recommend a better alignment between academic
priorities and those of practitioners. This may be stimulated
through (a) strengthened partnerships between humanitarian
agencies and universities, (b) upgrading basic research skills of
humanitarian practitioners to strengthen information gathering as
part of program implementation, and (c) increased funding for the
research questions prioritized in this study. 2. Mental Health and Psychosocial Support Interventions 2. Mental Health and Psychosocial Support Interventions
In
this
category,
participants
prioritized
research
that
facilitates the adaptation of existing interventions to different
sociocultural settings (#5) and that evaluates the effectiveness of
family- and school-based interventions to prevent mental
disorders and promote and protect psychosocial well-being in
humanitarian settings (#6 and #8, respectively). In addition to
addressing
the
importance
of
attention
to
the
differing
sociocultural contexts in which humanitarian settings occur,
these questions call specifically for more research on prevention
and promotion. Third, we note the particular dominance (four out of the top ten
questions) of problem analysis research. Although there is already
a very large body of research that has focused on establishing
prevalence rates of PTSD and depression in post-conflict and
natural disaster settings [3,23], the prioritized questions on
problem analysis cover much broader ground, in that they
concern major stressors faced, mental health and psychosocial
problems as defined by populations affected by humanitarian
crises, protective factors, and an open question on what the most
common mental health and psychosocial problems in humanitar-
ian settings are. The focus on problem analysis research may
reflect the lack of systematic use of needs assessments in the design
of mental health and psychosocial support programs [10,24]. In
accordance with the results of this study and recommendations in
international policy [7,8] we recommend a stronger emphasis on
needs assessments as a structural element of practice. PLoS Medicine | www.plosmedicine.org September 2011 | Volume 8 | Issue 9 | e1001096 1. Problem Analysis Research questions in this category concerned the stressors
faced
by
populations
in
humanitarian
settings
(#1),
local
perceptions of the mental health and psychosocial impact of
humanitarian crises (#3), major protective factors for mental
health and psychosocial problems (#7), and the most common
mental health and psychosocial problems in the general popula-
tion in humanitarian settings (#10). Rather than assume the
importance of specific mental disorders or psychosocial constructs,
the prioritization of these questions seems to favor a research
agenda that takes a step back and examines the most important
stressors, problems, and protective factors from the perspective of
affected populations. Second, we note that three of the ten most highly prioritized
research questions emphasize the inclusion of perspectives from
affected people and the promotion of sensitivity to the sociocul-
tural context. We recommend, in accordance with current
international policy [7,8], that researchers in humanitarian settings
more strongly emphasize these aspects in their work. In our
experience,
prioritizing
the
strengthening
of
local
research
infrastructure in humanitarian settings—especially in low- and
middle-income settings—as an integrated goal in research projects
may form an important contribution to this end. Study Limitations Finally, one prioritized question from this category focused on
whether current interventions address locally perceived needs
(#9). Similar to questions #3 and #5, this question highlights the
importance of considering local perspectives on the appropriate
methods of addressing psychosocial and mental health problems in
humanitarian settings. We point to a number of limitations of this exercise. First,
although we achieved targeted representation from four of eight
regions, we had smaller than intended representation from two
regions (Eastern Asia and the Pacific; Latin America and the
Caribbean) and stronger representation than targeted from two September 2011 | Volume 8 | Issue 9 | e1001096 September 2011 | Volume 8 | Issue 9 | e1001096 4 Jeannie Annan, Naimeh Baidun, Denfred P. Balleza, Zenaida Paez
Beltejar, Theresa S. Betancourt, Subhasis Bhadra, Alice Behrendt, Cecille
Bizouerne, Paul Bolton, Ivelina Borisova, Jorge Castilla, Sotheara Chhim,
Satyabrata Dash, Douki Dedieu, Gaithri Fernando, Tanja Franciskovic,
Rita Giacaman, Manuel Fontaine, David Alan Harris, Yvonne Suzy
Handajani, Takashi Izutsu, Ebun James, Eranda Jayawickreme, Davidson
Jonah, Joop T. V. M. de Jong, Mark J. D. Jordans, G. Krishnakumar,
Barbara Lopes Cardozo, Maryanne Loughry, Nagendra Prasad Luitel,
Crick Lund, Jihad Makhoul, Emilie Medeiros, Amanda Melville, Ken
Miller, Harry Minas, Andrew Mohanraj, Steven Muncy, Ma. Suzette
Munoz-Agcaoili, R. Srinivasa Murthy, Chee Ng, Isa M. Noor, Davaasuren
Oyunsuren, Duncan Pedersen, Isabel Perez-Olmos, Pau Perez Sales,
Benjamin Porter, Joseph O. Prewitt Diaz, Susan Prosser, Robert S. Pynoos, Sabine Rakotomalala, Beverley Raphael, Susan Rees, Miryam
Rivera Holguin, Khalid Saeed, Gameela Samarasinghe, Sujata Satapathy,
Ahmed Farah Shadoul, Bhogendra Sharma, Karuna Prasad Shrestha,
Xiao Shuiyuan, Abdul Majeed Siddiqi, Sambasivamoorthy Sivayokan,
Leslie Snider, Daya Somasundaram, Shobna Sonpar, Renato Souza,
Lindsay Stark, Hans Stolk, Alison Strang, Shishir Subba, Derek
Summerfield, Ninik Supartini, Dessy Susanty, Yuriko Suzuki, A. J. W. Taylor, Sivaprashanthi Thambaiah, Rangaswamy Thara, Matthias
Themel, Anavarathan Vallipuram, Peter Ventevogel, Wendy Wheaton,
John Williamson, and William Yule. regions (Middle East and North Africa; industrialized countries). The higher representation from industrialized countries may be
due to the inclusion of global practitioners, who work in the
countries where they reside as well as in other regions. We did,
however, achieve a sample that consisted of two-thirds of
participants originating in LMICs, and who worked in 47 different
languages. Second, we had an attrition rate of 53% between the
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7. Inter-Agency Standing Committee [IASC] (2007) IASC Guidelines on Mental
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and psychosocial support in humanitarian settings: linking practice and research. Lancet. In press. 23. Steel Z, Chey T, Silove D, Marnane C, Bryant RA, et al. (2009) Association of
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71–75. 12. Horwitz AV (2007) Transforming normality into pathology: the DSM and the
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Looking for and Why? Assessing Psychosocial Needs in Sri Lanka Post-Tsunami. Edinburgh: Institute of International Health and Development, Queen
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after conflict or other large disaster. Am J Prev Med 35: 284–286. 14. Tol WA, Patel V, Tomlinson M, Baingana F, Galappatti A, et al. Relevance or
Excellence? Setting Research Priorities for Mental Health and Psychosocial
Support in Humanitarian Settings. Harvard Review of Psychiatry. In press. 25. Conclusions Our research priority setting initiative—the first of its kind in
this particular field—showed promising points of agreement
between diverse stakeholders on research priorities for mental
health and psychosocial support in humanitarian settings. There
was a strong endorsement of research that achieves tangible
benefits for programming and that gives emphasis to participation
with and sensitivity to the specific sociocultural context of the
populations living in humanitarian settings. In addition, we would like to thank Beki Langford for her help in
compiling the generated research options, and Tim Williams for assistance
in analyzing focus group discussion data. Text S1
Supplementary information.
(DOC) Conceived and designed the experiments: WAT VP MT FB AG CPB DS
ES MW MVO. Performed the experiments: WAT VP MT FB AG CPB
DS ES MW MVO. Analyzed the data: WAT. Wrote the first draft of the
manuscript: WAT. Contributed to the writing of the manuscript: WAT VP
MT FB AG CPB DS ES MW MVO. ICMJE criteria for authorship read
and met: WAT VP MT FB AG CPB DS ES MW MVO. Agree with
manuscript’s results and conclusions: WAT VP MT FB AG CPB DS ES
MW MVO. Study Limitations Although we
did not find any differences between responders and nonrespond-
ers on sociodemographic and occupational variables, the pattern
of nonresponse may have contained other biases that were not
measured. Acknowledgments We would like to thank all of the advisory group members who provided
their inputs in setting research priorities, including: Sharon Abramowitz,
Alastair Ager, Grace Akello, Francis Alumai, Kennedy Amone-P’Olak, PLoS Medicine | www.plosmedicine.org References Sharan P, Gallo C, Gureje O, Lamberte E, Mari JJ, et al. (2009) Mental health
research priorities in low- and middle-income countries of Africa, Asia, Latin
America and the Caribbean. Br J Psychiatry 195: 354–363. PLoS Medicine | www.plosmedicine.org September 2011 | Volume 8 | Issue 9 | e1001096 5
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Mice Expressing a "Hyper-Sensitive" Form of the Cannabinoid Receptor 1 (CB1) Are Neither Obese Nor Diabetic
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PloS one
| 2,016
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cc-by
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Mice Expressing a "Hyper-Sensitive" Form of
the Cannabinoid Receptor 1 (CB1) Are
Neither Obese Nor Diabetic David J. Marcus1,2,3☯, Michael L. Zee3☯, Brian J. Davis1,2☯, Chris P. Haskins1,2,3☯, Mary-
Jeanette Andrews1,2,4, Randa Amin1,2,4, Angela N. Henderson-Redmond3, Ken Mackie1,2,4,
Traci A. Czyzyk3*, Daniel J. Morgan1,2,3,4* 1 Gill Center for Biomolecular Science, Indiana University, Bloomington, Indiana, 47405,United States of
America, 2 Department of Psychological and Brain Sciences, Indiana University, Bloomington, Indiana,
47405, United States of America, 3 Department of Anesthesiology, Penn State College of Medicine,
Hershey, Pennsylvania, 17033, United States of America, 4 Center for the Integrative Study of Animal
Behavior, Indiana University, Bloomington, Indiana, 47405, United States of America a1111 ☯These authors contributed equally to this work. ☯These authors contributed equally to this work. * tczyzyk@hmc.psu.edu (TAC); djm68@psu.edu (DJM) OPEN ACCESS Citation: Marcus DJ, Zee ML, Davis BJ, Haskins CP,
Andrews M-J, Amin R, et al. (2016) Mice Expressing
a "Hyper-Sensitive" Form of the Cannabinoid
Receptor 1 (CB1) Are Neither Obese Nor Diabetic. PLoS ONE 11(8): e0160462. doi:10.1371/journal. pone.0160462 Multiple lines of evidence implicate the endocannabinoid signaling system in the modulation
of metabolic disease. Genetic or pharmacological inactivation of CB1 in rodents leads to
reduced body weight, resistance to diet-induced obesity, decreased intake of highly palat-
able food, and increased energy expenditure. Cannabinoid agonists stimulate feeding in
rodents and increased levels of endocannabinoids can disrupt lipid metabolism. Therefore,
the hypothesis that sustained endocannabinoid signaling can lead to obesity and diabetes
was examined in this study using S426A/S430A mutant mice expressing a desensitization-
resistant CB1 receptor. These mice display exaggerated and prolonged responses to acute
administration of phytocannabinoids, synthetic cannabinoids, and endocannabinoids. As a
consequence these mice represent a novel model for determining the effect of enhanced
endocannabinoid signaling on metabolic disease. S426A/S430A mutants consumed equiv-
alent amounts of both high fat (45%) and low fat (10%) chow control diet compared to wild-
type littermate controls. S426A/S430A mutants and wild-type mice fed either high or low fat
control diet displayed similar fasting blood glucose levels and normal glucose clearance fol-
lowing a 2 g/kg glucose challenge. Furthermore, S426A/S430A mutants and wild-type mice
consumed similar amounts of chow following an overnight fast. While both THC and
JZL195 significantly increased food intake two hours after injection, this increase was simi-
lar between the S426A/S430A mutant and wildtype control mice Our results indicate that
S426A/S430A mutant mice expressing the desensitization-resistant form of CB1 do not
exhibit differences in body weight, food intake, glucose homeostasis, or re-feeding following
a fast. Editor: Daniela Cota, "INSERM", FRANCE
Received: November 13, 2014
Accepted: July 20, 2016
Published: August 8, 2016
Copyright: © 2016 Marcus et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited Editor: Daniela Cota, "INSERM", FRANCE Editor: Daniela Cota, "INSERM", FRANCE
Received: November 13, 2014
Accepted: July 20, 2016
Published: August 8, 2016
Copyright: © 2016 Marcus et al This is an open Published: August 8, 2016 Copyright: © 2016 Marcus et al. RESEARCH ARTICLE OPEN ACCESS This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited. Data Availability Statement: All relevant data are
within the paper and its Supporting Information files. Introduction study design, data collection and analysis, decision to
publish, or preparation of the manuscript. study design, data collection and analysis, decision to
publish, or preparation of the manuscript. The consequences of dysregulated metabolism, including obesity and diabetes, are among the
most devastating health crises facing the developed world today. In the United States, diabetes
mellitus is the 7th leading cause of death, with more than 73,282 deaths in 2011 from the dis-
ease [1]. In addition, the prevalence of obesity among adults has increased from 12% in 1990
to 35.6% in 2010 [2]. The CDC estimated the annual cost of obesity in 2008 at $147 billion
[3]. Competing Interests: The authors have declared
that no competing interests exist. One intriguing therapeutic target for the treatment of metabolic disease is the endocannabi-
noid system (ECS). In the central nervous system, the endocannabinoid signaling system is
comprised of the cannabinoid receptor 1 (CB1), its two primary endogenous ligands, N-arachi-
donoylethanolamide (also referred to as anandamide, AEA) and 2-arachidonoyl glycerol
(2-AG), and also the biosynthetic and hydrolytic enzymes that synthesize and degrade endo-
cannabinoids. Genetic and pharmacological manipulation has established the endocannabi-
noid system as a major player in metabolic regulation, ranging from the control of food intake
to the regulation of adipogenesis, fatty acid oxidation and energy expenditure. Mice lacking
CB1 are resistant to diet-induced obesity, are lean on a regular chow diet, and exhibit reduced
food intake [4,5]. CB1 knock-out (KO) mice do not develop diet-induced insulin resistance [4]. In mice lacking CB1, food intake is decreased while energy expenditure is increased suggesting
that both aspects could be involved in the leanness observed in CB1-deficient mutant mice [5]. Pharmacological blockade with a selective CB1 inverse agonist, rimonabant (SR141716), has
been widely studied in rodents and humans as a potential treatment for obesity. Treatment of
diet-induced obese rodents with daily rimonabant causes a transient reduction in food intake,
with sustained decreases in body weight, adiposity, and fasting glycemia [6,7]. Rimonabant
also prevents hepatic steatosis, a phenomenon where excess fat storage in the liver leads to cir-
rhosis [8]. Several studies have suggested that endocannabinoid modulation of the peripheral
elements of metabolic homeostasis such as energy expenditure might be responsible for the
weight-reducing effects of rimonabant [9–11]. Conversely, treatment with low doses of Δ9-THC increases the consumption and preference
for highly-palatable foods [12–14]. CB1 Desensitization Does Not Impact Metabolic Homeostasis Data Availability Statement: All relevant data are
within the paper and its Supporting Information files. Funding: This work was funded by National
Institutes of Health grants DA011322 (KM),
DA021696 (KM), and DA036385 (DJM). This project
is funded, in part, under a grant with the Pennsylvania
Department of Health using Tobacco CURE Funds. Additional support was provided by the Indiana
Academy of Sciences, the Indiana University
METACyt Initiative (funded in part by a grant from the
Lilly Foundation), and the Linda and Jack Gill Center
for Biomolecular Science. The funders had no role in 1 / 15 PLOS ONE | DOI:10.1371/journal.pone.0160462
August 8, 2016 PLOS ONE | DOI:10.1371/journal.pone.0160462
August 8, 2016 Re-feeding Following an Overnight Fast Re-feeding following an 18 hour overnight fast (5:00 PM-11:00 AM) was measured in 25 week
old S426A/S430A mutant and wild-type littermate control mice fed HFD or LFD starting at
weaning. Access to HFD or LFD was restored and food consumption was measured at 1, 4, 6
and 24 hours following resumption of food access. Insulin Tolerance Test Insulin tolerance was examined in 30 week old wild-type and S426A/S430A mutant littermate
mice given either HFD or LFD from weaning until 30 weeks of age. Testing was done in mice
that were fasted overnight for 18 hours (5:00 PM-11:00 AM). After fasting, mice received an IP
injection of 1U/kg of insulin (Eli Lilly, Indianapolis, IN). Blood was collected via lateral tail
vein nick prior to, and 15, 30, 60, and 120 minutes following Insulin injection. Blood glucose
levels were assessed using a glucometer and blood glucose test strips as described for glucose
tolerance experiments. Glucose Tolerance Test Fasting glucose levels were measured in S426A/S430A mutant and wild-type littermate control
mice that were given either HFD or LFD from weaning until 20 weeks of age. Glucose tolerance
testing was done at 20 weeks of age. Prior to testing, mice were fasted overnight (5:00 PM-
11:00 AM) for 18 hours. Fasting glucose levels were measured and then mice were given an
intraperitoneal (IP) injection of 2g/kg D-glucose (Fisher Scientific, St. Louis, MO). Blood glu-
cose was measured from blood collected via lateral tail vein nick just prior to injection (i.e. fast-
ing blood glucose levels), and 15, 30, 60, and 120 minutes following glucose injection. Blood
glucose levels were measured using a glucometer and Comfort Curve blood glucose test strips
from Accu-Chek (Roche Diagnostics, Indianapolis, IN). Food Intake and Body Weight Separate cohorts of male S426A/S430A and wild-type littermate mice were placed on either a
high fat diet (HFD) (D12451, 45% kcal from fat) from Research Diets Inc. (New Brunswick,
NJ), or a low fat control diet (LFD) (10% kcal from fat), at weaning. Body weight was measured
weekly and food intake was measured every 1–3 days from weaning until 20 weeks of age. Food
intake was calculated as the number of grams of food consumed per kilogram of body weight
per day. Animals All animal care and experimental procedures used in this study were approved by the Institu-
tional Animal Care and Use Committees of Indiana University Bloomington and Penn State
University College of Medicine and conform to the Guidelines of the National Institutes of
Health on the Care and Use of Animals. Mice were maintained on a 12 hour light dark cycle
with lights on at 7AM and lights off at 7PM. Introduction Several studies in mice have demonstrated endocannabi-
noids are increased in diet-induced obesity (DIO). Higher levels of 2-AG have been reported in
the lateral hypothalamus and in visceral adipose tissue of DIO mice [15–17]. Similar to mice,
visceral fat collected from obese patients had higher levels of 2-AG [15]. Blocking 2-AG synthe-
sis with a DAG lipase inhibitor acutely decreased high-fat diet (HFD) intake in mice [18]. Additionally, acute increases in endocannabinoid levels impair clearance of plasma triglycer-
ides by apolipoprotein E [19]. Collectively, these findings suggest that an overactive endocan-
nabinoid system may be obesogenic by influencing food choice and consumption, altering
energy expenditure, and disrupting lipid metabolism. Furthermore, these effects may be exac-
erbated with exposure to high-fat diets. In the present study, we have examined the effects of “chronically” enhanced endocannabi-
noid signaling on body weight regulation, food intake, and glucose homeostasis using S426A/
S430A mutant mice. The S426A/S430A mutants express a knock-in mutation of serines 426
and 430 on the C-terminus of the CB1 receptor to alanines. These residues are putative G pro-
tein-coupled receptor kinase (GRK) targets and are required for β-arrestin2-mediated desensi-
tization of CB1. Expression of these mutations blocked desensitization of CB1 in oocytes,
transfected cells, and in mice [20–24]. In mice, this mutation results in increased acute
responses to endocannabinoids, phytocannabinoids, and synthetic cannabinoids making these
mice a novel model for studying the effects of “over-active” endocannabinoid signaling in vivo
[24]. 2 / 15 PLOS ONE | DOI:10.1371/journal.pone.0160462
August 8, 2016 CB1 Desensitization Does Not Impact Metabolic Homeostasis Δ9-THC-induced Feeding Dose Response Curve Individually housed mice were habituated to HFD for one week prior to testing. Daily food
intake and body weight measurements were recorded to establish a stable HFD consumption. Food consumption was calculated and normalized to body weight as grams of food per 3 / 15 PLOS ONE | DOI:10.1371/journal.pone.0160462
August 8, 2016 CB1 Desensitization Does Not Impact Metabolic Homeostasis kilogram for each mouse. A dose response curve for Δ9-THC-induced feeding was done using
a Latin-square design in which a complete dose-response curve was generated during each test-
ing session across multiple mice (e.g. mice were divided into four groups A thru D and each
group was administered a different IP Δ9-THC dose; 0,1,3 or 10mg/kg). Multiple testing ses-
sions that were spaced at least 72 hours apart were performed to generate a complete dose
response for each mouse. Food was removed one hour before onset of the dark cycle and Δ9-
THC injections were given immediately prior to the dark cycle. Thirty minutes post-injection,
five pellets of HFD were weighed and placed in each cage. Food pellets were weighed 1, 2 and
24 hours after HFD was given. Δ9-THC was dissolved in an 18:1:1 saline:Cremophor:ethanol
vehicle. Food Intake and Body Weight Daily food intake and weekly body weight were measured in S426A/S430A mutant and wild
type mice from weaning until 20 weeks of age. S426A/S430A mutant and wild-type mice given
HFD displayed nearly identical body weights throughout the course of this experiment (Fig
1A). Average body weight of S426A/S430A mutant mice given control LFD also did not differ
from diet-matched wild-type controls at any age (Fig 1B). Daily food intake was calculated and
plotted as average daily food consumption in grams per day for each week. Mice expressing the
S426A/S430A mutation did not exhibit any difference in consumption of either HFD or con-
trol LFD at any age (Fig 2A and 2B). Thus we saw no evidence of abnormal body weight regula-
tion or feeding behavior in S426A/S430A mutant mice compared to wild-type littermate
controls. Similar results were found in male and female mice. JZL195-induced Feeding Mice were habituated to HFD, and food intake and body weight measurements were taken
daily as described above for the Δ9-THC-induced feeding experiments. In this cohort, body
composition was determined using TD-NMR (Bruker minispec LF90II) prior to, and after,
14 days of once-daily (IP) injections of 8mg/kg JZL195. Food consumption and body weights
were measured daily. For each test day, HFD was removed one hour before the onset of the
dark cycle and mice were injected with JZL195 immediately before the start of the dark cycle. Five pellets of HFD were pre-weighed and placed in each cage 90 minutes post-injection. HFD
was measured at 2 hours after placement into the food hopper and again 24 hours later. JZL195
was dissolved in a vehicle comprised of 90% saline, 5% Cremophor, 3.4% ethanol and 1.6%
DMSO. Data Analysis Food intake, body weight, and blood glucose data were analyzed using two-way ANOVA
(genotype x time or genotype x dose) with Bonferroni post-hoc tests. For experiments involv-
ing JZL195 treatment, food intake data were analyzed using two-way ANOVA with repeated
measures. Area under the curve values (AUC) were calculated for blood glucose levels during
glucose and insulin tolerance tests, and for JZL195 treatment. All statistics testing was done
using GraphPad Prism 6 (GraphPad Software, La Jolla, CA). Values are expressed as ± SEM. Glucose Homeostasis Glucose homeostasis was also examined in 20 week old S426A/S430A mutant and wild-type
mice given either HFD or LFD diet starting at weaning. Fasting blood glucose levels were 4 / 15 PLOS ONE | DOI:10.1371/journal.pone.0160462
August 8, 2016 CB1 Desensitization Does Not Impact Metabolic Homeostasis Fig 1. S426A/S430A mutant mice have normal body weight. Body weight was measured each week for
S426A/S430A mutant (red line and circles) and wild-type mice (WT; black line and squares) given HFD (A) or
LFD (B) starting from weaning until 20 weeks of age. Data are expressed as mean ± S.E.M and were
analyzed using two-way ANOVA. The number of animals tested in each group is indicated in parentheses. doi:10.1371/journal.pone.0160462.g001 Fig 1. S426A/S430A mutant mice have normal body weight. Body weight was measured each week for
S426A/S430A mutant (red line and circles) and wild-type mice (WT; black line and squares) given HFD (A) or
LFD (B) starting from weaning until 20 weeks of age. Data are expressed as mean ± S.E.M and were
analyzed using two-way ANOVA. The number of animals tested in each group is indicated in parentheses. doi:10.1371/journal.pone.0160462.g001 doi:10.1371/journal.pone.0160462.g001 measured after an overnight 18 hour fast in S426A/S430A mutants and were not significantly
different than fasted wild-type control mice (Fig 3). Glucose tolerance was measured in S426A/
S430A mutant and wild-type mice given an IP injection of 2 g/kg D-glucose. Glucose tolerance
was not significantly different between HFD-fed S426A/S430A mutant and wild-type mice (Fig
3A). Surprisingly, S426A/S430A given LFD displayed a slight improvement in glucose toler-
ance relative to diet-matched wild-type mice (F1, 60 = 4.265; P = 0.043; Fig 3B). However, area
under the curve (AUC) analysis failed to find any significant differences between the genotypes
(S426A/S430A, 29,821 ± 2634; WT, 37,500 ± 2477, p = 0.12). Fed-Fast Re-Feeding Re-feeding after an overnight fast was examined to assess feeding in a nutrient-deficient state. Consumption of HFD or LFD in S426A/S430A mutant and wild type mice was assessed 1, 4, 6,
and 24 hours after the restoration of food access following an overnight fast. Consumption of
HFD during re-feeding was not changed in S426A/S430A mutants compared to wild-type con-
trol mice for any time point (Fig 4A). Similarly, post-fast re-feeding values for LFD control diet Fig 2. S426A/S430A mutant and wild-type mice consume equal amounts of food. Average daily food
consumption was assessed for each week in S426A/S430A mutant mice (red line and circles) and wild-type
(WT; black line and squares) mice given either HFD (A) or LFD (B) starting from weaning until 20 weeks of
age. Data are expressed as mean ± S.E.M and were analyzed using two-way ANOVA. The number of
animals tested in each group is indicated in parentheses. doi:10.1371/journal.pone.0160462.g002 Fig 2. S426A/S430A mutant and wild-type mice consume equal amounts of food. Average daily food
consumption was assessed for each week in S426A/S430A mutant mice (red line and circles) and wild-type
(WT; black line and squares) mice given either HFD (A) or LFD (B) starting from weaning until 20 weeks of
age. Data are expressed as mean ± S.E.M and were analyzed using two-way ANOVA. The number of
animals tested in each group is indicated in parentheses. 5 / 15 PLOS ONE | DOI:10.1371/journal.pone.0160462
August 8, 2016 CB1 Desensitization Does Not Impact Metabolic Homeostasis Fig 3. S426A/S430A mutant mice given LFD but not HFD exhibit greater glucose tolerance compared
to wild-type controls. Fasting blood glucose (t = 0) and blood glucose levels at 15, 30, 60, and 120 minutes
following intraperitoneal administration of 2g/kg glucose were measured in 20 week old S426A/S430A
mutant (red circles and line) and wild-type (WT) littermates (black squares and line) fed either HFD (A) or LFD
(B). Data are expressed as mean ± S.E.M and were analyzed using two-way ANOVA. The number of animals
tested in each group is indicated in parentheses. Fig 3. S426A/S430A mutant mice given LFD but not HFD exhibit greater glucose tolerance compared g
g
g
g
p
to wild-type controls. Fed-Fast Re-Feeding Fasting blood glucose (t = 0) and blood glucose levels at 15, 30, 60, and 120 minutes
following intraperitoneal administration of 2g/kg glucose were measured in 20 week old S426A/S430A
mutant (red circles and line) and wild-type (WT) littermates (black squares and line) fed either HFD (A) or LFD
(B). Data are expressed as mean ± S.E.M and were analyzed using two-way ANOVA. The number of animals
tested in each group is indicated in parentheses. doi:10.1371/journal.pone.0160462.g003 in S426A/S430A mutant and wild-type mice were also not significantly different at any time
point (Fig 4B). However, S426A/S430A mutant mice trended to eat more than wildtypes at 1
hour, and this value approached significance (p = 0.10, unpaired t-test). Overall, these data
mirror the results of experiments examining ad libitum HFD and LFD consumption under
non-fasting conditions (Fig 2) and suggest that HFD and LFD consumption is not altered in
S426A/S430A mutant mice, even after fasting. Insulin Tolerance Sensitivity to 1 U/kg insulin was examined in HFD and LFD-fed S426A/S430A mutant and
wild-type mice fasted overnight for 18 hours. Insulin tolerance testing was conducted at 30
weeks of age in S426A/S430A mutant and wild-type mice given either HFD or LFD control
diet starting immediately after weaning. Insulin sensitivity was assessed by measuring the
decrease in blood glucose levels occurring after treatment with 1 U/kg insulin. S426A/S430A
mutant and wild-type mice exhibit pronounced decreases in blood glucose levels following Fig 4. S426A/S430A mutants exhibit normal re-feeding following an overnight fast. Re-feeding was
assessed in 20 week old S426A/S430A mutant (red bars) and wild-type (WT; black bars) mice given HFD (A)
or LFD (B) from weaning to 20 weeks of age. Consumption of HFD or LFD was measured in fasted mice at 1,
4, 6, and 24 hours following restoration of food access. Data are expressed as mean ± S.E.M and were
analyzed using two-way ANOVA. The number of animals tested in each group is indicated in parentheses. doi:10.1371/journal.pone.0160462.g004 Fig 4. S426A/S430A mutants exhibit normal re-feeding following an overnight fast. Re-feeding was
assessed in 20 week old S426A/S430A mutant (red bars) and wild-type (WT; black bars) mice given HFD (A)
or LFD (B) from weaning to 20 weeks of age. Consumption of HFD or LFD was measured in fasted mice at 1,
4, 6, and 24 hours following restoration of food access. Data are expressed as mean ± S.E.M and were
analyzed using two-way ANOVA. The number of animals tested in each group is indicated in parentheses. doi:10.1371/journal.pone.0160462.g004 6 / 15 PLOS ONE | DOI:10.1371/journal.pone.0160462
August 8, 2016 CB1 Desensitization Does Not Impact Metabolic Homeostasis Fig 5. S426A/S430A mutants possess normal insulin sensitivity. Fasting blood glucose (t = 0) and blood
glucose levels at 15, 30, 60, and 120 minutes after intraperitoneal administration of 1 U/kg insulin were
measured in HFD (A) or LFD (B) fed S426A/S430A mutant (red circles and line) and wild-type (WT)
littermates (black squares and line) at 30 weeks of age. Data are expressed as mean ± S.E.M and were
analyzed using two-way ANOVA. The number of animals tested in each group is indicated in parentheses. doi:10.1371/journal.pone.0160462.g005 Fig 5. S426A/S430A mutants possess normal insulin sensitivity. Insulin Tolerance Fasting blood glucose (t = 0) and blood
glucose levels at 15, 30, 60, and 120 minutes after intraperitoneal administration of 1 U/kg insulin were
measured in HFD (A) or LFD (B) fed S426A/S430A mutant (red circles and line) and wild-type (WT)
littermates (black squares and line) at 30 weeks of age. Data are expressed as mean ± S.E.M and were
analyzed using two-way ANOVA. The number of animals tested in each group is indicated in parentheses. doi:10.1371/journal.pone.0160462.g005 Fig 5. S426A/S430A mutants possess normal insulin sensitivity. Fasting blood glucose (t = 0) and blood
glucose levels at 15, 30, 60, and 120 minutes after intraperitoneal administration of 1 U/kg insulin were
measured in HFD (A) or LFD (B) fed S426A/S430A mutant (red circles and line) and wild-type (WT)
littermates (black squares and line) at 30 weeks of age. Data are expressed as mean ± S.E.M and were
analyzed using two-way ANOVA. The number of animals tested in each group is indicated in parentheses. doi:10.1371/journal.pone.0160462.g005 doi:10.1371/journal.pone.0160462.g005 administration of 1 U/kg Insulin. However, there were no differences in insulin sensitivity at
any time point between S426A/S430A mutant and wild-type mice subjected to either HFD (Fig
5A) or LFD (Fig 5B). Δ9-THC-induced Feeding Dose Response Curve In order to determine if S426A/S430A mutant mice are more sensitive to the ability of Δ9-THC
to stimulate food-intake, dose-response curves were generated for S426A/S430A mutant and
wildtype mice. Food intake was measured 1, 2, and 24 hours after an injection of Δ9-THC. 1
and 3 mg/kg Δ9-THC stimulated food intake at both 1 hour (F3, 68 = 53.57, p<0.0001) and 2
hours (F3, 68 = 46.48, p<0.0001) after injection (Fig 6A and 6B). 10 mg/kg caused significant
hypolocomotion and thus food intake was minimal 1 and 2 hours after this dose. At 24 hours,
there was a persistent reduction in food intake in mice injected with 10 mg/kg Δ9-THC (Fig
6C; F3, 68 = 9.904; P<0.0001). No differences were observed between intakes of S426A/S430A
mutant and wildtype mice indicating that both genotypes responded similarly to Δ9-THC-
induced food intake. All mice were fasted for 1 hour prior, injected with 0, 1, 3, and 10 mg/kg
Δ9-THC, and presented with food 30 minutes later. Of note, the ability 1 and 3 mg/kg Δ9-THC
to stimulate food intake was not observed unless mice were fasted prior to Δ9-THC treatment. Body weights were measured prior to each injection and they remained unchanged throughout
the study. JZL195-induced Feeding Fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) catalyze the break-
down of AEA and 2-AG, respectively. JZL195 is a dual FAAH/MAGL inhibitor that has previ-
ously be shown to raise endocannabinoid levels, but has yet to be tested for its effect on food
intake in mice [25]. Repeated daily injections of 8 mg/kg JZL195 significantly increased 2 hour
food intake over the 14 day treatment period (Fig 7A) (F3, 14 = 11.89; P = 0.0004), but did not
increase 24 hour food intake levels in either S426A/S430A mutant or wildtype mice (Fig 7B). No differences in food intake were found between the two genotypes at any timepoint. How-
ever, an overall decline in both 2 hour and 24 hour food intakes were observed from day 1 to
day 14. Similar results were obtained with area under the curve analysis where there was a PLOS ONE | DOI:10.1371/journal.pone.0160462
August 8, 2016 7 / 15 CB1 Desensitization Does Not Impact Metabolic Homeostasis significant effect of JZL195 (F1, 14 = 31.24; P<0.0001), but no effect of genotype (Fig 7C). Anal-
ysis of body composition using TD-NMR showed no differences in the initial percent fat mass
f S 26A/S 30A
d
ild
i
F
h
h
i
f
Fig 6. S426A/S430A mutants have similar sensitivity to Δ9-THC-induced feeding compared to wildtype control mice. Individual food intake
values were measured 1, 2 and 24 hours after a 30 minute pretreatment with 0, 1, 3, or 10 mg/kg Δ9-THC. Δ9-THC increased food intake in S426A/
S430A mutant (red circles and line) and wild-type (WT) littermates (black squares and line) 1 hour (A) and 2 hours (B) after injection. 24 hour food intake
is shown in (C). 10 mg/kg Δ9-THC suppressed food intake similarly in both genotypes at all time points (A-C). Mice were fasted for 1 hour prior to
intraperitoneal injections of Δ9-THC. Food intakes are expressed as grams of food per kilogram of body weight (BW). Data are expressed as mean ± S. E.M and were analyzed using two-way ANOVA. The number of animals tested in each group is indicated in parentheses. doi:10.1371/journal.pone.0160462.g006 Fig 6. S426A/S430A mutants have similar sensitivity to Δ9-THC-induced feeding compared to wildtype control mice. Individual food intake
values were measured 1, 2 and 24 hours after a 30 minute pretreatment with 0, 1, 3, or 10 mg/kg Δ9-THC. PLOS ONE | DOI:10.1371/journal.pone.0160462
August 8, 2016 JZL195-induced Feeding JZL195 was
administered daily for 14 consecutive days at a dose of 8 mg/kg. Food intake was increased 2 hours after a 90 minute pretreatment with JZL195 in
S426A/S430A mutant (KI+; red circles and dotted line) and wild-type littermates (WT+; black circles and dotted line) (A), but not after 24 hours (B). Responses from vehicle treated control mice are also shown in A and B (KI-; red triangles and solid line and WT-; black triangles and solid line). Day 4
data was omitted from analysis in A and B. Food intakes are expressed as grams of food per kilogram of body weight (BW). Shown in C is the area under
the curve (AUC) analysis for 2 hour food intake data from A. Shown in D are the values for the total percent of fat mass gained during the 14 day treatment
period. Data are expressed as mean ± S.E.M and were analyzed using two-way ANOVA with repeated measures (A and B) or two-way ANOVA (C and
D). The number of animals tested in each group is indicated in parentheses. doi:10.1371/journal.pone.0160462.g007 JZL195-induced Feeding Δ9-THC increased food intake in S426A/
S430A mutant (red circles and line) and wild-type (WT) littermates (black squares and line) 1 hour (A) and 2 hours (B) after injection. 24 hour food intake
is shown in (C). 10 mg/kg Δ9-THC suppressed food intake similarly in both genotypes at all time points (A-C). Mice were fasted for 1 hour prior to
intraperitoneal injections of Δ9-THC. Food intakes are expressed as grams of food per kilogram of body weight (BW). Data are expressed as mean ± S. E.M and were analyzed using two-way ANOVA. The number of animals tested in each group is indicated in parentheses. doi:10.1371/journal.pone.0160462.g006 significant effect of JZL195 (F1, 14 = 31.24; P<0.0001), but no effect of genotype (Fig 7C). Anal-
ysis of body composition using TD-NMR showed no differences in the initial percent fat mass
of S426A/S430A mutant and wildtype mice. Furthermore, no change in percent fat mass
occurred between the genotypes after 14 days of 8 mg/kg JZL195 treatment (Fig 7D). 8 / 15 PLOS ONE | DOI:10.1371/journal.pone.0160462
August 8, 2016 CB1 Desensitization Does Not Impact Metabolic Homeostasis Fig 7. Repeated injections of JZL195 increases 2 hour food intake similarly in S426A/S430A mutant and wildtype control mice. JZL195 was
administered daily for 14 consecutive days at a dose of 8 mg/kg. Food intake was increased 2 hours after a 90 minute pretreatment with JZL195 in
S426A/S430A mutant (KI+; red circles and dotted line) and wild-type littermates (WT+; black circles and dotted line) (A), but not after 24 hours (B). Responses from vehicle treated control mice are also shown in A and B (KI-; red triangles and solid line and WT-; black triangles and solid line). Day 4
data was omitted from analysis in A and B. Food intakes are expressed as grams of food per kilogram of body weight (BW). Shown in C is the area under
the curve (AUC) analysis for 2 hour food intake data from A. Shown in D are the values for the total percent of fat mass gained during the 14 day treatment
period. Data are expressed as mean ± S.E.M and were analyzed using two-way ANOVA with repeated measures (A and B) or two-way ANOVA (C and
D). The number of animals tested in each group is indicated in parentheses. doi:10.1371/journal.pone.0160462.g007 Fig 7. Repeated injections of JZL195 increases 2 hour food intake similarly in S426A/S430A mutant and wildtype control mice. PLOS ONE | DOI:10.1371/journal.pone.0160462
August 8, 2016 Discussion The endocannabinoid system has been shown to be an important regulator of feeding control
and metabolic function. Anecdotally, the ability of recreationally consumed cannabis to stimu-
late intake of palatable food in humans has been widely reported. In pre-clinical models, treat-
ment with endocannabinoids such as AEA or exogenous cannabinoids such as Δ9-THC can
stimulate feeding behavior [13,26–30]. Synthetic Δ9-THC, known commercially as Dronabinol,
has been approved for the treatment of cachexia and anorexia in AIDS and cancer patients by
the United States Food and Drug Administration [31–33]. 9 / 15 PLOS ONE | DOI:10.1371/journal.pone.0160462
August 8, 2016 CB1 Desensitization Does Not Impact Metabolic Homeostasis Conversely, the CB1-selective inverse agonist, SR141716 (Rimonabant) has been shown to
improve multiple parameters associated with normal metabolic physiology. In rodent models
these beneficial effects include reduced consumption of palatable food, improved glucose
homeostasis, increased insulin sensitivity, increased energy expenditure, and improved levels
of circulating lipids [7–11,34–36]. These results with Rimonabant are supported by CB1 knock
out (KO) mice which display decreased body mass and adiposity, decreased food intake, and
resistance to diet-induced obesity [4,5]. In clinical trials, Rimonabant was found to decrease
body weight and waist circumference, and reduced circulating glucose, leptin, and triglycerides
in obese humans [37]. Further work has demonstrated that circulating AEA and 2-AG are increased in obese
humans suggesting the possibility that the peripheral endocannabinoid system is activated in
obesity [38]. Work has shown that increasing endocannabinoid levels in mice leads to
increased levels of circulating triglycerides due to impaired apolipoprotein E-mediated lipid
clearance [19]. However, relative to the wealth of accumulated research on the effects of phar-
macological and genetic inactivation of CB1, much less is known about the effects of endocan-
nabinoid system activation on metabolism. Therefore, we undertook this study to address this
question using S426A/S430A mutant mice. These mice are more sensitive to endocannabinoids
as well as exogenous plant-derived cannabinoids such as Δ9-THC [24]. To this end, we exam-
ined food intake under both fed and fasted conditions, body weight, glucose clearance follow-
ing a glucose challenge, and insulin tolerance in S426A/S430A mutant mice. Surprisingly, the metabolic phenotype of these mice was modest: S426A/S430A mutant
mice given either HFD or control LFD displayed no differences in food consumption or body
weight compared to wild-type littermate controls. PLOS ONE | DOI:10.1371/journal.pone.0160462
August 8, 2016 Discussion Additionally, S426A/S430A mutant mice
given either HFD or LFD exhibited similar insulin tolerance as diet and age-matched wild-type
controls at 30 weeks of age. The only significant metabolic phenotype detected in S426A/
S430A mutant mice was the observation that mutants given LFD unexpectedly display
improved glucose tolerance compared to diet matched wild-type controls (Fig 3B). However,
this difference in glucose clearance between S426A/S430A mutants and wild-type littermates
did not extend to mutant mice given HFD. Early during the phenotypic characterization of S426A/S430A mutant mice we hypothe-
sized that the relative lack of metabolic phenotype might be due to down-regulation of CB1
and/or decreased amounts of endocannabinoids. This idea was based in part on the finding
that mice lacking CB1 exhibit improved glucose homeostasis relative to wild-type mice. Block-
ade of CB1 receptors has been shown to improve insulin resistance and glucose clearance in
obese rats while CB1 agonists have been found to induce glucose intolerance [39,40]. Therefore,
we wanted to examine glucose tolerance in S426A/S430A mutant mice expressing a form of
CB1 that is more sensitive to endocannabinoids and Δ9-THC. The finding that S426A/S430A
mutant mice exhibit a similar improvement in glucose tolerance compared to wild-type mice
suggested the possibility that the S426A/S430A mutation might result in compensatory
hypoactive endocannabinoid signaling. Our recent work demonstrated normal levels of AEA
and 2-AG throughout most regions of the brain and normal CB1 expression in the hippocam-
pus, striatum, and forebrain of S426A/S430A mutant mice [24]. The number of CB1 [3H]
CP55,940 binding sites (Bmax) was not different in S426A/S430A mutant mice compared to
wild-type mice. Additionally, the Emax for CP55,940-stimulated [35S]GTPγS binding in the spi-
nal cord was also similar in S426A/S430A mutant compared to wild-type mice demonstrating
that the mutant form of CB1 can be activated by agonist to an equivalent extent [24]. However,
lower levels of 2-AG in the cortex and CB1 in the cerebellum were detected in S426A/S430A
mutant mice. This raises the possibility that S426A/S430A mutants might possess region-spe-
cific dysregulation of endocannabinoid signaling in brain regions responsible for metabolic PLOS ONE | DOI:10.1371/journal.pone.0160462
August 8, 2016 10 / 15 CB1 Desensitization Does Not Impact Metabolic Homeostasis regulation such as the hypothalamus. Discussion Alternatively, CB1 receptor desensitization might be less
critical in some regions, a hypothesis that is supported by findings demonstrating that repeated
Δ9-THC administration resulted in significant CB1 desensitization in the prefrontal cortex,
amygdala and hippocampus but no CB1 desensitization was observed in the striatum [41]. Additional studies have also shown that the degree of tolerance to cannabinoids and CB1
desensitization is behavior and region specific [42–44]. A further study demonstrated that the
degree of CB1 desensitization in response to agonist is sex and age-specific [45], raising the pos-
sibility that female S426A/S430A mutant mice might have a distinct metabolic phenotype if
compared to males. Conditional S426A/S430A mutant mice might help in dissecting out any
region specific effects of CB1 receptor hyperactivity on specific metabolic parameters. Our previous work found no differences in baseline body temperature or tail-flick antinoci-
ception in untreated S426A/S430A mutants [24]. The increased cannabinoid response reported
in S426A/S430A mutant mice was detected only when exogenous Δ9-THC was administered
or endocannabinoid levels were increased using an inhibitor of endocannabinoid breakdown
[24]. Therefore, it is possible that changes in metabolic homeostasis such as food intake or
body weight might only occur in the presence of exogenous cannabinoid agonists in S426A/
S430A mutants. In order to address this, we generated a dose-response curve for Δ9-THC-
induced feeding. As expected, lower doses of Δ9-THC significantly and dose-dependently
increased food intake 1 and 2 hours after its administration (Fig 6). However, there was no evi-
dence that S426A/S430A mutant mice were more sensitive to an exogenously administered
cannabinoid agonist in regards to its ability to acutely stimulate food intake. These data collec-
tively suggest that the sensitivity of S426A/S430A mutant mice to the behavioral, metabolic
and other physiological effects such as hypothermia to exogenously administered cannabinoid
agonists are separable. These data also provide further evidence for region-specific dysregula-
tion of endocannabinoid signaling in distinct brain regions as described above. To further explore endocannabinoid-stimulated hyperphagia in S426A/S430A mutant
mice, the dual inhibitor of FAAH and MAGL JZL195 was administered daily for two weeks. To
our knowledge, this is the first study reporting the effects of JZL195 on food intake in mice. However, a prior study demonstrated that injection of the FAAH inhibitor AA5HT into the
ventral striatum of rats stimulated 1 and 4 hour food intake [46]. PLOS ONE | DOI:10.1371/journal.pone.0160462
August 8, 2016 Discussion In our mice, JZL195 signifi-
cantly increased food intake 2 hours after injection suggesting that overall elevation of AEA
and 2-AG endocannabinoid levels acutely stimulates food intake in mice. This dose of 8mg/kg
JZL195 has previously been shown to raise AEA and 2-AG levels 4–6 times above baseline lev-
els, respectively, [25] and thus supports the overarching hypothesis that elevated endocannabi-
noid levels promotes hyperphagia. However, similar to our findings with Δ9-THC, there were
no differences in food intake between S426A/S430A mutant and wildtype control mice suggest-
ing that at least for cannabinoid–induced hyperphagia S426A/S430A mutants were no more
sensitive than wildtype controls to this dose of JZL195. Interestingly, we have previously shown
that doses up to 10mg/kg caused very minimal analgesic and hypothermic responses in both
wildtype and S426A/S430A mutant mice. However, S426A/S430A mutant mice were signifi-
cantly more sensitive to both the analgesic and hypothermic effects of 30 mg/kg JZL195 [24]. Therefore, it is possible that S426A/S430A mutant mice would have increased food intake com-
pared to wildtype controls if higher doses of JZL195 were given. Furthermore, this work pro-
vides additional evidence that the analgesic, hypothermic and food intake effects of
cannabinoids are separable. Previous studies have shown that peripherally-restricted CB1 antagonists are as effective as
rimonabant in reducing body weight, reducing lipids and improving insulin sensitivity in
rodents [47,48] that are due, in part, to direct effects on peripheral tissues including adipose,
liver, skeletal muscle and pancreas [9,49]. A major limitation of our study is that we have not PLOS ONE | DOI:10.1371/journal.pone.0160462
August 8, 2016 11 / 15 CB1 Desensitization Does Not Impact Metabolic Homeostasis measured circulating levels of endocannabinoids or their levels in any of these peripheral tis-
sues in S426A/S430A mutant mice. Moreover, CB1 expression levels and functionality have not
been assessed nor has the levels of endocannabinoid synthetic and degradative enzymes. Thus,
we cannot rule out that compensatory changes in peripheral tissues, including a decrease in cir-
culating endocannabinoid levels, might be attenuating the obesogenic effects that would be
expected in our mutant mice. To summarize, our previous phenotypic characterization of S426A/S430A mutant mice
revealed an exaggerated and prolonged response to both exogenous and endogenous cannabi-
noids [24]. Discussion Due to the extensive literature surrounding the involvement of the endocannabi-
noid system in metabolic control we anticipated that these mutant mice would display
metabolic abnormalities such as increased food intake and body weight and reduced glucose
tolerance and insulin sensitivity. However, we do not observe evidence of metabolic abnormali-
ties in S426A/S430A mutant compared to wild-type controls with the exception of modestly
improved glucose tolerance in mutants given LFD. One possible explanation for the lack of
metabolic phenotype could be that the S426A/S430A mutation isn’t sufficient to confer signifi-
cantly enhanced responses to relatively low basal levels of endocannabinoids in tissues or circu-
lation. Furthermore, the S426A/S430A mutation does not confer additional sensitivity to the
hyperphagia promoted by pharmacologically-induced increases in endocannabinoid signaling
pathways. Therefore, we conclude that this model is likely to have limited utility for studying
endocannabinoid system roles in metabolic regulation. PLOS ONE | DOI:10.1371/journal.pone.0160462
August 8, 2016 Supporting Information S1 File. Fig 1A Data Files. (PZFX) References 1. Hoyert DL, Xu J (2012) Deaths: preliminary data for 2011. Natl Vital Stat Rep 61: 1–51. 2. May AL, Freedman D, Sherry B, Blanck HM (2013) Obesity—United States, 1999–2010. MMWR Sur-
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(PZFX) S2 File. Fig 1B Data Files. (PZF) S2 File. Fig 1B Data Files. (PZF) S2 File. Fig 1B Data Files. (PZF) S3 File. Fig 2A Data Files. (PZF) S4 File. Fig 2B Data Files. (PZF) S5 File. Fig 3A Data Files. (PZFX) S6 File. Fig 3B Data Files. (PZFX) S7 File. Fig 4A Data Files. (PZF) S8 File. Fig 4B Data Files. (PZF) S9 File. Fig 5A Data Files. (PZFX) S10 File. Fig 5B Data Files. (PZF) S11 File. Fig 6 Data Files. (PZFX) S11 File. Fig 6 Data Files. 12 / 15 PLOS ONE | DOI:10.1371/journal.pone.0160462
August 8, 2016 CB1 Desensitization Does Not Impact Metabolic Homeostasis S12 File. Fig 7 Data Files. (PZFX) S12 File. Fig 7 Data Files. Acknowledgments The authors wish to thank Ben Cornett and James Wager-Miller for technical assistance. Writing - original draft: D. J. Morgan CPH D. J. Marcus KM TC. Writing - original draft: D. J. Morgan CPH D. J. Marcus KM TC. Writing - original draft: D. J. Morgan CPH D. J. Marcus KM TC. Conceptualization: D. J. Morgan KM AHR TC. Formal analysis: MJA D. J. Morgan BJD AHR TC. Investigation: D. J. Marcus MZ BJD D. J. Morgan RA. Investigation: D. J. Marcus MZ BJD D. J. Morgan RA. PLOS ONE | DOI:10.1371/journal.pone.0160462
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PMID: 26880114 15 / 15
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Near infra-red labelling and tracking of corneal endothelial cells in-vivo
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Scientific reports
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cc-by
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www.nature.com/scientificreports www.nature.com/scientificreports Near infra‑red labelling
and tracking of corneal endothelial
cells in‑vivo
Maninder Bhogal1,2*, Heng‑Pei Ang1, Shu‑Jun Lin1, Chan N. Lwin1, Khadijah Adnan1,
Gary Peh1,3 & Jodhbir S. Mehta1,3,4,5*
OPEN Following corneal transplantation, there is an initial, rapid decline in corneal endothelial cells
(CECs) following surgery. Direct imaging of post-transplantation endothelial cells is only possible
weeks after surgery and with a limited field of view. We have developed a labelling approach using
1,1′-dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide (DIR) dye solution, that enables
tracking of labelled CECs in vivo for at least 1 month. Initial in vitro optimization, with assessments
of dye concentration on fluorescence, cellular toxicity and cell migration, performed in propagated
primary CECs. Subsequently, in vivo evaluation of cellular labelling was assessed within a rabbit
wound healing model. Finally, real-time visualization of human cadaver donor tissue incubated in
DIR transplanted into rabbits was achieved using a clinical confocal microscope. Results revealed
detectable fluorescence increased with concentration to a plateau of 100 µg/ml, with no toxicity of
CECs at any concentration evaluated. DIR-labelled CECs were detectable in vivo up to 1 month, and
transplanted labelled donor graft could be visualized and were trackable in vivo. Acute endothelial
rejection in 1 rabbit was evidenced by detectable DIR positive cells within the anterior chamber. DIR
imaging allowed for detailed imaging of the transplanted human corneal endothelium, and enabled
non-invasive observation of the corneal endothelial morphology following transplantation. Abbreviations
CECs
Corneal endothelial cells
ECD
Endothelial cell density
CCT
Central corneal thickness
OCT
Optical coherence tomography
EK
Endothelial keratoplasty
NIR
Near-infrared
DIR
1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide
DM
Descemet’s membrane
DMEK
Descemet membrane endothelial keratoplasty
DSAEK
Descemet stripping automated endothelial keratoplasty
ICG
Indocyanine green Abbreviations
CECs
Corneal endothelial cells
ECD
Endothelial cell density
CCT
Central corneal thickness
OCT
Optical coherence tomography
EK
Endothelial keratoplasty
NIR
Near-infrared
DIR
1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide
DM
Descemet’s membrane
DMEK
Descemet membrane endothelial keratoplasty
DSAEK
Descemet stripping automated endothelial keratoplasty
ICG
Indocyanine green After corneal transplantation, a rapid decline in corneal endothelial cell density (ECD) is observed in the early
post-operative period1,2. We have previously shown that iatrogenic cell loss occurs during the preparation3,
storage4, insertion and unfolding of endothelial grafts5. In addition, factors such as air tamponade6,7, post-
operative inflammation and co-morbidities such as glaucoma8,9, are likely to affect early endothelial cell loss. Immediately after transplantation, areas of dead or denuded endothelial cells are present5. A process of cell
migration and restoration of the endothelial monolayer must be completed in order for corneal clarity and
thickness to be restored10. Near infra‑red labelling
and tracking of corneal endothelial
cells in‑vivo
Maninder Bhogal1,2*, Heng‑Pei Ang1, Shu‑Jun Lin1, Chan N. Lwin1, Khadijah Adnan1,
Gary Peh1,3 & Jodhbir S. Mehta1,3,4,5*
OPEN Monitoring of endothelial cells can either be performed directly, by imaging the cells10, or indirectly, by using
measurements of endothelial function: central corneal thickness (CCT) and transparency11. Presently, endothelial
cells are imaged in vivo in the clinical setting using specular or confocal microscopy12, although newer modalities, 1Tissue Engineering and Cell Therapy, Singapore Eye Research Institute, Singapore, Singapore. 2Cornea Unit, Guy’s
& St Thomas’, London, UK. 3Eye‑ACP, Duke-NUS Graduate Medical School, Singapore, Singapore. 4Singapore
National Eye Centre, Singapore, Singapore. 5School of Material Science and Engineering, Nanyang Technological
University, Singapore, Singapore. *email: manibhogal@aol.com; jodmehta@gmail.com Scientific Reports | (2022) 12:6338 | https://doi.org/10.1038/s41598-022-09677-w www.nature.com/scientificreports/ Figure 1. Optimization of DIR concentration in vitro. (a) Graph showing average (mean and standard
deviation) DIR fluorescence in human CECs as a function of concentration. (b) Bar graph comparing toxicity of
DIR at different concentrations in cells derived from the same donor cornea (n = 6) per concentration. Figure 1. Optimization of DIR concentration in vitro. (a) Graph showing average (mean and standard
deviation) DIR fluorescence in human CECs as a function of concentration. (b) Bar graph comparing toxicity of
DIR at different concentrations in cells derived from the same donor cornea (n = 6) per concentration. based on optical coherence tomography (OCT) imaging, are being developed13. Typically, only a small number
of cells, between 30 to 100, are imaged at high magnification14. Image quality can be poor following endothelial
keratoplasty (EK), especially with non-contact methods15. Poor image acquisition from off-axis portions of the
cornea, and the lack of automated methods for stitching multiple images together, mean these methods do not
allow for global assessment of the entire graft16. Consequently, tracking small areas of interest over time can be
very difficult17. In both specular and confocal microscopy, image quality is reliant on a clear corneal stroma,
meaning images obtained through oedematous or scarred corneas are often of poor quality. The presence of an
intra-cameral gas bubble can also impact image quality. As such, it is often not possible to use these methods in
the first 2 weeks following EK, nor is it possible to use these methods to examine endothelial healing following
wounding studies, which result in significant swelling of the cornea5.fi g
gi
g
Wounding studies are commonly used to assess the efficacy of surgical/pharmaceutical interventions aimed
at promoting endothelial healing15–17. In our previous studies, in vivo endothelial wound healing was assessed
using repeated intracameral trypan blue injection10. Near infra‑red labelling
and tracking of corneal endothelial
cells in‑vivo
Maninder Bhogal1,2*, Heng‑Pei Ang1, Shu‑Jun Lin1, Chan N. Lwin1, Khadijah Adnan1,
Gary Peh1,3 & Jodhbir S. Mehta1,3,4,5*
OPEN Whilst this was a reliable and reproducible technique, it is
an invasive procedure that carries the risk of damaging healing endothelial cells or introducing infections18. The
method cannot be used in the early post-operative periods following EK or Descemet’s membrane transplanta-
tion, whilst an intracameral gas bubble remains. Caution needs to be taken early after bubble resolution to avoid
dislodging the recently transplanted material. An additional limitation of the trypan blue technique is that it only
allowed global imaging of endothelial wound healing. This meant animals had to be sacrificed at staggered time
intervals in order to perform histological evaluation of wound healing at a cellular level. p
g
g
We previously developed and validated a method to image endothelial cells, in vivo, using Calcein AM; a
green-fluorescent viability stain5. Whilst capable of global and cell-level imaging in the immediate post-operative
period, our in vitro experiments showed that Calcein AM was rapidly cleared from endothelial cells indicating
that any useful fluorescence signal will be extinguished by 24 h post-incubation. As such, it is not useful for
longer-term tracking of endothelial cells in vivo. The fate of the transplanted cells can only truly be determined if
methods to quantify transplanted corneal endothelial cell death immediately post-operatively can be combined
with techniques to track endothelial cells through early post-operative phase, which we define as 1 month based
on previous reports19,20. p
p
In this study, we describe a technique for labelling and tracking corneal endothelial cells in vivo, both globally
across the entire cornea and at an individual cell level. We compared these in vivo images to immuno-histology
following animal sacrifice. We show this technique is suitable for tracking both native and transplanted endothe-
lial cells and can be used in wound healing studies as well as xenotransplantation of human EK grafts into rabbits. Results
i
i Optimizing DIR concentration in vitro. Average DIR fluorescence increased with concentration in a
linear-quadratic fashion. Comparison of average fluorescence levels at different doses, derived from macroscopic
images taken from individual wells, revealed a significant difference between cells stained with 50 µg/ml and
100 µg/ml (repeated measures ANOVA, corrected p < 0.0001, N = 15 per group) but not between 100 and 200 µg/
ml (p = 0.78) (Fig. 1a). Based on these findings a dose of 100 µg/ml was chosen for in vitro subsequent labelling. No significant differences were found on Calcein AM/ethidium homodimer toxicity assessment between DIR at
any concentration and matched controls from the same donor (N = 6) (Fig. 1b). Wound healing kinetics follow-
ing standardized scratch wounding in HCEC’s were similar between control and treated groups (Fig. 2). There
was no significant difference in wound area remaining at any time point (n = 6, paired t-test) (Fig. 2d). In vivo labelling of rabbit endothelial cells. Direct injection of DIR diluted in the rabbit aqueous to
a final concentration of 100 µm/ml was successful in labelling rabbit endothelial cells. No adverse effects were
observed following DIR injection, with no significant inflammation (conjunctival hyperemia, iris vascular dila-
tion, anterior chamber cellular activity) seen at any time point. Slit lamp microscopy and infra-red imaging https://doi.org/10.1038/s41598-022-09677-w Scientific Reports | (2022) 12:6338 | www.nature.com/scientificreports/ Figure 2. Wound healing in DIR-labelled primary human CECs. (a) Human CECs were plated and allowed to
stabilize. A standard scratch wound was made with a 10 µl pipette tip. Th original wound area was delineated
(yellow border) and the wound area measured. Time-lapse images were taken every hour after wounding. (b)
The wound area was reassessed serially (yellow border), and the percentage wound area remaining calculated. (c) Imaging was continued until complete closure of the scratch wound was observed. (d) The wound area
remaining (mean and standard deviation) was compared between paired CEC controls vs CEC exposed to DIR
every 3 h for 15 h. (N = 6, paired t-test). No significant difference between controls and DIR stained cells was
seen at any time point Scale bar: 250 µm Figure 2. Wound healing in DIR-labelled primary human CECs. (a) Human CECs were plated and allowed to
stabilize. A standard scratch wound was made with a 10 µl pipette tip. Th original wound area was delineated
(yellow border) and the wound area measured. Results
i
i Time-lapse images were taken every hour after wounding. (b)
The wound area was reassessed serially (yellow border), and the percentage wound area remaining calculated. (c) Imaging was continued until complete closure of the scratch wound was observed. (d) The wound area
remaining (mean and standard deviation) was compared between paired CEC controls vs CEC exposed to DIR
every 3 h for 15 h. (N = 6, paired t-test). No significant difference between controls and DIR stained cells was
seen at any time point. Scale bar: 250 µm. on day 1 showed no change in corneal clarity (Fig. 3a). DIR stained endothelial cells were visible on infra-red
imaging, with significant cell-to-cell variation in staining intensity observed (Fig. 3b). There was no increase in
corneal thickness (Fig. 3c,f) at any time point after surgery, with CCT remaining < 400 µm throughout the obser-
vation period. At day 28, all corneas remained clear (Fig. 3d). DIR labelled cells were still visible, but with some
reduction in observable fluorescence (Fig. 3e). DIR uptake was prominent in the iris, meaning that pupil dilation
was necessary to reduce background fluorescence in subsequent examinations. Background fluorescence was
further reduced by imaging using the higher magnification lenses, as these reduced the depth of focus (Fig. 3e). In vivo corneal wounding: comparison between in vivo DIR imaging, confocal microscopy and
immunohistology. DIR staining was compared to conventional confocal microscopy and post-sacrifice
immunohistology in 6 rabbits (2 sacrificed at day 3, 2 at day 5 and 2 at day 28 post wounding). By using additional
magnifying lenses attached to the HRA/Spectralis, it was possible to image individual DIR stained endothelial
cells, although significant variability in the fluorescence of adjacent endothelial cells was observed (Fig. 4). Dur-
ing early wound healing (day 3), elongated, migrating endothelial cells were seen (Fig. 4a). This same morphol-
ogy was observed on immunofluorescence staining performed after sacrifice of the same animal (Fig. 4d). At
this stage, no endothelial imaging was possible with conventional confocal microscopy (Fig. 4g). There was https://doi.org/10.1038/s41598-022-09677-w Scientific Reports | (2022) 12:6338 | www.nature.com/scientificreports/ Figure 3. The effect of DIR on rabbit corneal endothelium following intracameral injection in vivo. DIR (final
concentration 100 µg/ml) was injected into the anterior chamber of 3 rabbits to determine if this had any side-
effects. (a) Photomicrograph of the rabbit anterior segment taken 1 day post DIR intracameral injection. Results
i
i Conventional confocal imaging was still unable to produce useful images, largely
because applanation of the cornea resulted in undulations of the posterior corneal surface (Fig. 4h). After initial
wound closure, continuing maturation of central endothelial cells was observed. As time progressed, cell borders
became narrower and cells became more circular (Fig. 4c). The regular endothelial mosaic observed with DIR
staining at day 28 corresponded to small, largely hexagonal cells, with a continuous ring of ZO-1 staining seen
on immunofluorescence (Fig. 4f). Conventional in-vivo confocal images could be obtained from day 14 onward
as seen at day 28 (Fig. 4i). Analysis at day 28 showed no difference in cell circularity (0.71 vs 0.74, t-test, p = 0.87)
or the number of hexagonal cells as assessed by histology and DIR imaging. (50.9% (histology) vs 49.1% (HRA/
Spectralis), chi-squared, p = 0.9). In vivo corneal wounding: comparison between in vivo DIR imaging and trypan blue stain‑
ing. Good agreement was seen between macroscopic, non-invasive DIR imaging and intracameral trypan
blue staining, both at the time of wound creation (Fig. 5a,b) and at day 3 after wounding (Fig. 5c,d). Importantly,
it was possible to acquire these images through corneas that were thickened or irregular in shape, as observed
on the simultaneously acquired OCT (Fig. 5e). Bland–Altman plots were produced to compare the area and
circularity of wounds (Fig. 6). Good agreement was seen between both methods, with less than 2% difference in
area seen in any sample (Fig. 6a). Circularity was within 4% for all samples (Fig. 6b). In vivo tracking of migrating endothelial cells following endothelial wounding in the rab‑
bit. The wounding of the rabbit corneal endothelium in vivo was followed for 14 days using infra-red (Fig. 7a–
d), OCT (Fig. 7e–h), and DIR imaging (Fig. 7i–l) with their respective magnified region (Fig. 7m–p). Complete
closure of an 8 mm endothelial scrape wound occurred by day 5 (Fig. 7k–o). CCT (as assessed by OCT) returned
to pre-wound levels by day 14 in all cases (Fig. 7h). These findings are in keeping with our previously published
results10 obtained from scrape wounding in unstained corneas. Taken together, these findings suggest that DIR
staining does not have a deleterious impact on endothelial healing or migration in vivo. Labelling of ex vivo human corneas. Double staining using Calcein AM and DIR allowed multiplexed
image acquisition of donor human corneas within the donor viewing chamber (Fig. Results
i
i The
cornea remains transparent, with easy visualization of the iris details. (b) Infra-imaging using, the ICG filters on
the Spectralis HRA, shows variable fluorescence across the endothelium, with speckled hyperfluorescent seen
at areas of increased cellular dye uptake. (c) OCT image showing an optical cross-section of the central rabbit
cornea. No increase in corneal thickness or inflammation is seen (CCT. Images from the same rabbit cornea
taken on day 28 post DIR injection. (d) A photomicrograph of the rabbit anterior segment showing the cornea
remains clear. (e) Infra-imaging using the ICG filters shows residual fluorescence at day 28, although maximal
fluorescence intensity is reduced. (f) An OCT image taken at day 28 showing corneal thickness remains normal
and unchanged throughout the study period (CCT 377 µm). Figure 3. The effect of DIR on rabbit corneal endothelium following intracameral injection in vivo. DIR (final
concentration 100 µg/ml) was injected into the anterior chamber of 3 rabbits to determine if this had any side-
effects. (a) Photomicrograph of the rabbit anterior segment taken 1 day post DIR intracameral injection. The
cornea remains transparent, with easy visualization of the iris details. (b) Infra-imaging using, the ICG filters on
the Spectralis HRA, shows variable fluorescence across the endothelium, with speckled hyperfluorescent seen
at areas of increased cellular dye uptake. (c) OCT image showing an optical cross-section of the central rabbit
cornea. No increase in corneal thickness or inflammation is seen (CCT. Images from the same rabbit cornea
taken on day 28 post DIR injection. (d) A photomicrograph of the rabbit anterior segment showing the cornea
remains clear. (e) Infra-imaging using the ICG filters shows residual fluorescence at day 28, although maximal
fluorescence intensity is reduced. (f) An OCT image taken at day 28 showing corneal thickness remains normal
and unchanged throughout the study period (CCT 377 µm). no difference in cell circularity measured from DIR and immunofluorescence images, with an average value
of 0.51 ± 0.14 for histology derived circularity and 0.58 ± 0.12 for DIR imaging (t-test, p = 0.34). After wound
closure, a confluent monolayer of cells with broad cell borders was seen with in vivo DIR imaging (Fig. 4b). This corresponded to confluent cells with incomplete tight junction formation, as assessed by ZO-1 immuno-
fluorescence staining (Fig. 4e). Results
i
i 8a,b), as well as immediately
post operatively inside the rabbit eye (Fig. 8c,d). Initially, staining with DIR showed large degrees of variation,
with adjacent areas of the corneal endothelium showing markedly different degrees of fluorescence (Fig. 8b,d,
yellow arrows). Calcein AM staining was able to detect large areas of cell loss (Fig. 8c, blue arrow) and differenti- https://doi.org/10.1038/s41598-022-09677-w Scientific Reports | (2022) 12:6338 www.nature.com/scientificreports/ Figure 4. In vivo tracking of corneal endothelial cells. (a–c) The top row shows DIR stained rabbit corneal
endothelial cells at day 3, 5 and 28 post wounding, imaged in vivo. (d–f) The middle row shows the same
cells imaged with immunohistology at the same time points, after the animal was sacrificed. (g–i) The bottom
row shows images acquired with conventional in vivo confocal microscopy. Each column represents images
taken from the same cornea at the same time point (a) In vivo DIR imaging at day 3 showing migrating
endothelial cells at the leading edge of the wound. Cells show an elongated morphology with incomplete
monolayer formation. Marked variation in DIR uptake is seen between adjacent endothelial cells, with some
staining intensely (b) At day 5, a complete monolayer is seen with high degrees of pleomorphism and broad
intercellular junctions. (c) By day 28 cells have regained a more regular morphology and tight junctions have
become narrower. Fixed cells stained with phalloidin, ZO-1 and Hoechst show similar morphological features to
in vivo DIR imaging. (d) At day 3 cells are elongated with no junctional ZO-1 staining. (e) At day 5 a complete
monolayer is seen with incomplete junctional ZO-1 staining and pleomorphic cells. (f) At day 28, regular cells
with mature borders are seen. (g) Conventional in vivo confocal imaging failed to acquire useful images at day
3, and (h) day 5. (i) At day 28 a stable monolayer of cells was visible on conventional confocal microscopy, but
nuclear morphology was more prominent than cell border morphology. Scale bar: 100 µm. Figure 4. In vivo tracking of corneal endothelial cells. (a–c) The top row shows DIR stained rabbit corneal
endothelial cells at day 3, 5 and 28 post wounding, imaged in vivo. (d–f) The middle row shows the same
cells imaged with immunohistology at the same time points, after the animal was sacrificed. (g–i) The bottom
row shows images acquired with conventional in vivo confocal microscopy. Results
i
i (a) Intra-operative trypan
blue staining shows bare DM where endothelial cells have been removed by scraping. (b) The same eye was
imaged immediately post-operatively. The DIR imaging of the area of denuded endothelial cells shows good
concordance with intraoperative trypan blue (yellow outline), where DIR fluorescence is blocked by the
nictitating membrane. At 3 days post-wounding, significant closure of the wound can be seen. (c) Injection of
intracameral trypan blue stains a small residual area bare DM (yellow outline). A feathered edge is observed,
representing migrating endothelial cells. (d) Infrared imaging of the same eye shows the same area of bare DM,
surrounded by fluorescent endothelial cells (yellow outline). (e) An OCT image of the cornea at day 3 post
wounding confirms DIR images are successfully acquired through thickened and irregular corneas. Scale bar: Figure 5. Comparison between in vivo DIR imaging and trypan blue staining. (a) Intra-operative trypan
blue staining shows bare DM where endothelial cells have been removed by scraping. (b) The same eye was
imaged immediately post-operatively. The DIR imaging of the area of denuded endothelial cells shows good
concordance with intraoperative trypan blue (yellow outline), where DIR fluorescence is blocked by the
nictitating membrane. At 3 days post-wounding, significant closure of the wound can be seen. (c) Injection of
intracameral trypan blue stains a small residual area bare DM (yellow outline). A feathered edge is observed,
representing migrating endothelial cells. (d) Infrared imaging of the same eye shows the same area of bare DM,
surrounded by fluorescent endothelial cells (yellow outline). (e) An OCT image of the cornea at day 3 post
wounding confirms DIR images are successfully acquired through thickened and irregular corneas. Scale bar:
1 mm. rescence for the entire study period. DIR images of the graft were taken at the pre-implantation (Fig. 9a) and
post-implantation (Fig. 9b), and at intervals from day 1 to day 28 (Fig. 9c–e) in conjunction with infra-red
(Fig. 9f–j), and OCT (Fig. 9k–o). The yellow bordered figures (Fig. 9p–q) is the magnified area denoted by the
yellow arrows (Fig. 9a,b,d,e), whereas the blue bordered figures (Fig. 9r,s) is the magnified area denoted by the
blue arrows (Fig. 9c–e). At day 1 post implantation, areas of bare DM devoid of endothelial cells were visible
(Fig. 9c; red arrows). A triangular orientation mark (yellow arrow) used to confirm the correct position was also
clearly visible. Results
i
i Each column represents images
taken from the same cornea at the same time point (a) In vivo DIR imaging at day 3 showing migrating
endothelial cells at the leading edge of the wound. Cells show an elongated morphology with incomplete
monolayer formation. Marked variation in DIR uptake is seen between adjacent endothelial cells, with some
staining intensely (b) At day 5, a complete monolayer is seen with high degrees of pleomorphism and broad
intercellular junctions. (c) By day 28 cells have regained a more regular morphology and tight junctions have
become narrower. Fixed cells stained with phalloidin, ZO-1 and Hoechst show similar morphological features to
in vivo DIR imaging. (d) At day 3 cells are elongated with no junctional ZO-1 staining. (e) At day 5 a complete
monolayer is seen with incomplete junctional ZO-1 staining and pleomorphic cells. (f) At day 28, regular cells
with mature borders are seen. (g) Conventional in vivo confocal imaging failed to acquire useful images at day
3, and (h) day 5. (i) At day 28 a stable monolayer of cells was visible on conventional confocal microscopy, but
nuclear morphology was more prominent than cell border morphology. Scale bar: 100 µm. ate between living and dead individual cells and cells clusters, when a magnifying objective lens was attached
to the imaging set-up (Fig. 8e, red arrow). DIR staining was not able to differentiate between living cells and
dead endothelial cells that remained attached to DM (DIR +’ve/Calcein am −‘ve) (Fig. 8d,f). As predicted by our
in vitro assessment5, Calcein AM fluorescence diminished quickly, and no useful fluorescent images could be
obtained at 24 h post-implantation (Fig. 8g). Conversely, DIR fluorescence tended to become more uniform by
24 h post graft insertion. At 24 h post implantation, macroscopic areas devoid of cells could be seen, and cor-
responded to the areas of cell death seen immediately following transplantation using Calein AM (Fig. 8h, blue
arrow c.f. Calcein AM Fig. 8c). This suggests that non-vial endothelial cell have been ejected from the transplant
by this stage. Tracking transplanted human endothelial cells in vivo. Human EK grafts labelled with DIR were
implanted into the rabbit anterior chamber. Stained endothelial cells within the graft maintained strong fluo- https://doi.org/10.1038/s41598-022-09677-w Scientific Reports | (2022) 12:6338 | www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 5. Comparison between in vivo DIR imaging and trypan blue staining. Results
i
i Over the first week, cells migrated to cover the areas of the bare DM (Fig. 9c; red arrows). Areas of https://doi.org/10.1038/s41598-022-09677-w Scientific Reports | (2022) 12:6338 | www.nature.com/scientificreports/ Figure 6. Bland–Altman Plots comparing in vivo wound size as assessed by DIR staining and intra-cameral
trypan blue injection. Cornea endothelial wounds were measured using trypan blue and DIR in 6 eyes. The
dotted black line represents the average bias, with the wound area measured by trypan blue tending to be
marginally larger. The red dotted lines represent the 95% percent confidence interval. (a) Residual wound area
as measured by DIR and trypan blue was within 0.3 mm for all samples. (b) Residual wound circularity as
measured by DIR and trypan blue was within 4% for all individual samples. Figure 6. Bland–Altman Plots comparing in vivo wound size as assessed by DIR staining and intra-cameral
trypan blue injection. Cornea endothelial wounds were measured using trypan blue and DIR in 6 eyes. The
dotted black line represents the average bias, with the wound area measured by trypan blue tending to be
marginally larger. The red dotted lines represent the 95% percent confidence interval. (a) Residual wound area
as measured by DIR and trypan blue was within 0.3 mm for all samples. (b) Residual wound circularity as
measured by DIR and trypan blue was within 4% for all individual samples. cell migration from the donor to host could be seen on DIR imaging. In the case illustrated, this resulted in loss
of the straight edge of the triangular orientation mark (yellow arrows). Following animal sacrifice, endothelial
cells were stained with anti-human nuclear antibodies (Fig. 9q,s, FITC channel). Migration of human endothe-
lial cells (stained green) across the graft border was visible (Fig. 9q) and the pattern of donor cell distribution
showed good concordance between DIR imaging and immunohistological assessment (Fig. 9p,q). Transfer of
fluorescence to neighboring rabbit endothelial cells was not observed. Areas of rabbit endothelial migration onto
the host DM were seen in DMEK cases (Fig. 9p,r) but not in DSAEK. These cells were distinguishable, by the lack
of staining with both DIR (Fig. 9p,r) and anti-human nuclear antibodies (Fig. 9q,s). g
g
p
g
q
In one animal that had DMEK, clinical features of rejection (corneal oedema, ciliary injection) was observed
at day 28 (Fig. 10). Compared to day 21 images (Fig. Results
i
i 10a,b), DIR imaging showed central loss of fluorescence
at lower magnification (Fig. 10c) and patchy attachment of small, circular endothelial cells at higher powers
(Fig. 10d). This was associated with the onset of circulating, DIR positive, cells in the anterior chamber (Video
S1). An anterior chamber tap confirmed the presence of cellular material, but the yield was too low to confirm
cell origin or DIR positivity using FACS/immunofluorescence. High resolution cell imaging. By attaching the Rostock module (Heidelberg Engineering, Heidelberg,
Germany) to the HRA/Spectralis, we were able to acquire detailed, high-resolution in vivo images. This allowed
us to observe subtle biological processes at a cellular level, including nuclear fragmentation and cell detachment
in both rabbit (Fig. 11a,b) and human (Fig. 11c) corneal endothelium; podocyte extension from neighboring
cells (Fig. 11d); nuclei in anaphase (Fig. 11e) and daughter cell formation (Fig. 11f). Discussion At higher magnification levels,
migrating cells closing the wound at the edge are seen (n). By day 5, although some residual corneal oedema
and thickening persists on OCT imaging (g), complete wound closure can be observed (k,o). By day 14, corneal
thickness has returned to normal (h) and a stable layer of DIR positive cells was observed with macroscopic (i)
and higher magnification imaging (p). Figure 7. In vivo tracking of endothelial cells at pan-corneal and single-cell level. Images taken from an
individual, DIR stained rabbit cornea. Following standard endothelial wounding, infra-red (a–d), OCT (e–h)
and DIR images (i–p) were acquired using the HRA/Spectralis. Use of the anterior segment lens supplied
by the manufacturer allowed macroscopic imaging of the whole cornea. By coupling additional lenses to the
infinity optics retinal lens, higher magnification imaged could be captured (m–p). The images in the bottom
row correspond to the areas seen in the white square outline in the macroscopic images. By sequentially
imaging with increasing magnification, it was possible to track areas of interest. Immediately after scraping no
DIR positive cells are seen in the wound area (i,m). At day 3 the cornea is still significant thicker that pre-
wounding (f). The wound area can be seen to be much smaller (j, white box). At higher magnification levels,
migrating cells closing the wound at the edge are seen (n). By day 5, although some residual corneal oedema
and thickening persists on OCT imaging (g), complete wound closure can be observed (k,o). By day 14, corneal
thickness has returned to normal (h) and a stable layer of DIR positive cells was observed with macroscopic (i)
and higher magnification imaging (p). this is the first time biological processes such as healing of iatrogenic wounds and migration of endothelial cells
from donor to host have been observed in vivo. At the macroscopic level, we found DIR imaging to be comparable to repeated trypan blue injection when
assessing endothelial wound healing following standard injury; a model commonly used to assess pharmacologi-
cal therapies aimed at enhancing endothelial regeneration22–24. High magnification assessment of the healing
edge of the wound showed that comparable morphological information could be obtained from in vivo imag-
ing as compared to immuno-histology following animal sacrifice. A temporal relationship between closure of
endothelial wounds, as observed by DIR imaging, and restoration of corneal clarity/CCT was also seen in all
cases. Discussion Tracking of CECs in vivo is not widely performed to date due to a lack of suitable imaging techniques and paucity
of data on labelling efficiency, duration of fluorescence, general toxicity, and effects of labelling on functional-
ity of transplanted tissue21. The aim of this study was to determine whether NIR dye, and specifically for this
study, DIR, could be used to label and track CECs, in vivo, without impacting cell viability or cellular function. We began by assessing the effect of DIR staining on wound healing, cell migration and cell viability, in vitro,
using cultured primary human CEC’s. At the concentrations required for in vivo imaging, DIR stained CECs
migrated at the same rate as unlabeled CECs. At 24 h post-labelling, there was no significant difference in cell
death between labelled and unlabeled cells. Taken together, these results suggest that DIR was a viable candidate
for use as a tracker in CECs. We subsequently assessed whether use of DIR had any deleterious effects in vivo. A single intracameral DIR injection was sufficient to label rabbit endothelial cells and was not associated with
any apparent inflammation or acute disturbance of corneal endothelial function. We subsequently used DIR
imaging to track the endothelial cells on EK transplants from human tissue. We found it was possible to obtain
high quality images of human endothelial cells within the anterior chamber following EK. To our knowledge, Scientific Reports | (2022) 12:6338 | https://doi.org/10.1038/s41598-022-09677-w www.nature.com/scientificreports/ Figure 7. In vivo tracking of endothelial cells at pan-corneal and single-cell level. Images taken from an
individual, DIR stained rabbit cornea. Following standard endothelial wounding, infra-red (a–d), OCT (e–h)
and DIR images (i–p) were acquired using the HRA/Spectralis. Use of the anterior segment lens supplied
by the manufacturer allowed macroscopic imaging of the whole cornea. By coupling additional lenses to the
infinity optics retinal lens, higher magnification imaged could be captured (m–p). The images in the bottom
row correspond to the areas seen in the white square outline in the macroscopic images. By sequentially
imaging with increasing magnification, it was possible to track areas of interest. Immediately after scraping no
DIR positive cells are seen in the wound area (i,m). At day 3 the cornea is still significant thicker that pre-
wounding (f). The wound area can be seen to be much smaller (j, white box). www.nature.com/scientificreports/ www.nature.com/scientificreports/ We have previously shown that rabbit endothelial cells are capable of rapidly migrating over bare DM with
subsequent restoration of corneal clarity and thickness to pre-transplantation levels10. As such, it is important
to be certain that any restoration of function is attributable to the transplanted cells and not a consequence of
cellular regeneration in models where in vivo endothelial replication occurs25. Until now, detailed assessment of
wound healing at different time points has relied either on repeated surgical interventions, in the case of trypan
blue injection10, or on the sacrifice of animals at multiple time points26. Our assessments showed that a DIR fluo-
rescence signal, allowing for high quality image acquisition, was retained for at least 28 days and in subsequent
experimentation we have found fluorescence to be retained up to 60 days (data not shown). pl
p
y (
)
With high resolution imaging, biological processes typically observed on histological examination could be
seen in vivo; including cell spreading, apoptosis and cell division. In one animal, a reduction in corneal clarity,
together with a development of corneal edema and an increase in corneal thickness occurred between days 21
and 28. DIR imaging showed numerous endothelial cells had detached from the DM and were freely floating
in the aqueous. The cells remaining attached to DM displayed features consistent with an acute insult13. In the
setting of xenotransplantation, these features were consistent with acute graft rejection27,28. In keeping with this,
small DIR positive cells, consistent with previous reports of inflammatory cells, were observed interspersed with
the endothelial cells were seen in both in the rabbit endothelial monolayer and transplanted human endothelial
cells29.t Graft rejection remains a leading cause of transplant failure. Currently, models of rejection rely on indirect
measures of endothelial function, and it is not always possible to separate rejection from other forms of endothe-
lial trauma11,30. Methods of imaging inflammatory cell changes that proceed acute rejection in vivo will allow a
greater understanding of the mechanisms at play31. Being able to assess the degree of cell death occurring during
a rejection episode will provide information on the likelihood of recovery following rejection and effectiveness
of anti-rejection strategies.hhil j
g
The use of NIR markers for tracking has several advantages. These tracers benefit from very low autofluores-
cence at NIR wavelengths and have excellent tissue penetration. www.nature.com/scientificreports/ This combination gives a high signal-to-noise
ratio and allows non-invasive tracking of cells through the skin in organs such as the spleen or heart32. NIR tracers
have recently been used in surgical trials to aid tumor clearance33 and in clinical ophthalmic trials to assess cell
loss in patients with early glaucoma34. Using an 800 nm band pass, sufficient emitted light is captured for high
quality imaging, and this is possible even through opaque corneas with significant edema. The NIR dye, indo-
cyanine green (ICG), has been used in ophthalmology for over 5 decades. This means that dedicated equipment
for imaging of NIR tracers in the eye are widely available in clinical and research settings. Our imaging protocol
uses clinical equipment, designed for ICG imaging, with minimal adjustments. Given that DIR staining is a simple, non-toxic, one step process, it is possible that this method could be
applied to graft tissue destined for human use if a good manufacturing practice form of the dye were made
available. For organ cultured tissue, the point at which graft tissue is transferred to dextran containing media
may provide an ideal opportunity for this35. It is possible that DIR may also be useful to track other cells used in
corneal regenerative therapy such as limbal epithelial21 or stromal stem cells36.lh Our study showed that DIR fluorescence was initially quite heterogenous. This meant multiplexing with the
viability dye, Calcein AM, was needed to assess immediate post-op graft viability. Fluorescence peaked at 24 h
post transplantation making imaging easier, although some heterogeneity remained, meaning appropriately
exposing the images was challenging. It may be possible to improve the use of DIR by combining several images
taken at different sensitivities (iso settings); high dynamic range imaging37. f
g
g
y
g
g
g
We believe that DIR imaging provides a powerful new tool for studying CECs in vivo. More detailed informa-
tion about endothelial cell behavior following transplantation will improve our understanding of phenomena
such as accelerated cell loss in high risk grafts and may lead to targeted therapeutics and improved graft survival. Its use in a research setting should ultimately lead to a reduction in the number of research animals needed and
there is a potential for easy adoption in the clinical setting. Further studies confirming the exact length of time
that DIR fluorescence persists in-vivo are warranted. Discussion In vivo wound healing kinetics were similar to those we previously reported in unlabeled cells10. Images
could be acquired through opaque/edematous corneas, something not possible with conventional confocal
imaging. Combined, these findings suggest that DIR staining has no significant effect on endothelial function or
healing and allows continuous, in vivo, assessment at all time points post-wounding. Assessment of endothelial
transplants stained with DIR showed no diffusion of DIR across cell membranes from adjacent endothelial cells,
meaning that staining of rabbit endothelial cells did not occur following xenotransplantation with human DMEK
grafts (Fig. 8). This allowed for a clear distinction between transplanted human endothelial cells and native rabbit
endothelial cells, even if cells had migrated on or off the transplant itself. https://doi.org/10.1038/s41598-022-09677-w Scientific Reports | (2022) 12:6338 Methods There is marked variation in fluorescence across the graft meaning portions become either over or under
exposed on global imaging. The yellow arrow points to an area of high DIR fluorescence. Comparison with
the Calcein AM staining shows that most of the graft, including this area is cover by viable cells. (c) Global
Calcien AM imaging of the graft, in vivo, immediately following transplantation shows a new area of cell death
attributable to implantation trauma (blue arrow) (d) Global DIR imaging of the graft, in vivo, immediately
following transplantation shows the same variation of fluorescence (e) High magnification imaging acquired
by coupling a ×40 air objective microscope lens to the retinal imaging lens of the HFA/Spectralis. Individual
Calcein AM negative cells are seen. (d) DIR imaging using the same lens shows DIR fluorescence in the cells
that appeared non-fluorescent on the global image, suggesting the level of fluorescence is variable, but present
across the entire graft. At this time point, comparison between DIR and Calcein AM imaging shows DIR does
not differentiate between living and dead, but attached, cells. (f) At 24 h post transplantation, Calcein AM
fluorescence has diminished to the point where now useful images can be obtained. (h) By contrast, at 24 h
post-implantation, DIR fluorescence has become more uniform and areas corresponding to pre-identified dead
cells can be seen to be devoid of DIR staining, suggesting the cells have been ejected from the monolayer (blue
arrow). Scale bar (a–d and g–h): 1 mm, Scale bar (e,f): 250 µm. by a single surgeon (MB). Transplant grade tissue with endothelial cell counts of > 2200 cells/mm2 and a storage
time < 14 days in Optisol-GS was used for all experimentation. y a single surgeon (MB). Transplant grade tissue with endothelial cell counts of > 2200 cells/mm2 and a storage
ime < 14 days in Optisol-GS was used for all experimentation. Research animals. Animals procured for this study were treated in accordance with the ARVO State-
ment for Use of Animal in Ophthalmic and Visual Research as well as the ARRIVE guidelines40, and all experi-
ments involving these animals were approved by the Singhealth Institutional Animal Care and Use Commit-
tee (IACUC; 2016/SHS/1212), Singapore. Interventions were performed in New Zealand white rabbits aged
16–20 weeks, weighing between 3 and 3.5 kg. Methods All surgical procedures and clinical evaluations were performed in
a single eye of each rabbit under general anesthesia from intra-muscular xylazine 5 mg/kg (Troy Laboratories,
Smithfield, Australia) and ketamine 40 mg/kg (Parnell Laboratories, Alexandria, Australia) with additional topi-
cal anesthesia (lidocaine 1%, Bausch and Lomb, Rochester) as previously described10.i p
y
Animals underwent phacoemulsification at least 1 week prior to endothelial keratoplasty to deepen the
anterior chamber. Vancomycin was used in the irrigating fluid (20 µg/ml) for surgical procedures and rabbits
received subconjunctival dexamethasone (1 mg) and gentamcin (0.4 mg) at the end of all surgical procedures. Prior to EK, rabbits were injected with intravenous unfractionated heparin (1000 units in 1 ml of normal
saline). Heparin was also added to the balanced salt solution used to irrigate the eye at a concentration of 50
units/ml. For all experiments, rabbits were examined daily for the first 5 days and then on days 7, 14, 21, and 28. Examinations consisted of slit lamp biomicroscopy, tonometry, optical coherence tomography, confocal fluo-
rescence imaging using the Heidelbreg HRA/Spectralis (Heidelberg Engineering GmbH, Heidelberg, Germany)
and conventional corneal confocal imaging using the Heidelberg HRT-II with the Rostock module (Heidelberg
Engineering GmbH, Heidelberg, Germany). Optimizing
DIR
concentration
in
vivo. A
stock
solution
of
1,1′-dioctadecyl-3,3,3′,3′-
tetramethylindotricarbocyanine iodide (DIR) dye was made by dissolving 10 mg of DIR powder in 1 ml of pure
ethanol (ThermoFisher Scientific, USA).l hi
Primary cultured CECs were seeded at a concentration of 2 × 105 cells/cm2 in a Greiner 48-well flat bot-
tom polystyrol microplate (Greiner, Frickenhausen, Germany), pre-coated with FNC coating mix. Cells were
incubated with DIR diluted in M5 media39 at a concentration of 10–200 µg/ml overnight at 37 °C (N = 15 per
concentration). The following day, cells were washed thrice in fresh culture media and imaged using the HRA/
Spectralis at a standardized sensitivity of 100. Images were exported to ImageJ (National Institute of Health,
Bethesda, USA) and converted to 8-bit. Mean grey values were measured in three separate regions of each plate
and average fluorescence for each dose calculated. A comparison between groups was made using ANOVA. Toxicity of DIR in vitro. DIR-labelled endothelial cells and controls were cultured for a further 48 h in
fresh media. Calcein AM/ethidium homodimer/Hoechst were added to the cultured cells to achieve final con-
centrations of 2 µM, 4 µM and 10 µM respectively. Methods Study tissue. The use of cadaveric donor corneas for this study was approved by Singhealth centralized
institutional review board (Ref: 2016/2839), and all cell-based research performed with human derived tissue
was carried out in accordance with the tenets and the principles outlined in the Declaration of Helsinki. For
the expansion of primary CECs, research grade donor corneas with endothelial cell counts of > 1800 cells/mm2
with a storage time of < 14 days in Optisol-GS (Bausch & Lomb Rochester, New York) were procured from
either Lions Eye Institute for Transplant and Research (Tampa, Florida, USA) or Saving Sights (Kansas City,
Missouri, USA), with informed consent from the next of kin. The isolation and subsequent propagation of the
primary CECs was achieved using a dual media approach as described38,39. Briefly the DM is peeled from the
donor cornea, and subsequently subjected to a two-step enzymatic treatment to release the CECs from the DM. Isolated cells were seeded onto pre-coated collagen culture plate at a seeding density of at least 1.0 × 104 cells/
cm2 and established in a maintenance medium (M5-Endo; Human Endothelial-SFM supplemented with 5%
serum) overnight. Subsequently, CECs were cultured in the proliferative medium (M4-F99; Ham’s F12/M199,
5% serum, 20 μg/ml ascorbic acid, 1× ITS, and 10 ng/ml HrFGF) until they are approximately 80% confluent,
before being re-exposed to M5-Endo for at least two days. The expanded CECs were then sub-cultured via
single-cell dissociation using TrypLE Select, and plated at the desired seeding density for optimization studies. All cultured were carried out in a humidified atmosphere at 37 °C and 5% CO2. i
p
2
For transplantation study, human corneo-scleral buttons with consent for research use were obtained from
Miracles in Sight (Winston Salem, North Carolina, USA). Tissue preparation and transplantation was performed https://doi.org/10.1038/s41598-022-09677-w Scientific Reports | (2022) 12:6338 | www.nature.com/scientificreports/ Figure 8. Labelling of ex vivo human corneas. Human corneas were dual stained with Calcein AM and
DIR dyes. (a) After preparation for implantation, the DMEK graft, resting on stroma, is returned to the
viewing chamber and imaged using the FFA filter set to detect Calcein AM stained cells. Note the presence
of a triangular orientation mark. Uniform green fluorescence is seen as areas of healthy cells and black areas
represent dead or absent cells. (b) The same transplant is imaged using the ICG filters to detect DIR staining. Methods Linear, parallel wounds associated with graft insertion can be seen following DMEK (red arrow)
as can the notch in the transplanted graft (yellow arrow). (c–e) The graft is repeatedly imaged over 28 days. Central areas, previously devoid of cells, become DIR positive suggesting cells migration over DM has occurred
(red arrow). The triangular notch fades suggesting cells have migrated off the graft and onto the rabbit DM
at this point (yellow arrow). Areas in the periphery of the graft are not DIR fluorescent, suggesting they are
devoid of human endothelial cells. (f–j) Images of the rabbit eye show at the time of imaging show that DIR
fluorescence is detectable when the anterior chamber contains air (g,h) and that cornea clarity is restored
once a stable endothelial monolayer has been established (i,j). OCT images of cornea at various time points
following transplantation (k–o). Scale bar (a–j): 1 mm. (p) The triangular notch shows created for orientation
identification shows a loss of the straight edge (red dashed line) with apparent migration of endothelial cells
off the graft. (q) Images of the same area are acquired after staining with human nuclear antibody (FITC). The
presence of human endothelial cells beyond the border of the DMEK graft (red dashed line) is confirmed. Scale
bar: 250 µm. (r) Areas that do not stain with DIR are seen in the graft periphery. (s) Cells staining positively
with DAPI, but not staining with anti-human nuclear antibody (FITC) can be seen, suggesting these cells are of
rabbit origin and that transfer of fluorescence between cells does not occur. Scale bar: 250 µm. Figure 9. Tracking of transplanted human corneal endothelial cells in vivo. (a) A DIR stained DMEK graft
imaged in the viewing chamber pre-implantation. A triangular notch is cut out the circular graft and used to
confirm the correct side is up during surgery (yellow arrow). (b) The same graft is imaged immediately after
surgery. Linear, parallel wounds associated with graft insertion can be seen following DMEK (red arrow)
as can the notch in the transplanted graft (yellow arrow). (c–e) The graft is repeatedly imaged over 28 days. Central areas, previously devoid of cells, become DIR positive suggesting cells migration over DM has occurred
(red arrow). The triangular notch fades suggesting cells have migrated off the graft and onto the rabbit DM
at this point (yellow arrow). Methods The plate was gently agitated, incubated at 37 °C for 30 min
and imaged on the fluorescence microscope. The viable ECD was calculated and compared between control and
treated cells. A positive control was performed by using the same viability assessment in cells incubated with
70% ethanol for 1 min prior to assessment. A comparison between groups was made using ANOVA. Effect of DIR on endothelial cell migration. As the primary aim of DIR tracking was to examine cell
migration, a scratch wound assay was performed. Primary cultured CEC’s from the same donors were seeded
onto different wells within the same plate. After reaching confluence, cells were cultured in M5 culture media
with DIR 100 µg/ml or M5 media alone (controls). Each well received a single scratch using the tip of a 10 µl
pipette. Plates were placed within the incuCyte imaging incubator (Sartorius, Essen Bioscience, Ann Arbor, https://doi.org/10.1038/s41598-022-09677-w Scientific Reports | (2022) 12:6338 | www.nature.com/scientificreports/ Michigan, USA). The incuCyte is a multiplex imaging system that is incorporated into an incubator, allowing
continuous imaging in multiple wells without alterations in temperature or atmosphere. Image registration to
define the scratch area was performed and serial phase-contrast images were taken every hour for 24 h. The
wound area was measured every 3 h and expressed as a percentage of that measured at T = 0 h. Wound size was
compared between stained cells and unstained controls using multiple t-tests. Labelling of rabbit endothelial cells in vivo. 0.2 ml of DIR, diluted in BSS at a concentration of 200 µg/
ml was drawn into an insulin syringe and the needle inserted into the anterior chamber at the limbus, taking care
to tunnel the entry for at least 1 mm. A 1.0 mm paracentesis was made using a diamond knife. 0.2 ml of aqueous
was drawn into the insulin syringe achieving a final concentration of 100 µg/ml DIR. The posterior lip of the https://doi.org/10.1038/s41598-022-09677-w Scientific Reports | (2022) 12:6338 | www.nature.com/scientificreports/ Figure 9. Tracking of transplanted human corneal endothelial cells in vivo. (a) A DIR stained DMEK graft
imaged in the viewing chamber pre-implantation. A triangular notch is cut out the circular graft and used to
confirm the correct side is up during surgery (yellow arrow). (b) The same graft is imaged immediately after
surgery. Methods Areas in the periphery of the graft are not DIR fluorescent, suggesting they are
devoid of human endothelial cells. (f–j) Images of the rabbit eye show at the time of imaging show that DIR
fluorescence is detectable when the anterior chamber contains air (g,h) and that cornea clarity is restored
once a stable endothelial monolayer has been established (i,j). OCT images of cornea at various time points
following transplantation (k–o). Scale bar (a–j): 1 mm. (p) The triangular notch shows created for orientation
identification shows a loss of the straight edge (red dashed line) with apparent migration of endothelial cells
off the graft. (q) Images of the same area are acquired after staining with human nuclear antibody (FITC). The
presence of human endothelial cells beyond the border of the DMEK graft (red dashed line) is confirmed. Scale
bar: 250 µm. (r) Areas that do not stain with DIR are seen in the graft periphery. (s) Cells staining positively
with DAPI, but not staining with anti-human nuclear antibody (FITC) can be seen, suggesting these cells are of
rabbit origin and that transfer of fluorescence between cells does not occur. Scale bar: 250 µm. paracentesis was depressed causing expression of the aqueous all of the aqueous, after which the DIR was slowly
injected thus performing a complete DIR/aqueous exchange and ensuring a concentration of not greater than
100 µg/ml. It is possible the final concentration was a little lower due to the inability to remove all of the aqueous
and its rapid replenishment. The rabbits were kept with the treated eye facing down until the anaesthesia had
worn off (approximately 45 min). Imaging of endothelial cells post‑operatively. Imaging was performed using the HRA/Spectralis set
in the ICG mode. In the ICG mode, the HRA/Spectralis utilizes a 790 nm diode excitation laser and 800 nm
long-pass filter. Images were captured at varying levels of magnification by attaching microscope objective lenses
(Nikon, Japan) or the Rostock module to the standard fundus imaging lens by using custom built collars, as pre-
viously described3. Use of multiple lenses allowed both macroscopic and individual cell level imaging. Comparison of DIR imaging and established methods in a wound healing model. Two weeks
following DIR injection, 9 rabbits had 8 mm endothelial scrape wounds as described in our previous work10. Methods 6
rabbits were sacrificed at day 3, one at day 5 and the remaining two animals were sacrificed at day 28. Pan-cor- https://doi.org/10.1038/s41598-022-09677-w Scientific Reports | (2022) 12:6338 | www.nature.com/scientificreports/ Figure 10. Spectralis images depicting an event of acute graft rejection. (a) Low magnification HRA/Spectralis
image of a central area of a human DMEK graft, 21 days after transplantation into a rabbit eye. This shows a
confluent monolayer of DIR positive cells. Scale bar: 1 mm. (b) On day 28 corneal oedema was noted associated
with a significant reduction in DIR fluorescence. (c) At higher magnifications, a monolayer DIR positive, human
endothelial cells were seen at day 21. Scale bar: 50 µm. (d) On day 28, the stable monolayer has been replaced
with sparse cells that appear rounded and are in the process of detaching from the DM. Figure 10. Spectralis images depicting an event of acute graft rejection. (a) Low magnification HRA/Spectralis
image of a central area of a human DMEK graft, 21 days after transplantation into a rabbit eye. This shows a
confluent monolayer of DIR positive cells. Scale bar: 1 mm. (b) On day 28 corneal oedema was noted associated
with a significant reduction in DIR fluorescence. (c) At higher magnifications, a monolayer DIR positive, human
endothelial cells were seen at day 21. Scale bar: 50 µm. (d) On day 28, the stable monolayer has been replaced
with sparse cells that appear rounded and are in the process of detaching from the DM. eal images were acquired using the manufacture supplied anterior segment lens for the HRA/spectralis. These
llowed tracking of the entire wound areas, macroscopically.i For higher magnification imaging, the inferior edge of the wound was located by moving the lens array up
from the inferior limbus until the leading edge of migrating cells was seen, allowing a consistent area to be sam-
pled for DIR and conventional in vivo confocal microscopy. In rabbits sacrificed at day 3, the endothelial wound
was assessed by injecting trypan blue into the anterior chamber as previously described5. Trypan blue and DIR
images were exported to ImageJ and the wound outlined using the polygon selection tool. This was converted to
a smoothed spline and measurements of wound area and circularity were taken. Methods The measurements for imaging
technique were compared using a Bland–Altman plots.t q
p
g
p
After killing, the inferior cornea was marked by passage of a 6-0 silk suture through the limbus at the 6 o’clock
osition and used as a reference for immunohistology.hi The corneas were harvested, washed in BSS and fixed in 1% paraformaldehyde at room temperature for
5 min. Samples were permeabilized in Triton-X 1% for 5 min and then blocked using and 2 step process, first in
10% goat serum for 45 min followed by 3% BSA for 45 min. Samples were incubated with primary antibody to
ZO-1 (1:300, Thermofischer) or Human Nuclear Antigen (1:100, Merck-Millipore) at 4 °C for 24 h in a solution
composed of phosphate buffered saline (PBS) with 0.1% Triton-X and 1% BSA. Samples were washed 3× with
PBS, each for 10 min, and then incubated with secondary antibody, Hoechst (1:1000) and phalloidin (1:500), as
required, overnight at 4 °C. To assess specificity of the immunostaining, corneas were processed without primary
antibody. Specimens were washed 3× in PBS, re-fixed in 3% PFA, washed again and flat mounted in ProLong
gold. The entire cornea was flat mounted and the inferior edge of the wounded area, in line with the silk suture Scientific Reports | (2022) 12:6338 | https://doi.org/10.1038/s41598-022-09677-w www.nature.com/scientificreports/ Figure 11. High-resolution images achieved with Spectralis and Rostock module. High-reso
of rabbit endothelial cells (stained with DIR by intracameral injection) and human endothelia
EK transplants stained with DIR) are achievable with the HRA/Spectralis and Rostock modu
endothelial cell can be seen detaching from the monolayer. (b) Small, possibly inflammatory,
positive for DIR are visible interspersed with the rabbit endothelial cells. (c) A human endoth
undergoing apoptosis exhibits a pyknotic nucleus and separation from adjacent cells. (d) Pod Figure 11. High-resolution images achieved with Spectralis and Rostock module. High-resolution images
of rabbit endothelial cells (stained with DIR by intracameral injection) and human endothelial cells (from
EK transplants stained with DIR) are achievable with the HRA/Spectralis and Rostock module. (a) A rabbit
endothelial cell can be seen detaching from the monolayer. (b) Small, possibly inflammatory, cells staining
positive for DIR are visible interspersed with the rabbit endothelial cells. (c) A human endothelial cell
undergoing apoptosis exhibits a pyknotic nucleus and separation from adjacent cells. (d) Podocyte extension to
fill a gap in the monolayer following cell loss can be seen. www.nature.com/scientificreports/ was identified for imaging. Individual cells were manually outlined and cell metrics compared between in vivo
images and flat-mounted, immunofluorescence images (stained with phalloidin, Hoechst and ZO-1). At day 3,
when cells were still migrating, a comparison between cell circularity was made (a minimum of 100 cells were
measured in each group). At day 28 when a stable monolayer was present, a comparison between cell circularity
and the percentage of hexagon cells was made (neighbor analysis function in the BioVoxxel toolbox for ImageJ). was identified for imaging. Individual cells were manually outlined and cell metrics compared between in vivo
images and flat-mounted, immunofluorescence images (stained with phalloidin, Hoechst and ZO-1). At day 3,
when cells were still migrating, a comparison between cell circularity was made (a minimum of 100 cells were
measured in each group). At day 28 when a stable monolayer was present, a comparison between cell circularity
and the percentage of hexagon cells was made (neighbor analysis function in the BioVoxxel toolbox for ImageJ). Labelling of ex vivo human corneas. Human corneas were labelled prior to tissue preparation for
Descemet Membrane Endothelial Keratoplasty (DMEK) and following mircokeratome cutting for Descemet
Stripping Automated Endothelial Keratoplasty (DSAEK). The procedure was optimized in a series of initial
experiments. To limit the amount of dye required and prevent corneal swelling, the corneas were kept within the
Optisol-filled viewing chamber. Optisol was aspirated from the corneal cup and the endothelial surface further
dried with cellulose sponges. DIR (100 µg/ml), diluted in M5 medium, was pipetted onto the endothelial surface
until the entire endothelium and trabecular meshwork were covered. The epithelial surface remained suspended
by the pillars of the viewing chamber and in contact with the Optisol, with the scleral rim acting as barrier to
prevent the two media for mixing. The lid for the viewing chamber was replaced loosely and the corneas were
incubated over night at 37 °C. For corneas in which a comparison between DIR and Calcein AM fluorescence
was performed, corneas were rinsed 3× in fresh M5 media and the incubated with Calcein AM 1.67 µM in BSS
for 30 min prior to surgery. p
g
y
Tissue was prepared and implanted using our previously described techniques for DMEK (n = 4)5,41 and
DSAEK (n = 6)42 and imaging for Calcein AM and DIR fluorescence performed alongside the standard examina-
tions, following the review schedule outlined above. Statistical analysis. References Air reinjection and endothelial cell density in Descemet membrane endo p
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(
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suggestive of paired daughter cells are suggestive of in vivo cell division. Scale bar, 50 µm. https://doi.org/10.1038/s41598-022-09677-w Scientific Reports | (2022) 12:6338 | www.nature.com/scientificreports/ www.nature.com/scientificreports/ Use of a single peripheral triangular mark to ensure correct graft orientation in Desc
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The Impact of Farmland Management Scale on Carbon Emissions
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The Impact of Farmland Management Scale on
Carbon Emissions 军 刘
College of Geography and Environmental Sciences, Northwest Normal University
Jing Bai
(
baij15@nwnu.edu.cn
)
College of Geography and Environmental Sciences, Northwest Normal University
LiBang Ma
College of Geography and Environmental Sciences, Northwest Normal University
WenBo Zhang
College of Geography and Environmental Sciences, Northwest Normal University Research Article Keywords: Counties in Gansu Province, Farmland management scale, Intermediary effect, Agricultural carbon
emission
Posted Date: March 29th, 2023
DOI: https://doi.org/10.21203/rs.3.rs-2735088/v1
License:
This work is licensed under a Creative Commons Attribution 4.0 International
License. Read
Full License Page 1/27 Abstract In rural China, the conversion between fine-grained farmland and large-scale farmland is a widespread
phenomenon, with different impacts on agricultural carbon emissions. Therefore, how to define the optimal
scale of farmland management to achieve " reducing CO2" has become an urgent problem to be solved. Based on the agricultural panel data of Gansu Province for 2000–2020, taking the scale of agricultural land
management as the breakthrough point and the consumption intensity of agricultural materials as the
intermediary factor, this paper discusses the driving mechanism of agricultural carbon emissions by the scale
of agricultural land management. The results including: (1) From the perspective of the intermediary effect,
large-scale farmers pay more attention to input efficiency of chemicals such as fertilizers than small-scale
farmers, which can effectively promote the development of low-carbon agriculture. (2) A “U-shaped”
relationship existed between agricultural land management scale and agricultural carbon emissions. The
optimal agricultural land management scale was 7.413 mu/person without considering intermediary
variables, and the agricultural carbon emissions were the lowest when agricultural land management scale in
Gansu was 9.12 mu/person after adding the intermediary variables. (3) Dividing Gansu into different regions
and considering intermediary factors, the carbon emission intensity reached its peak when the scale of
farmland management in the Hexi region was 2.14 mu/person. When the optimal scale of farmland
management in the Longdong and Longnan regions was 2.14 and 5.22 mu/person, respectively, intermediary
variables such as chemical fertilizers in these regions all showed complete intermediary effects. 1 Introduction According to data released by the Food and Agriculture Organization of the United Nations, of the 54 billion
tons of global anthropogenic carbon emissions in 2019, agricultural and food system emissions represented
31% [17]. making this the second largest source of global carbon emissions. Effective control and reduction
of agricultural carbon emissions is the consensus reached for global climate change action[25]. As a largely
agricultural country, agriculture accounts for more than 17% of China’s carbon emissions[16].With the
development of urbanization, China’s rural areas are facing problems such as labor loss and arable land
desertion, which adversely affect the development of agricultural production[5]. To improve this situation, the
No. 1 document of the Central Committee from 2013 to 2020 proposed “the development of moderate-scale
operation”, pointing out that moderately expanding the scale of farmland management is needed for China’s
agricultural development[13]. This paper discusses the impact of farmland management scale on
agricultural carbon emissions, identifies the farmland management scale suitable for low-carbon agricultural
development, and provides reference data for the expansion of farmland management scale. Farmland scale
management is an important mechanism to eliminate carbon dioxide (negative emissions) in the future[6]. At
present, scholars at home and abroad have studied the relationship between agricultural land management
scale and agricultural carbon emissions, and produced different views. Some scholars have compared the
results of agricultural land scale policies implemented by the United States and other developed countries,
and believe that the development of agricultural land scale management will increase social and economic
benefits and, at the same time, have significant effects in reducing agricultural carbon emissions[10]. Small-
scale operations can make more effective use of resources[8] and large-scale agricultural management has a
greater negative impact on the agricultural ecological environment[2]. Some scholars believe that expanding Page 2/27 Page 2/27 the scale of agricultural land management positively impacts agricultural carbon emission reduction[9]. Empirical research has shown that expanding the scale of agricultural land management can reduce the
carbon dioxide emissions of crops such as wheat and rice and improve resource utilization efficiency[22, 29]. For example, Duan[3] et al. used the undesirable slacks-based measurement model to calculate the moderate
size of agricultural land as 1.20–1.53 hm2 for Changwu County, Shaanxi Province. Using a literature search,
Zhang [24] et al. 1 Introduction concluded that the low-carbon require ements of agricultural product inputs, the efficient
utilization of agricultural mechanization, and the reuse of agricultural waste are the main reasons that large-
scale farmland contributes to low-carbon agriculture. Liu and Xiao [14]used the intermediary effect model to
conclude that the change in agricultural land management scale induces a change in chemical and
mechanical input intensities, and so affects agricultural carbon emissions. However, most research focuses
on the macro-scale, of country and province, and less on the micro-scale of the county. In addition, most
research has concentrated on the agricultural provinces in the central and eastern regions, with less attention
paid to the arid areas in the northwest. Therefore, taking counties in Gansu Province as the research unit,
starting from the scale of agricultural land management and taking the input factors of agricultural
production as the intermediary factors, it is of great theoretical reference significance to explore the
mechanisms influencing agricultural carbon emissions for further promoting agricultural emission reduction
and high-quality development of agriculture. Gansu Province is in an arid area of northwest China. Affected by the special landform environment and
urbanization, the problems of rural labor loss and land abandonment in Gansu are serious. In order to
increase the output of agricultural grain and cash crops, farmers have to strengthen the use of input factors
such as chemical fertilizers, agricultural films, and pesticide[7, 26]. Excessive use of agricultural chemicals
such as fertilizers produces a lot of greenhouse gases, which will affect agricultural carbon emissions; at the
same time, there are differences in the impact of farmers with different farmland scales on agricultural
carbon emissions, mainly because changes in farmland scale will lead to changes in land use structure and
affect agricultural carbon emissions(Fig. 1). The utilization efficiency of small-scale farmland operators on
input factors such as chemical fertilizers is lower than that of large-scale operators. Appropriately increasing
the scale of farmland management can strengthen the management of farmland and efficiency of resource
utilization. This paper selects counties in Gansu as the research area and analyzes the influence of the
change of agricultural land management scale on agricultural carbon emission intensity, with the optimal
value of scale resulting in the lowest agricultural carbon emission intensity, in order to provide a decision-
making reference for the development of low-carbon agriculture in Gansu. 2.1 Study Area Gansu Province is in northwest China, which is the intersection of the Qinghai-Tibet Plateau, Inner Mongolia,
and the Loess Plateau – the territory is a narrow strip oriented northwest–southeast(Fig. 2). The territory is
vast, although the total land area is 4.54×107 hm2, the terrain and geomorphology are complex and the
available cultivable area is 2.73× 107 hm2. The study area is dry all year round, and more than 70% of the
cultivated land in the area is dry land. The agricultural ecological environment is fragile and highly dependent Page 3/27 on natural resources. Increasing the output of agricultural products depends on the extensive use of chemical
fertilizers, pesticides, and agricultural films, which leads to increased agricultural carbon emissions and
seriously restricts the sustainable development of rural areas. It is a great challenge for agriculture in Gansu
to control the generation of agricultural carbon emissions while ensuring that farmers increase production
and income. 2.2 Data Sources The data in this study are mainly derived from two sources: (1) spatial data, including the topographic map
of Gansu (1:100,000), from the Gansu Provincial Bureau of Surveying and Mapping and (2) data of grain
crops, sown area of cash crops, cultivated area, irrigated area, chemical fertilizers, the total power of
agricultural machinery, and planting area of agricultural products in counties and districts of Gansu were all
from the Gansu Statistical Yearbook and the Gansu Rural Yearbook from 2001 to 2021. Among these,
chemical fertilizer was calculated by the pure amount of chemical fertilizer in the current year, and some
missing data were supplemented by interpolation. Many panel data were missing for Lanzhou New District
and Maqu County, and so these areas were not included in this study. 2.3.1 Calculation method of carbon emission Agricultural carbon emission refers to greenhouse gases such as methane and nitrous oxide produced in the
process of agricultural production, and carbon dioxide produced by fossil energy consumed by agricultural
machinery and rural life. In agricultural production activities, the main sources of agricultural carbon
emissions are chemical fertilizers, pesticides, agricultural films, plowing, irrigation, and consumption of
agricultural inputs by machinery. The carbon emission conversion coefficient method recommended by the
United Nations Climate Committee was adopted for calculations. The calculation formulas for agricultural
carbon emission and agricultural carbon emission intensity in each county follow: Cm = ∑Cmn = ∑Tmn × ϵn 1 CEIm = Cm
A CEIm = Cm
A 2 where, Cm is the carbon emission of agricultural land in area m, Cmn is the carbon emission of n-type carbon
sources in area m, Tmn is the amount of n-type carbon source in area m, εn is the carbon emission coefficient
of n-type carbon sources, CEIm is the agricultural carbon emission intensity, and A is cultivated land area. Carbon emission coefficients are shown in Table 1. where, Cm is the carbon emission of agricultural land in area m, Cmn is the carbon emission of n-type carbon
sources in area m, Tmn is the amount of n-type carbon source in area m, εn is the carbon emission coefficient
of n-type carbon sources, CEIm is the agricultural carbon emission intensity, and A is cultivated land area. Carbon emission coefficients are shown in Table 1. Page 4/27 Page 4/27 Table 1
Carbon emission coefficient of agricultural input factors
Carbon
source
Carbon
emission
expression
Explain
Reference source
Chemical
fertilizer
Tm1 × ε1
Tm1 is the pure amount of chemical fertilizer
application; The coefficient ε1 is 0.8956 kg/kg. Zhi and Gao [28]
Pesticide
Tm2 × ε2
Tm2 is the amount of pesticide used, and the
coefficient ε2 is 4.93 kg/kg. ORNL (Oak Ridge
National Laboratory)
Agricultural
plastic
sheeting
Tm3 × ε3
Tm3 is the amount of agricultural film used, and
the coefficient ε3 is 5.18 kg/kg. ORNL (Oak Ridge
National Laboratory)
Agricultural
plowing
Tm4 × ε4
Tm4 is the total sown area of crops; The
coefficient ε4 is 312.60 kg/km2. Nanjing Agricultural
University Institute of
Resources and
Environment
Agricultural
irrigation
Tm5 × ε5
Tm5 is the irrigation area; The coefficient ε5 is
25kg/ hm2. 2.3.1 Calculation method of carbon emission Dubey and Lal[4]
Production
and use of
agricul-ture
machinery
Am×B +
Wm×C
Am is the total sown area of crops; Wm total
power of agricultural machinery; B and C are
coefficients, with B being 16.47kg/ hm2 and C
being 0.18kg/ hm2. Zhao RQ[27] Table 1
Carbon emission coefficient of agricultural input factors Note: The original carbon emission coefficient of agricultural irrigation is 25 kg/hm2. In the process of
electricity consumption, some of the electricity will come from thermal power generation, and the
consumption of fossil energy indirectly causes carbon emissions.This paper is based on Wu Guoyong. [21] The average thermal power coefficient calculated by others is 0.7943, and the carbon emission
coefficient of agricultural irrigation is 19.8575 kg/hm2 by multiplying the two coefficients. 2.3.2 Intermediate variables In this paper, the test of stepwise regression of the mediating effect proposed by Baronet al[1]. and the
mediation effect model proposed by Wen et al[20]. were used to analyze the logical relationship between
farmland operation scale, intermediary factors, and agricultural carbon emissions. The specific models
follow: Yi,t = a1Xi,t + a2Zi,t + a0 + ϵi,t 3 Mi,t = b1Xi,t + b2Zi,t + b0 + ϵi,t Mi,t = b1Xi,t + b2Zi,t + b0 + ϵi,t 4 Yi,t = c1Xi,t + c2Zi,t + dMi,t + c0 + ϵi,t 5 Page 5/27 where, Yi,t are the explained variables, which represent the agricultural carbon emissions of county i in period
t; Xi,t are the core explanatory variables, which indicate per capita cultivated land area of county i in period t;
Mi,t are intermediate variables, which indicate the intensity of chemical fertilizer input, pesticide input, and
agricultural film use in county i during period t; Zi,t represent the control variables that may affect agricultural
carbon emissions and intermediary factors in county i during the period t; a1, b1, c1, a2, b2, and c2 are
regression coefficients; and εi,t are random errors. (1) The variable setting of farmland operation scale refers to the research of Liu Qiong[15] and takes the
logarithmic reflection of the ratio of cultivated land area in counties to the number of agricultural employees
in Gansu. At the same time, considering that production behavior of farmers at different scales may differ,
the square term of per capita arable land area was introduced in each model. (2) The selection of
intermediate variables focused on the impact of inputs such as chemical fertilizers, agricultural films, and
pesticides on agricultural carbon emissions, and represented the unexpected output in the process of
agricultural production, which is characterized by the pure amount of chemical fertilizers applied per unit
agricultural area and the consumption of agricultural films and pesticides. (3) To further control the influence
of other variables on the analysis results, the indicators comprehensively consider the characteristics of
agricultural production, farmers’ income, and urbanization development, and five indicators were selected as
control variables: per capita disposable income, planting structure, multiple cropping index, rural labor force,
and urbanization rates. See Table 2 for the description of the variables. 3.1 Time trend of county carbon emissions in Gansu For 2000–2020, the agricultural carbon emissions in Gansu showed a trend of initial increase and then
declining (Fig. 3), with an average annual growth rate of 2.60%. In 2015, the carbon emissions in Gansu
reached a peak of 2.04 million tons, and a downward trend was obvious after 2016. From the perspective of
annual growth rate, there was a fluctuating trend during 2000–2020. In 2016, carbon emissions began to
decline, with the largest decline in 2018, and the annual growth rate reached the lowest value of − 6.0%. Note
that agricultural carbon emissions in Gansu began to decline significantly in 2017, mainly because the Gansu
provincial government issued and implemented the Implementation Plan for Controlling Greenhouse Gas
Emissions in the 13th Five-Year Plan of Gansu, and put forward the goal that “the carbon dioxide emissions
in Gansu will peak around 2030 and reach the peak as soon as possible”. After agricultural carbon emissions
and carbon emission intensity reached a peak in 2017, they showed a downward trend, indicating that the
action plan showed initial results in agricultural carbon emission reduction. The contributions of various carbon sources to agricultural carbon emissions in Gansu differed, with the
largest contribution from the use of chemical fertilizers and agricultural films (Fig. 4). Among them, the
carbon emission of chemical fertilizer accounted for 34.26–52.95% of total carbon emissions, and
agricultural film accounted for 31.35–53.95%, followed by pesticides, machinery production consumption,
plowing, and irrigation in descending order. Overall, carbon emissions of chemical fertilizers, pesticides, and
agricultural films showed a trend of initial increase and then decrease, increasing from 527.67, 328.46, and
54.63 thousand tons in 2000 to 1008.55, 737.95, and 171.60 thousand tons in 2016, respectively, and
decreasing to 749.20, 684.81, and 160.58 thousand tons in 2020. Carbon emissions from machinery
consumption, plowing, and irrigation increased from 62.55, 11.52, and 18.72 thousand tons in 2000,
respectively, to 66.93, 11.93, and 24.00 thousand tons in 2020, showing a fluctuating growth trend. It is
noteworthy that carbon emission of agricultural films in Gansu was relatively high, indicating relatively high
consumption of agricultural film, mainly due to the dry climate in most parts of Gansu, and its use in
agriculture can achieve the goal of “saving water and increasing production”. Fertilizers, pesticides, and
plastic film are the main carbon emission sources, and all showed a downward trend after 2016. 2.3.2 Intermediate variables Page 6/27 Table 2
Descriptive statistics of variables
Variable
type
Variable
name
Variable
symbol
Variable
interpretation
Sample
number
Average/mean
value
Standard
deviation
Explained
variable
Agricultural
carbon
emission
intensity
CI
Agricultural carbon
emission/cultivated
land area
(kg/ mu)
1806
3.18
0.84
Core
Explanatory
variable
Farmland
management
scale
Scale
Cultivated land
area/number of
rural employees
(mu /person)
1806
0.90
0.68
Mediator
variable
Fertilizer
input
intensity
AIF
Pure amount of
fertilizer
application/total
sown area of crops
(kg/mu)
1806
2.37
0.97
Agricultural
film input
intensity
AIM
Usage of plastic
film/total sown
area of crops (kg/
mu)
1806
-1.12
0.91
Pesticide
input
intensity
AIP
Pesticide
consumption/total
sown area of crops
(kg/ mu)
1806
-1.18
1.62
Control
variable
Per capita
disposable
income
INC
Per capita
disposable income
of rural families
(yuan)
1806
5395.29
4595.90
Planting
structure
Stru
Sowing area of
grain crops/total
sown area of crops
(kg/ mu)
1806
-0.28
0.73
Multiple
cropping
index
MCI
Crop sown
area/cultivated
land area
1806
0.91
0.24
Rural labor
force
FAL
Number of rural
laborers (10,000)
1806
8.15
5.59
Urbanization
rate
UR
Urban
population/total
population at the
end of the year
1806
0.33
0.24
A stepwise regression method was adopted to test the relationship between intermediary variables and
i
l
l
b
i
i
Fi
h
h
i
l
l l
d
l h d
l ff Descriptive statistics of variables A stepwise regression method was adopted to test the relationship between intermediary variables and
agricultural carbon emissions. First, whether agricultural land management scale had a total effect on
agricultural carbon emissions was tested using a regression model of agricultural land management scale to Page 7/27 Page 7/27 agricultural carbon emissions. Second, the scale of agricultural land management and intermediary variables
were used as explanatory variables to analyze the synthetic model to test whether the scale of agricultural
land management and the input intensity of agricultural chemicals had a direct effect on agricultural carbon
emissions. Then, the effect of intermediary variables on the impact of agricultural land management scale on
agricultural carbon emissions was calculated step by step. Finally, the results of the mediation effect were
tested in turn. 3.2 Spatial differences of county carbon emissions in Gansu The spatial distribution map of carbon emission intensity of counties in Gansu for 2000–2020 was obtained
using Arcgis and the natural discontinuity method, and showed clear spatial concentration characteristics of
agricultural carbon emission intensity (Fig. 5). Compared with 2000, the carbon emission intensity in most
counties in 2020 showed an increasing trend, concentrated in Huan, Huating, and Zhuanglang Counties, with
the largest growth rate in Huan County; Zhuoni, Anning District, Honggu District, Yongchang County, and
other counties with negative carbon emission intensity. The regions with relatively small changes in carbon
emission intensity included Xiahe County, Cheng County, and Xigu District, with the smallest change rate for
Cheng County. The high-value areas with carbon emission intensity in the range of 63.51–335.50 kg/mu
were concentrated in the east and north, mainly due to the large planting area of wheat, corn, and other crops
in the central, east and north of Gansu, and the higher consumption of chemicals such as fertilizers and
pesticides and power of agricultural machinery. At the same time, due to the expansion of cultivated land, the
use of chemicals such as fertilizers and pesticides in some areas has increased, resulting in these areas
becoming high-value areas of agricultural carbon emission intensity in Gansu. Although the location of high-carbon emission regions in Gansu has not changed much, the range of middle-
and high-value regions had a shrinking trend from 14 to seven counties during 2015–2020. Note that the
cultivated land area in Bai Yin City is relatively small, but it is rich in water resources, and the planting area of
crops and the rural population account for a large proportion. Every year, agricultural film covers about
2.2 million mu, the amount used is about 20,000 tons about 20,000 t and resulting in high agricultural carbon
emissions. Therefore, in the past 20 years, the carbon emission intensity of agriculture in Bai Yin City has
always been in the high-value area. 3.3 Influencing factors of agricultural carbon emissions Before data processing, we first carried out two unit root tests, LLC (Levin-Lin-Chu Tested)and
ADF(Augmented Dickey-Fuller Tested), which showed that the data were a stationary series at 1% level. In this
paper, the mixed regression model was tested by F-test to determine the optimal regression model, with
significance at 1% level, indicating that the original hypothesis was strongly rejected. That is, the fixed effect
model (FE) was superior to the mixed regression model. In addition, this paper used the data of 86 counties in
Gansu (except Lanzhou New District and Maqu County) for the most recent 20 years. In order to ensure the
validity of the analysis results and avoid the problem of "pseudoregression" caused by using nonstationary
data for analysis, the FE model was used for analysis. 3.1 Time trend of county carbon emissions in Gansu On the one
hand, the cultivated land area and rural employees in Gansu have decreased compared with before and, on
the other hand, agricultural carbon sources have been included in the scope of emission reduction during the
Twelfth Five-Year Plan period in Gansu, and emission reduction measures have been taken, with observable
results. Page 8/27 Page 8/27 3.3.1 Impact of farmland management scale on agricultural
carbon emission intensity Firstly, we measured the total impact of agricultural land management scale on carbon emission intensity. Considering that there may be differences in farmers’ production behavior at different scales, the square term
of farmland management scale was introduced into the model. According to the principle of Baron and
Kenny’s stepwise regression method, Model 1 (Table 3) showed that the coefficient of farmland management Page 9/27 Page 9/27 Page 9/27 scale was significantly negative (− 1.181) and the square term coefficient of farmland management scale
was significantly positive (0.325). For Model 2, the regression coefficients of farmland management scale
and the square term of farmland management scale were significantly negative and positive, respectively,
and the total effect of the core explanatory variable farmland management scale on agricultural carbon
emission intensity was significant, indicating that when farmland management scale was small, the
expansion of scale had an inhibitory effect on agricultural carbon emission. When the scale of agricultural
land management was large, the expansion of scale promoted agricultural carbon emission, that is, there
was an “optimal management scale” between agricultural land management scale and carbon emission,
resulting in the lowest agricultural carbon emission intensity. After solving, the optimal farmland
management scale for Models 1, 2, and 3(Table 3) was 6.153, 7.806, and 8.281 mu/person, respectively, with
an average of 7.413 mu/person. Model 2(Table 3) had relatively strong multicollinearity in per capita disposable income and urbanization
rate, so the square of per capita disposable income was added to Model 3 and the urbanization rate was
removed. The regression coefficients of per capita disposable income and its square term on agricultural
carbon emissions were 0.484 and 0.081, respectively, indicating that a “U-shaped” relationship between per
capita disposable income and agricultural carbon emission intensity. There may be a negative correlation
between the INC of rural families and CEI. When INC is low, with the accumulation of capital, farmers will
maximize profits and increase external investment in agriculture, thus promoting CEI. With the rising of INC,
the increase of opportunity cost of agricultural production promotes the extensive management of farmland,
which leads to the decline of CEI. Planting structures, MCI, rural labor force, and UR all had significant effects
on agricultural carbon emission intensity. 3.3.1 Impact of farmland management scale on agricultural
carbon emission intensity Page 10/27 Page 10/27 Table 3 Table 3 Benchmark regression results
Explanatory variable
Total sample
Model 1(lnCEI)
Model 2(lnCEI)
Model 3(lnCEI)
Scale
-1.181***
(-11.551)
-0.711***
(-8.097)
-0.795***
(-8.975)
Square of scale
0.325***
(10.568)
0.173***
(6.654)
0.188***
(7.128)
stru
-0.365***
(-15.475)
-0.421***
(-17.294)
MCI
-0.188**
(-2.931)
-0.160**
(-2.458)
INC
0.484***
(17.411)
-0.706
(-1.819)
Square of INC
0.081***
(3.441)
FAL
0.251***
(17.411)
0.229***
(11.988)
UR
0.795***
(8.464)
-cons
4.150***
(45.118)
-0.790***
(-3.306)
3.831**
(2.415)
N
1806
1806
1806
R2
0.069
0.397
0.378
F
67.114***
171.269***
151.576***
present significance levels of 10%, 5% and 1%, respectively. The numbers in brack Benchmark regression results *, * * and * * represent significance levels of 10%, 5% and 1%, respectively. The numbers in brackets are t-
values. *, * * and * * represent significance levels of 10%, 5% and 1%, respectively. The numbers in brackets are t- 3.3.2 Impact of farmland management scale on intermediary
variables 3.3.2 Impact of farmland management scale on intermediary
variables For Model 4 (Table 4), under the control of other variables, the regression coefficients of the agricultural land
management scale and its square term to the intensity of chemical fertilizer use were − 0.2568 and 0.1238, Page 11/27 Page 11/27 respectively. Thus, farmers’ investment in chemical fertilizer will decrease when agricultural land
management scale expands, but will increase with expansion of scale when it exceeds the “optimal
agricultural land management scale”. This shows that the scale of operation can regulate the use of
agricultural production materials. For Models 4 and 5(Table 4), under the control of other variables, the regression coefficients of the farmland
management scale and its square term for the intensity of fertilizer use were − 0.245 and 0.122, respectively,
and for the intensity of agricultural film use were − 9.896 and 2.406. When the scale of farmland operation
was small, the scale of farmland operation had a significant negative effect on the input of chemical fertilizer
and agricultural film. On the contrary, when the scale of farmland operation was large, the input of chemical
fertilizer and agricultural film had a significant positive effect. For Model 6(Table 4), the scale of agricultural land management had a significant positive effect on input
intensity of pesticides, while the square term of agricultural land management scale had a significant
negative effect on input intensity of pesticides, with regression coefficients of 0.518 and − 0.262, respectively. As the scale of farmland increases, farmers will increase their input to pesticides to ensure the output of food
or cash crops. However, beyond a certain scale, with the expansion of farmland scale, farmers tend to be
more specialized in management of farmland, and in order to save costs, will replace the use of pesticides in
other ways, thereby reducing the input of pesticides. The results of Model 6 are contrary to those of Models 4
and 5, indicating that the scale of agricultural land management has regulation and rationality on the use of
agricultural production materials, will also have different effects on the input of different agricultural factors,
and different regional climatic conditions will have a certain impact on input of factors. 3.3.2 Impact of farmland management scale on intermediary
variables Page 12/27 Page 12/27 Table 4 Table 4
Regression results of agricultural land management scale and intermediary
variables Regression results of agricultural land management scale and intermediary
variables
Explanatory variable
Model 4 (AIF)
Model 5 (AIM)
Model 6 (AIP)
Scale
-0.245***
(-2.222)
-9.896***
(-27.601)
0.518*
(1.992)
Square of scale
0.122**
(3.630)
2.406***
(22.691)
-0.262***
(-3.410)
Stru
-0.429***
(-13.848)
-1.005***
(-10.439)
-0.624***
(-8.940)
MCI
-0.296***
(-3.515)
0.816***
(3.121)
-1.121***
(-5.915)
INC
0.464***
(12.484)
1.016***
(8.954)
0.566***
(6.881)
FAL
0.328***
(12.480)
0.587***
(7.659)
0.185***
(3.324)
UR
0.650***
(5.277)
3.220***
(8.405)
2.199***
(7.921)
-cons
-2.044***
(-6.523)
0.497
(0.510)
-6.164***
(-8.717)
N
1806
1806
1806
R2
0.252
0.203
0.200
F
78.504***
251.065***
64.126*** Explanatory variable
Model 4 (AIF)
Model 5 (AIM)
Model 6 (AIP) *, * * and * * represent significance levels of 10%, 5% and 1%, respectively. The numbers in brackets are t-
values. 3.3.3 Impact of farmland management scale and
intermediate variables on agricultural carbon emission
i
i Page 13/27
When the intermediary variables were chemical fertilizer, agricultural film, and pesticides, the direct impact of
agricultural land management scale on agricultural carbon emission intensity was still significant, indicating
that the three intermediary variables played a partial intermediary role. Substituting the regression results of
Models 7 and 8(Table 5) into the intermediary effect model relationship, the optimal farmland scale in Gansu
was 18.56 and 6.10 mu/person, respectively. The farmland management scale corresponding to the carbon emission intensity value obtained by Model 9 was 2.69 mu/person, with an average of 9.12 mu/person. The
regression coefficients of chemical fertilizer, agricultural film, and pesticides were 0.639, 0.155, and 0.198,
respectively, which had significant positive impacts on agricultural carbon emission intensity, because the
use of chemical fertilizer, agricultural film, and pesticides was the main factor promoting agricultural carbon
emissions, and the input of these agrochemicals not only guaranteed crop yield, but also posed a great threat
to the agricultural ecological environment. emission intensity value obtained by Model 9 was 2.69 mu/person, with an average of 9.12 mu/person. The
regression coefficients of chemical fertilizer, agricultural film, and pesticides were 0.639, 0.155, and 0.198,
respectively, which had significant positive impacts on agricultural carbon emission intensity, because the
use of chemical fertilizer, agricultural film, and pesticides was the main factor promoting agricultural carbon
emissions, and the input of these agrochemicals not only guaranteed crop yield, but also posed a great threat
to the agricultural ecological environment. emission intensity value obtained by Model 9 was 2.69 mu/person, with an average of 9.12 mu/person. The
regression coefficients of chemical fertilizer, agricultural film, and pesticides were 0.639, 0.155, and 0.198,
respectively, which had significant positive impacts on agricultural carbon emission intensity, because the
use of chemical fertilizer, agricultural film, and pesticides was the main factor promoting agricultural carbon
emissions, and the input of these agrochemicals not only guaranteed crop yield, but also posed a great threat
to the agricultural ecological environment. *, * * and * * represent significance levels of 10%, 5% and 1%, respectively. The numbers in brackets are t-
values. 3.4 Regional Analysis Because the scale of agricultural land management is influenced by mountainous terrain, the available land
area in plain areas is larger than that in plateau and mountainous areas. Gansu was divided into five county-
level administrative regions: Hexi, Longzhong, Longdong, Longnan, and Gannan. The five sub-samples were
used for regionalization analysis. 3.3.3 Impact of farmland management scale and
intermediate variables on agricultural carbon emission
i
i Page 14/27 Table 5 Table 5
Regression results of the influence of agricultural land management scale and
intermediary variables on carbon emissions
Explanatory variable
Model 7(lnCEI)
Model 8(lnCEI)
Model 9(lnCEI)
Scale
-0.555***
(-11.545)
0.818***
(10.051)
-0.814***
(-11.408)
Square of scale
0.095***
(6.681)
-0.199***
(-8.695)
0.225***
(10.631)
AIF
0.639***
(65.023)
AIM
0.155***
(34.446)
AIP
0.198***
(30.601)
Stru
-0.093***
(-6.832)
-0.210***
(-11.122)
-0.242***
(-12.357)
MCI
0.006
(0.175)
-0.314***
(-6.294)
0.034
(0.649)
INC
0.195***
(12.063)
0.327***
(14.827)
0.372***
(16.289)
FAL
0.041**
(3.781)
0.160***
(10.786)
0.214***
(14.005)
UR
0.380***
(7.360)
0.297***
(3.998)
0.359***
(4.640)
-cons
0.516***
(3.910)
-0.867***
(-4.672)
0.429*
(2.169)
N
1806
1804
1804
R2
0.821
0.637
0.604
F
917.05***
396.995***
344.876***
epresent significance levels of 10%, 5% and 1%, respectively. The numbers in bracke egression results of the influence of agricultural land management scale and
intermediary variables on carbon emissions Page 15/27 Regarding the influence of agricultural land management scale on agricultural carbon emission, when all
three intermediate variables were included in the regression model, the input intensity of chemical fertilizers,
agricultural film, and pesticides all played a part in the intermediary effect, with input intensity in the
following order: chemical fertilizer > pesticide > agricultural film(Table 5). Therefore, adjusting the scale of
agricultural land management can optimize the input intensity of agricultural external input factors, such as
chemical fertilizers, agricultural film, pesticides, and improve the efficiency of input, to achieve the goal of
agricultural carbon emission reduction under the premise of ensuring output of food or cash crops. 3.4.1 Impact of regional agricultural land management scale
on agricultural carbon emission intensity Because of the strong collinearity between per capita disposable income and urbanization rate in the control
variables, two variables were excluded from the regionalization analysis. The square term of agricultural land
management scale in the Hexi and Longdong regions had a significant impact on agricultural carbon
emissions, with regression coefficients of − 0.945 and 0.265, respectively (Table 6); that is, the relationship
between agricultural land management scale and agricultural carbon emissions in this region was “U-
shaped” and “inverted U-shaped”. The regression coefficients of the agricultural land management scale and
its square term to agricultural carbon emission intensity in the Gannan area were 1.716 and − 0.489,
respectively, showing an “inverted U” trend. The regression coefficients of the square term of agricultural land
management scale in the Longnan area were 1.095 and − 0.263, respectively, but the results were non-
significant, indicating a linear trend between scale and agricultural carbon emissions in this area. Substituting the results into the regression model showed that the scale of agricultural land management in
the Hexi and Gannan regions should exceed 2.05 and 5.78 mu/person, respectively, and the optimal scale in
the Longdong region was 3.28 mu/person and resulted in the least impact on agricultural carbon emissions. The significant difference between central and southern Gansu may be due to the influence of topography
and natural conditions. Substituting the results into the regression model showed that the scale of agricultural land management in
the Hexi and Gannan regions should exceed 2.05 and 5.78 mu/person, respectively, and the optimal scale in
the Longdong region was 3.28 mu/person and resulted in the least impact on agricultural carbon emissions. The significant difference between central and southern Gansu may be due to the influence of topography
and natural conditions. 3.4.1 Impact of regional agricultural land management scale
on agricultural carbon emission intensity Page 16/27 Table 6
Regional benchmark regression results
Explanatory
variable
Hexi region
Longzhong
area
Longdong
area
Longnan
region
Gannan area
Model
1(lnCEI)
Model
2(lnCEI)
Model
3(lnCEI)
Model
4(lnCEI)
Model 5(lnCEI)
Scale
3.880***
(9.572)
-0.241***
(-8.097)
-1.738***
(-2.630)
1.095
(1.214)
1.716**
(2.142)
Square of scale
-0.945***
(-9.297)
-0.007
(-0.257)
0.265*
(1.826)
-0.263
(-0.964)
-0.489*
(-1.884)
Stru
-0.158***
(-6.683)
-0.016
(-0.480)
0.402***
(7.883)
0.229***
(4.835)
0.107*
(1.875)
MCI
-0.842***
(-6.252)
0.065
(0.799)
0.028
(0.242)
-0.381***
(-2.993)
-1.178***
(-8.369)
FAL
0.025***
(2.687)
-0.464***
(-17.602)
-0.263***
(-5.797)
0.252***
(6.001)
0.911***
(13.083)
-cons
0.550
(1.461)
8.890***
(30.250)
8.511***
(9.338)
-0.754
(-0.904)
0.473
(0.773)
N
420
735
315
210
147
R2
0.293
0.503
0.291
0.262
0.752
F
35.764***
149.693***
26.729***
15.857***
89.566***
* * *
d * *
t i
ifi
l
l
f 10% 5%
d 1%
ti
l
Th
b
i
b
k t
t Table 6
Regional benchmark regression results *, * * and * * represent significance levels of 10%, 5% and 1%, respectively. The numbers in brackets are t-
values. 3.4.2 Impact of agricultural land management scale and intermediary variables on agricultural carbon
emission intensity at the county level When three intermediary variables (chemical fertilizer, plastic film, and pesticide input intensity) were added,
the influence of agricultural land management scale on agricultural carbon emission in the Longdong and
Gannan areas was not significant (Table 7), while the benchmark regression results showed a significant
relationship between agricultural land management scale and carbon emission intensity (Table 6), indicating
that intermediary factors such as chemical fertilizer in the two areas play a complete intermediary role. In
contrast, intermediary factors such as chemical fertilizer in Hexi, Longzhong, and Longnan areas played a
partial intermediary role. The regression coefficients of the agricultural land management scale and its
square term to agricultural carbon emission in the Hexi region were 0.356 and − 0.083(Table 7), respectively,
showing an “inverted U” trend, and so management scale needed to exceed 2.14 mu/person to promote Page 17/27 Page 17/27 Page 17/27 agricultural carbon emission reduction. The regression coefficients in central Gansu were − 0.120 and
0.028(Table 7), respectively, and those in southern Gansu were − 1.221 and 0.369(Table 7), both of which
showed a “U-shaped” trend. When the optimal farmland management scale was 2.14 and 5.22 mu/person,
respectively, the agricultural carbon emission intensity values in both regions were the lowest. 4.1.1 Direct impact of farmland management scale on
agricultural carbon emissions In this paper, the agricultural carbon emissions in Gansu were analyzed in terms of the structure of
agricultural carbon emissions. The agricultural carbon emissions of counties in Gansu were calculated using
the carbon emission coefficient method, and the mechanism of agricultural land management scale on
agricultural carbon emissions at the county level was explored. The results showed that the scale of
agricultural land management had both direct and indirect effects on agricultural carbon emissions. The
literature review showed that the direct cause of the impact of agricultural land management scale on
agricultural carbon emission was that the change of scale leads to the change of land structure[11]. That is,
the transformation between woodland, farmland, grassland, construction land, and other lands, have
different abilities of “carbon source” and “carbon sink”[19], which in turn affects agricultural carbon
emissions. 3.4.1 Impact of regional agricultural land management scale
on agricultural carbon emission intensity Table 7
Regression results of regional agricultural land management scale and intermediary variables on carbon
emissions
Explanatory
variable
Hexi region
Longzhong
area
Longdong
area
Longnan
region
Gannan area
Model
1(lnCEI)
Model 2(lnCEI)
Model
3(lnCEI)
Model
4(lnCEI)
Model
4(lnCEI)
Scale
0.356***
(2.910)
-0.120***
(-4.259)
-0.212
(-1.104)
-1.221***
(-3.070)
1.190
(4.072)
Square of scale
-0.083***
(-2.746)
0.028***
(2.903)
0.029
(0.664)
0.369***
(3.087)
-0.292
(-3.259)
AIF
0.504***
(37.161)
0.307***
(27.462)
0.426***
(27.850)
0.468***
(17.969)
0.199(13.275)
AIM
0.108***
(30.028)
0.428***
(52.566)
0.346***
(32.897)
0.332***
(23.155)
0.300(17.505)
AIP
0.122***
(14.583)
0.085***
(16.259)
0.040***
(8.528)
0.077***
(8.764)
-0.043
(-6.806)
Stru
0.015**
(2.130)
-0.005
(-0.507)
0.045***
(2.724)
0.062***
(2.990)
-0.024
(-1.209)
MCI
-0.208***
(-5.247)
0.089***
(3.446)
0.024
(0.607)
-0.292
(-5.022)
-0.473
(-7.016)
FAL
0.007**
(1.986)
-0.062***
(-6.346)
-0.046***
(-3.224)
-0.061***
(-2.897)
0.335
(8.431)
-cons
1.701***
(15.412)
3.086***
(26.755)
2.844***
(12.222)
3.618***
(9.449)
0.885
(3.617)
N
420
735
315
210
147
R2
0.949
0.952
0.945
0.954
0.973
F
971.502***
1828.726***
725.814***
871.352***
664.123*** Table 7 Page 18/27 *, * * and * * represent significance levels of 10%, 5% and 1%, respectively. The numbers in brackets are t-
values. *, * * and * * represent significance levels of 10%, 5% and 1%, respectively. The numbers in brackets are t-
values. The significant regression results for the five sub-regions were quite different from the overall results of
counties in Gansu. There are three reasons: (1) there was a big difference between the number of regional
samples and the total number of samples, and the estimation accuracy decreased, which affected model
analysis results; (2) the landform and different natural climate characteristics between regions, that is,
heterogeneity between regions, led to great differences in the scale of agricultural land management; and (3)
uneven regional economic development and different investment in agricultural scientific training led to
different scientific management levels of agricultural land among operators, and the efficiency of investment
in chemicals such as fertilizers or external factors such as machinery. 4.2 Carbon emission reduction path under the scale of
agricultural land management At present, agriculture in Gansu is characterized by high input, low output, and high pollution. Although
agricultural carbon emissions in Gansu have shown a downward trend in recent years, agricultural carbon
emissions are still high, among which the emissions of agricultural chemicals such as fertilizers and
agricultural films account for the largest proportion, indicating that there is an overuse problem of agricultural
chemicals in Gansu. This study investigated the relationship between agricultural land management scale
and carbon emissions, and showed that adjusting agricultural land management scale and improving
resource utilization efficiency can effectively control agricultural carbon emissions. Below we propose some
suggestions on the research conclusion. (1) Appropriately increase the scale of agricultural land management. The present farmland scale in Gansu is
below optimal, so it is necessary to appropriately increase farmland scale, which will aid development of low-
carbon agriculture. Therefore, on the one hand, relevant departments in Gansu should encourage farmers
who have been planting for a long time to appropriately expand their scale, and concentrate finely-divided
farmland into large-scale farmland to avoid the problem of agricultural intensification. Skill training
institutions should be established to guide farmers to scientifically manage agricultural land, adopt improved
techniques and farming methods, broaden the sales market of agricultural products, formulate and
promulgate policy documents and methods for rational land transfer and increase financial subsidies for
large-scale management of agricultural land, and actively introduce technical talent for agricultural land
management to promote large-scale management of agricultural land. On the other hand, excessive
concentration of agricultural land will also have a negative impact. If the scale of agricultural land is too
large, it will increase the cost of labor and farmland utilization, require greater supervision of agricultural land
management, increase management costs, withstand uncertainties such as drought and flood, and increase
the input of chemicals such as fertilizers and pesticides, thus increasing the environmental load. Therefore, it
is necessary to reasonably control the scale of agricultural land management and improve the input
efficiency of chemical fertilizers and other factors. (2) Standardize the use of agricultural film, and use scientific application of chemical fertilizers and
pesticides. The analysis of agricultural carbon emission sources showed that chemical fertilizers, agricultural
films, and pesticides were the main factors of agricultural carbon emission in Gansu. In recent years,
excessive use of agricultural films in some areas of Gansu has led to an increase in carbon emissions. mastery of the scientific application of fertilizers and other chemicals[15], so the scale of farmland
management can indirectly affect carbon emissions through the input of fertilizers and other chemicals. mastery of the scientific application of fertilizers and other chemicals[15], so the scale of farmland
management can indirectly affect carbon emissions through the input of fertilizers and other chemicals 4.1.2 Indirect impact of farmland management scale on
agricultural carbon emissions The impact of large-scale farmland management on carbon emissions is reflected in the different choices of
farmers in the use of chemical fertilizers and farmland irrigation, resulting in differences in agricultural
carbon emissions, among which the production and use of chemical fertilizers, pesticides, and other
chemicals had the greatest impact, Zhang Xiaojie[23] et al. reached similar conclusions using empirical
research. China’s agricultural production is highly dependent on chemical inputs[12], so the average farmer
may choose to change the scale of farmland operations and the input of chemicals such as fertilizers in
order to increase agricultural production. Large-scale farmers are often willing to accept new technologies for
efficient use of chemical fertilizers[18], while farmers with small-scale operations tend to have less technical Page 19/27 Page 19/27 Page 19/27 5 Conclusions Based on the proportion of sources of agricultural carbon emissions in Gansu Province, we selected the use
intensity of chemical fertilizers, agricultural films, and pesticides as intermediary variables, built an
intermediary effect model to explore the impact of agricultural land management scale on agricultural carbon
emission, and systematically analyzed the transmission mechanism of agricultural land management scale
on agricultural carbon emission. The main conclusions follow: (1) Analysis of the spatiotemporal characteristics of agricultural carbon emissions in Gansu for 2000–2020
showed that, in terms of time series, agricultural carbon emissions and emission intensity in Gansu generally
showed a downward trend, reaching a peak in 2015. After 2016, the agricultural carbon emission reduction
effect was remarkable. In spatial sequence, the high-value areas of carbon emissions in Gansu are mainly
distributed in the central, eastern, and southeastern regions, and the range of high-value areas showed a
decreasing trend. (1) Analysis of the spatiotemporal characteristics of agricultural carbon emissions in Gansu for 2000–2020
showed that, in terms of time series, agricultural carbon emissions and emission intensity in Gansu generally
showed a downward trend, reaching a peak in 2015. After 2016, the agricultural carbon emission reduction
effect was remarkable. In spatial sequence, the high-value areas of carbon emissions in Gansu are mainly
distributed in the central, eastern, and southeastern regions, and the range of high-value areas showed a
decreasing trend. (2) The indirect effects of agricultural land management scale on agricultural carbon emissions through
three intermediary channels (fertilizer, pesticides, and agricultural film use intensity) were verified at the
county scale in Gansu: the management scale showed a “U-shaped” relationship with fertilizer and
agricultural film input, and an “inverted U” relationship with pesticide. After adding intermediary factors, the
scale of farmland operation still has a significant impact on agricultural carbon emissions. (2) The indirect effects of agricultural land management scale on agricultural carbon emissions through
three intermediary channels (fertilizer, pesticides, and agricultural film use intensity) were verified at the
county scale in Gansu: the management scale showed a “U-shaped” relationship with fertilizer and
agricultural film input, and an “inverted U” relationship with pesticide. After adding intermediary factors, the
scale of farmland operation still has a significant impact on agricultural carbon emissions. 4.2 Carbon emission reduction path under the scale of
agricultural land management Therefore, relevant regulatory authorities need to vigorously control the application of chemical fertilizers and
pesticides, replace the original agricultural film with energy-saving agricultural film, increase the recycling of
agricultural film, and at the same time strengthen scientific research to study the number of chemicals such
as fertilizers needed by different crops to avoid excessive application and improve chemical fertilizers. 4.3 Innovation and Deficiency Page 20/27 Page 20/27 In terms of research results, the agricultural-related emission reduction measures in Gansu have achieved
initial results and began to show an obvious downward trend in 2017. By reasonably controlling the scale of
agricultural land management, the consumption of chemicals such as fertilizers can be effectively controlled
and regulated, and the ideal effect of “high output and low emission” in agricultural planting can be achieved. Calculation showed that the optimal farmland management scale in Gansu was 7.413 mu/person without
considering the intermediate variables, and the optimal scale when considering the intermediate variables
was 9.12 mu/person. Combined with the county-level panel data of Gansu, present farmland scale is 7.221
mu/person, so it is necessary to reasonably expand the scale, and at the same time, it is necessary to control
the input of chemical fertilizers, agricultural films, pesticides, and other chemicals to reduce carbon
emissions. Concerning the research scale, this study fills the gap in research of small areas such as counties
and verifies the results of Liu et al.[14], which has certain reference significance for the formulation of
regional agricultural carbon emission reduction measures. However, in this paper, the scale of agricultural
land management was expressed by per capita arable land area. Due to the lack of data on farmers, the
impact of agricultural land management scale on agricultural carbon emissions was not analyzed at the level
of farmers, and the impact of agricultural industrial structure, rural age structure, and education on
agricultural carbon emissions was not considered, and further research should be made on these aspects. Acknowledgments The work was supported by the National Natural Science Foundation of China (Project No: 42261033), the
Natural Science Foundation of Gansu Province (Project No.: 22JR5RA143), and the Gansu Higher Education
Innovation Project (Project No.༚2011A-14). 5 Conclusions In addition, the total effect of the agricultural land management scale on agricultural carbon emission had a
significant “U-shaped” relationship, and the existence of an “optimal scale of agricultural land” resulted in
agricultural carbon emission intensity having a minimum value of 7.41 mu/person. The optimal value of the
farmland scale was 9.12 mu/person when considering the intermediate variables. Page 21/27 Page 21/27 (3) The scale of agricultural land management in the Hexi region exceeded 2.05 mu/person, which would
promote reductions in agricultural carbon emission. The optimal scale of agricultural land management in
the Longdong region was 3.28 mu/person, and the scale of agricultural land management in the Longzhong
and Longnan regions had a linear trend. After adding intermediary variables, the scale of agricultural land
management in the Hexi region was at least 2.14 mu/person, and had a positive role in promoting
development of low-carbon agriculture. The optimal scale of agricultural land management in the Longdong
and Longnan regions was 2.14 and 5.22 mu/person, respectively, and intermediary variables such as
chemical fertilizers in the Longzhong region had a complete intermediary effect. (3) The scale of agricultural land management in the Hexi region exceeded 2.05 mu/person, which would
promote reductions in agricultural carbon emission. The optimal scale of agricultural land management in
the Longdong region was 3.28 mu/person, and the scale of agricultural land management in the Longzhong
and Longnan regions had a linear trend. After adding intermediary variables, the scale of agricultural land
management in the Hexi region was at least 2.14 mu/person, and had a positive role in promoting
development of low-carbon agriculture. The optimal scale of agricultural land management in the Longdong
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and rural areas of China. Progress in Geography, (3), 429–434. 29. Zhu, Y., Waqas, M. A., Zou, X., Jiang, D., Wilkes, A., Qin, X., … Hasbagan, G. (2018).Large-scale farming
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operations are win-win for grain production, soil carbon storage and mitigation of greenhouse gases. Journal of Cleaner Production, 172, 2143–2152. Figure 1 The impact path of farmland scale on carbon emissions Page 24/27 Page 24/27 Figure 2
Schematic diagram of the study area Figure 2 Figure 2 Schematic diagram of the study area Schematic diagram of the study area Page 25/27
Schematic diagram of the study area Page 25/27 Page 25/27 Figure 3 Agricultural carbon emissions in Gansu for 2000–2020 Agricultural carbon emissions in Gansu for 2000–2020 Agricultural carbon emissions in Gansu for 2000–2020
Figure 4
Carbon emissions of agricultural input factors in Gansu for 2000–2020 Figure 4 Figure 4 Carbon emissions of agricultural input factors in Gansu for 2000–2020 Page 26/27 Page 26/27 Page 26/27 Figure 5 Figure 5 Spatial pattern change of agricultural carbon emission intensity in various districts and counties Spatial pattern change of agricultural carbon emission intensity in various districts and counties Spatial pattern change of agricultural carbon emission intensity in various districts and counties Page 27/27
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Introduction to the Study of the Fundamental Cause of Splanchn Optosis — Abdominal Incompetence: A Developmental Factor
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changes; (3) girls and women wearing definitely
constricting clothing. audible with much greater intensity on passing up-
wards and inwards, being loudest in the third left
interspace near the sternum. No accentuation of the
aortic second was detected. The pulse was 46 to the
minute, regular in force and rhythm, of good volume
and tension. No sclerosis of the vessel wall was made
out. There was no edema of the extremities. h audible with much greater intensity on passing up-
wards and inwards, being loudest in the third left
interspace near the sternum. No accentuation of the
aortic second was detected. The pulse was 46 to the
minute, regular in force and rhythm, of good volume
and tension. No sclerosis of the vessel wall was made
out. There was no edema of the extremities. h constricting clothing. Gradually, it appeared evident that all girls
and women could be classified as conforming to
one or the other of two types, which types ap-
peared to develop spontaneously and in accord-
ance with natural laws. On Dec. 23, 1905, while at the railroad station, in
line to purchase a ticket for New York, she suddenly
felt as if something had hit her on the forehead with
a piece of steel. Then, while leaning over a bench
nearby, she experienced a queer, quivering sensation,
as if everything was giving away, coming from the
veins of her legs up, and suddenly she became uncon-
scious. Does not know how long this attack lasted. A week later she had a dizzy feeling which induced
her to go to bed, and again she lost consciousness. At this time she had no warning and the attack lasted
from two to three minutes. On recovery she
com-
plained of general
numbness. Shortly before this
attack she was seen by Dr. E. G. Janeway, who con-
curred
in the diagnosis
of
Stokes-Adams
disease. Efforts were made at this time to get a cardiagram and
a jugular and brachial sphygmogram, but with only
partial success. Pulsations, however, were observed
in the external jugular vein, which equaled 115 to the
minute, while the radial pulse varied from 46-48. The
maximum arterial pressure was 150 mm., the
mini-
mum 95 mm. On March 17 I endeavored to get simi-
lar pulse tracings at the Trinity College Psychological
Laboratory, with the kind assistance of Professor Urban
and Dr. *Read before the Section in Surgery of the Fifteenth Interna-
tional Medical Congress, at Lisbon, April 20, 1906. The Boston Medical and Surgical Journal as published by
The New England Journal of Medicine. Downloaded from nejm.org at WEILL CORNELL MEDICAL COLLEGE LIBRARY on June 27, 2016.
For personal use only. No other uses without permission. From the NEJM Archive. Copyright © 2010 Massachusetts Medical Society. INTRODUCTION
TO
THE
STUDY
OF
THE
FUNDAMENTAL
CAUSE
OF
SPLANCHNOP-
TOSIS
\p=m-\ ABDOMINAL
INCOMPETENCE:
A
DEVELOPMENTAL FACTOR.* A and,
,
y
y
Eventually, it became evident that boys and
men grouped themselves under the
same two
types as girls and women, i. e., the broad and the
retracted lower thorax types. d d th
b Swan, but met with similar results. Three
days prior to this, however, I was able to detect 80
visible, venous pulsations in the right external jugular
vein, while the beats at the heart and wrist registered
only 39 (2:1 rhythm). On April 2, when her pulse
rate was 39 to the minute, I made the following radial
tracing. Her urine examinations have been negative
throughout the course of the disease. These types may be called
as to the lower
thorax
" broad "
or
" retracted ";
though it
will be shown later that they can be also classified
according to the spinal column. g
p
The writer next observed that while a large
number of girls and women with retracted lower
thorax presented a displacement of one or more
of the abdominal viscera, yet apparently
an
equally
large
number
of
abdominal
visceral
displacements occurred in patients who had the
broad
lower
thorax. These
visceral displace-
ments seemed sometimes to be most easily demon-
strable in
the
retracted
thorax
class;
and
this at first made it appear as if they were more
frequent in this class. Further study showed
that many of them are as frequent in the broad
thorax class, but that they occur along somewhat
different lines, and at times require somewhat
different methods for demonstration. In endeavoring to discover the time of onset
and original course of these changes, they were
found already begun in childhood. y
g
In the study of children, these changes were
found to develop spontaneously in boys as well
as in girls, but at first they seemed to be
a
transient phase in boys, tending to disappear
towards puberty,
while tending to persist in
girls. b
i girls. Extending the field of her observations, the
writer next noted that the transiency of these
changes in boys was not uniform; that they per-
sisted in many boys beyond the age of puberty,
and, further, that they existed in many men. i
b
h
b
d The Boston Medical and Surgical Journal as published by Presentation. p
—
Evolution towards two main types of human
figures,
depending
upon
variations
in
these
correlations. The study of these records showed that her
clinical observations and deductions were correct,
and, further, that the two types of development
which she finds existing to-day are the two main
types along which the race has been developing
since the date of the earliest records she has yet
found. Further, at the Musée d'Anthropologie,
at the Jardin des Plantes, in Paris, she found
photographs and other records of contemporary
peoples of more primitive civilization, showing
development along the
same two main lines. Through the courtesy of Professor Halmy, she
is able to show copies of some of the photographs
of these peoples. Type 1. Broad lower thorax;
slight lumbo-
sacral anterior convexity; slight forward rota-
tion of pelvis. pelvis. Type
2. Retracted lower thorax;
marked
lumbo-sacral anterior convexity; marked forward
rotation of pelvis. (This type must be consid-
ered pathological
— a variety of developmental
deformity.) pelvis. Type
2. Retracted lower thorax;
marked
lumbo-sacral anterior convexity; marked forward
rotation of pelvis. (This type must be consid-
ered pathological
— a variety of developmental
deformity.) p
Type
2. Retracted lower thorax;
marked
lumbo-sacral anterior convexity; marked forward
rotation of pelvis. (This type must be consid-
ered pathological
— a variety of developmental
deformity.) deformity.)
Correlations towards which each type tends:
In abdomen, thorax, lower extremities, respira-
tory mechanics, and specialized activities. Presentation. yp
As the study of these cases proceeded, the prob-
lem developed complications in many directions. The spinal column, the pelvis, the lower extremi-
ties, and even the upper extremities and head,
all seemed to be contributing factors that could
not be eliminated. d
i
i
h In 1892, while teaching physical diagnosis at
the Woman's Medical College of the New York
Infirmary, the writer demonstrated to her stud-
ents several cases of girls and women who had
never worn corsets nor sufficiently constricting
clothing, and who yet presented what has been
called
the
corset
figure
retraction
of
the
lower thorax, with more or less marked lumbo-
sacro-thoracic anterior convexity. Di
i
h
thi
i t
i By slow degrees during the ensuing years the
clinical observations made appeared to correlate
themselves along certain lines, but the practical
difficulties in studying these correlations
were
for a long time insurmountable. li i
l
di
l i
ti
i y
Directing her attention to this point, an in-
creasing number of such patients continued to
come under observation. These patients grouped
themselves into three sub-classes:
(1) Girls and
women who had absolutely never worn corsets
or sufficiently constricting clothing;
(2) girls
and women who, while wearing corsets or other
constricting clothing, yet did not
wear these
garments, and had never worn them, sufficiently
tight to make it appear reasonable that such
garments could produce such marked anatomical g
A series of
clinical studies culminating in
1900-01 in that of Case LX, showed that of
the two types, one, the broad lower thorax, is
basic and physiological;
while the other, the
retracted lower thorax, is secondary and tends
toward the pathological. This
case not only
gave the key to the path followed in the spon-
taneous evolution
of
the retracted from the
broad lower thorax, but it also gave the key to
the later evolution of the abdomen. This case
seemed to show that the evolution of the abdo- men
and
lumbo-sacral
spine
was
especially
correlated with the evolution of the lower ex-
tremities, and, though to a less extent, also with
that of the upper extremity. This was followed
by the clinical demonstration that the evolution
of the thoracic cage depends largely on the evo-
lution of the abdomen. At the same time, evo-
lution of the abdomen was observed largely to
modify and determine the mechanics of respiration. Summary. An incompetent
abdominal
wall
is largely
compensated for in the supine position, for then
it is not called upon to share in the support of
the viscera and the rest of the trunk; gravity
in this position causes the viscera to rest on the
back, which, in turn, rests upon the bed or other
supporting surface. As soon
as the sitting
or
standing position (or even the lateral supine), is
assumed, an incompetent abdominal wall yields
to gravity and other displacing factors and it
becomes displaced. i (b) main lines of argument. p
p
In 1903-04, through the granting of a fellow-
ship by the Medical Board of the Hospital of
the Society of the Lying-In, in New York, she
was able to study in the newborn the anatomical
paths predisposing to the evolution of these types. To some extent, she was also able to follow these
paths backward in the fetus. At the same time
she was able to begin the study of the postnatal
evolution of the abdomen in the young infant,
and to study some of the modifications presented
in the pregnant woman. ( )
General argument leading
to
conclusion
that
question is one of evolution. h
bd
i
l quest o
o
In their highest development, the abdominal
walls not only contain the abdominal viscera,
but also retain them in position against gravity
and other opposing forces. opposing
The abdomen may then be said to be compe-
tent. A failure in the retaining power of the abdomi-
nal walls leads to visceral
displacement
as
a
result of the action of gravity and other displac-
ing factors. b
id
i p eg a t
In 1904, through the courtesy of Prof. Emmet
L. Holt, she was able to make some studies of
the young child at the New York Foundling
Hospital. ing
Such an abdomen may be said to be incompe-
tent. g
p
y
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For personal use only. No other uses without permission. From the NEJM Archive. Copyright © 2010 Massachusetts Medical Society. The Boston Medical and Surgical Journal as published by The Boston Medical and Surgical Journal as published by A'
Status at term.
B
P
t
t l A'
Status at term. B
P
t
t l —
B
Postnatal. S d
f
l ti Presentation. Outline of evolution of specific human abdomen. Correlated with evolution of spine, pelvis, thorax
and extremities; especially with extension, adduc-
tion and forward rotation of the lower extremi-
ties, so that the patella and toes point forward. Effects of abdominal incompetence on the me-
chanics of respiration. Anatomical and physio-
logical variations predisposing some of the viscera
to displacement, and relation of this predisposi-
tion to certain frequent pathological conditions. Spontaneous tendencies of the trunk and extrem-
ities towards such structural variations
as
are
called pathological
(developmental deformities). These developmental deformities include round
shoulders,
lateral
and antero-posterior
curva-
tures of the spine, modifications of weak foot,
including
pes planus; retracted lower thorax;
marked
lumbar
or
lumbo-thoracic
anterior
convexity;
marked
forward
rotation
of
the
pelvis;
abdominal
incompetence;
and visceral
ptosis
en masse and individual. Outline of evolution of specific human abdomen. Correlated with evolution of spine, pelvis, thorax
and extremities; especially with extension, adduc-
tion and forward rotation of the lower extremi-
ties, so that the patella and toes point forward. Effects of abdominal incompetence on the me-
chanics of respiration. Anatomical and physio-
logical variations predisposing some of the viscera
to displacement, and relation of this predisposi-
tion to certain frequent pathological conditions. Spontaneous tendencies of the trunk and extrem-
ities towards such structural variations
as
are
called pathological
(developmental deformities). These developmental deformities include round
shoulders,
lateral
and antero-posterior
curva-
tures of the spine, modifications of weak foot,
including
pes planus; retracted lower thorax;
marked
lumbar
or
lumbo-thoracic
anterior
convexity;
marked
forward
rotation
of
the
pelvis;
abdominal
incompetence;
and visceral
ptosis
en masse and individual. E
l ti
t
d
i
f h od y
respiration. In 1901-02, the writer was able to go to Europe,
to have access to many of the originals of the
historical records of the race,
as expressed in
mural drawings, sculpture and painting. She
studied the records of older Asia, of Egypt, of
Greece, of Rome, of medieval Europe, of more
modern Asia, and of modern and contemporary
civilizations, as found in the museums and art
galleries
of
Paris,
Dresden,
Leipzig,
Berlin,
Holland, Belgium and
London. The interven-
tion of
a serious illness prevented her visiting
the south of Europe, and compelled her to return
home. p
—
Evolution towards two main types of human
figures,
depending
upon
variations
in
these
correlations. (a) RÉSUMÉ. General argument leading to conclusion that
question is one of evolution. f
bd question
Outline of evolution of human abdomen. P
t l question
Outline of evolution of human abdomen. P
l A — Prenatal
A' The Boston Medical and Surgical Journal as published by
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p
y
nal of Medicine. Downloaded from nejm.org at WEILL CORNELL MEDICAL C
N
th
ith
t
i
i
F
th
NEJM A
hi
C
i ht © 20 The Boston Medical and Surgical Journal as published by
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B
Postnatal.
St d
f e ol tio —
Study of evolution of human abdomen leads
back to earlier vertebrate series and forward to
postnatal development. bd d sp aced
Given a cavity which contains no free space, and
whose
contents
are movable; whenever the
walls
of the cavity are displaced, in whole or in part,
its contents are displaced, in whole
or in part. postnatal development. Outline of evolution of vertebrate abdomen,
including man, showing continuity and correla-
tions. towards
excursion
of
the
abdominal
viscera
downward and forward. Hence, incompetence of the abdominal wall
always leads to
some displacement of
one
or
more of the abdominal viscera; and, in itself,
is sufficient to cause all stages of visceral ptosis,
up to the complete dislocation of all the abdomi-
nal viscera. Clinical observation shows that this excursion
is also towards the median line; this latter modi-
fication is explained by the observation of the
writer that the sides of the diaphragm descend
more than the center. (Illustrations of this law as manifested in areas
of partial incompetence (active or potential) of
the abdominal walls
— both physiological and
pathological.)
Th The
great
counterbalancing
factor
against
these two forces which make for movement of
the viscera downward, forward,
and towards
the median line of the abdomen, is the abdomi-
nal walls:
(1)
The anterior and antero-lateral
muscles of these walls,
which
make towards
antagonizing the descent of the diaphragm and
lungs, and towards pushing the abdominal viscera
backward, upward and laterally;
and (2) the
posterior and postero-lateral muscles and bones
of these walls and of the posterior wall of the
whole trunk, which make (a) for stability of the
region which furnishes a point of departure for
the action of all the abdominal muscles, as well
as for all the large extensor activities of the trunk
and lower and
upper extremities;
and which
make (b) for enlargement of the thoracic cage in
its posterior and postero-lateral
planes. This
favors backward and outward expansion of the
lungs during inspiration; and this action lessens
the downward and forward excursion of the dia-
phragm and lungs (with consequent en masse dis-
placement of the viscera), decreases stress on the
weaker anterior abdominal walls, and favors the
development and conservation in them of the con-
dition of reflex activity which the writer desig-
nates as competence. —
B
Postnatal.
St d
f e ol tio p
g
)
The viscera retain their " normal " positions
as the result of an equilibrium between gravity
and other displacing factors, on the one side, and
certain counterbalancing
forces
on the
other
side. The essential factors in visceral displacement
are gravity and altered respiratory equilibrium,
including unantagonized downward and forward
excursion of the diaphragm and lungs in in-
spiration. spiration. Unsupported abdominal viscera tend to travel
downward, forward and towards the median line. After traveling in this direction for
a certain
distance, the hollow viscera tend to travel also
upward and
forward,
this
latter
movement
being increased in proportion to the amount of
gas in their contents. g
The downward course is the result of the direct
action of gravity, which is constantly at work in
this direction, in the sitting and standing posi-
tions. It acts upon the abdominal wall as well
as upon the contained viscera. upo
The upward and forward course is due to the
distention of the hollow viscera by gaseous, liquid,
or solid contents. When these viscera are empty
and collapsed, they follow the influence of gravity
alone and travel downward. p
Exposure of the viscera to the action of gravity
is caused directly by incompetence of the ab-
dominal walls, and, indirectly, by this incompe-
tence of the abdominal walls modifying other
body conditions, and being in turn modified by
them, so that these body conditions themselves
still further expose the viscera to the action of
gravity and other displacing factors. Part of the downward course and of the ven-
trad and medad modification of the entire down-
ward course, are due to the unantagonized down-
ward and forward excursion of the diaphragm
and inspiring lungs, which are in action fourteen
to eighteen times every minute;
and this dis-
placing action is active in the supine as well as
in the sitting and standing positions. g a ty
d sp ac g
These body conditions are as follows:
(1) Erect (
ti
l)
iti
f th
t
k g
y
p
g
These body conditions are as follows:
(1) Erect (vertical) position of the trunk. g
y
p
g
These body conditions are as follows:
(1) Erect (vertical) position of the trunk. (2) E t
i
f h
l
i i
i
h body
(1) Erect (vertical) position of the trunk. g
p
y
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(B) Postnatal.
b y
In proportion as the abdomen fails to develop,
or loses after development, its competence, the
viscera tend to be displaced downward, forward
and towards the median line, and the hollow
viscera also upward and forward; in proportion
as the abdomen develops competency, the viscera
tend to be held upward, backward and laterally. Every abdomen is, as a whole, competent or
incompetent. This competence may vary greatly
in degree. positions. From the point of view of this retention of the
viscera in position, the abdomen, as a whole, may
be said to proceed in
an orderly development
from incompetence to competence. i incompetence
p
When
a competent abdomen is studied, its
principal characteristic is seen to be the intimate
and exact correspondence existing between the
viscera and the muscle walls, by which the latter
always move in the direction required to retain
and support the former. The muscle reflex is so
active that when the body is in motion, there is
a constant play of the muscles of the anterior,
lateral and posterior walls of
the abdomen,
adapting the latter to the changed conditions of
pressure and stress which would otherwise reach
the viscera; and against gravity, protecting these
viscera from such conditions by interposing
a
firm muscle plane. i id degree. Every abdomen possesses
areas of localized
incompetence;
this localized incompetence may
be only potential, or it may be actual. only potential,
may
General
incompetence
of
the
abdomen
is
physiological only in early prenatal life. p ys o og ca o y
early prenatal
Development of general competence of the
abdomen begins in later prenatal life;
but its
further
development
can
be
observed
and
studied
from the
moment
of birth, through
infancy and childhood to, in selected cases, its
completion before the age of puberty. th t co p et o
g
puberty. Clinical observation shows that in the majority
of eases this evolution is not completed at the
age of puberty; that incompetence of the abdo-
men, with more or less visceral ptosis, persists
through adolescence and early adult life
— pro-
ceeding, at times, towards greater competence,
or remaining stationary, or retrograding towards
greater
incompetence
and
more
pronounced
visceral ptosis, according to the activity of the
concomitant factors to be enumerated. And,
finally, that in the neighborhood of the fourth plane. —
B
Postnatal.
St d
f e ol tio (2) E t
i
f h
l
i i
i
h ( )
(
) p
(2) Extension of the lower extremities in the
plane of the trunk; this extension being, in its
highest form, accompanied by adduction and
forward rotation,
so that the patella and toes
point forward. s tt g
g p
Part of the ventrad and medad modification of
the entire downward course is also due to the
greater relative development, and the greater
bony content, of the posterior and postero-lateral
abdominal and pelvic walls,
as compared with
the anterior and antero-lateral walls. point
(3) Altered respiratory equilibrium,
includ-
ing unantagonized downward and forward
ex-
cursion of the diaphragm and inspiring lungs. The diaphragm is set obliquely from above
downward and from before backward, between
the thorax and the abdomen. When passive
and relaxed, it domes upward towards the thorax;
when active and contracting, it descends into
the abdomen. Whatever variations in activity
may occur in different parts of the diaphragm, the
oblique plane of its attachment makes its unmodi-
fied descent favor enlargement of the base of the
thoracic cavity, from above downward and from
behind forward, and diminution of the abdomi-
nal cavity in the same direction, i. e., from above
downwards and from behind forwards. d ap ag
inspiring lungs. (4) Retarded or uncompleted evolution of the
abdominal viscera. R
d d (5) Retarded
or uncompleted
evolution
of
the trunk and extremities (and even the head)
in the direction
of mutual independence for
the release of specialized activities. spec a ed
These conditions
can all be studied in their
earlier manifestations through comparative anat-
omy and physiology. In their advanced develop-
ment, their study becomes exceedingly involved
and intricate, because it enters the stage of evo-
lution with which the human race is at present
struggling. p
These conditions
can all be studied in their
earlier manifestations through comparative anat-
omy and physiology. In their advanced develop-
ment, their study becomes exceedingly involved
and intricate, because it enters the stage of evo-
lution with which the human race is at present
struggling. This action would make towards excursion of
the inspiring lungs downward and forward, and struggling. Hence, the conclusion that abdominal compe- active factor is introduced into relationship with
the
abdominal wall,
its
muscle
reflexes
are
immediately aroused. ( )
(B) Postnatal.
b ( )
Viscera appear before body walls. l
l pp
body
Body walls at first merely enclose the viscera. This
enclosure
at
first imperfect;
later,
the
enclosure, as a whole, becomes complete, but cer-
tain localized areas of incompleteness (actual 01
potential) remain. d ,
quite,
vanishing point. One of the earliest characteristics of an incom-
petent abdomen is seen in the respiratory reflex. The incompetent portion of the abdomen is re-
taxed
during
expiration,
as
well
as
during
inspiration;
and upon forced expiration,
as in
coughing, is protruded (as in the case of hernia)
downward and forward, as well as upward and
forward. In advanced
cases the whole abdo-
men acts in this manner. pote t a )
Viscera withdraw within the walls, and, by
an orderly progression, take up within the body
cavity certain positions which occur with suffi-
cient predominance
to
be
called
" normal."
These normal positions are antagonized by the
unbalanced downward and forward excursion
of the diaphragm and inspiring lungs, in the
supine as well as the sitting and standing posi-
tions; by extension of the lower extremities in
the plane of the trunk, in the supine or standing
positions; and by gravity as soon as the individ-
ual assumes a sitting or standing position. Again, the portion of the abdomen which tends
towards competence tends, in
these
cases, to
overaction or even spasm. This tendency is seen
in inspiration as well as in expiration. It affects
most strongly the upper abdominal zone;
and
clinical observation indicates that this is one of
the most potent factors
in producing the
re-
tracted lower thorax. sitting
sta d g pos t o
Coincident with the progression of the viscera
toward
such positions and relative
size and
volume as are called normal, the abdominal walls,
as a whole, pass through a series of developmental
changes, as a result of which they not only en-
close the viscera, but also, by counteracting the
displacing action of the diaphragm and inspiring
lungs, of extension of the lower extremities and
of gravity, they retain the viscera in their normal
positions. The same tendency to spasm is seen in the ex-
tensor muscles of the back, and is an important
factor
in increasing
the
sacro-lumbo-thoracic
anterior convexity. The Boston Medical and Surgical Journal as published by
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(A') Status at term.
(B) P
t
t l ( )
(A') Status at term. (B) P
t
t l ( )
(B) Postnatal. i
b The Boston Medical and Surgical Journal as published by
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B
Postnatal.
St d
f e ol tio Even during sleep, the
muscular response of the abdominal walls to the
visceral and other reflexes may be observed and
studied. The earliest characteristic of an incom-
petent abdomen is a change in this muscle reflex,
which becomes erratic, irregular,
or partly
or
entirely absent. Later, structural changes occur,
and portions of the abdominal walls may hang
as
more
or less flaccid pouches whose reflex
activity,
under
ordinary
circumstances,
has
diminished almost, or quite, to a vanishing point. f h
h
t i ti tence is
a developmental factor. This conclu-
sion is verified by finding that its earlier phase;
can be traced backward through the vertebrat«
series and in human prenatal life; while the evo-
lution of its later phases can be observed anc
studied throughout human postnatal life. throughout
postnatal
Outline of evolution of human abdomen. (A) Prenatal. (A) Prenatal. S ( )
(A') Status at term. (B) P
t
t l ( )
(B) Postnatal.
b The abdominal wall is neither a rigid nor a
flaccid structure, but one which is elastic and
contractile;
and this elasticity and contractility
is constantly controlled by visceral and other
reflexes. These reflexes are so co-ordinated that
a relaxation of one part of the wall is controlled
by the adjacent parts of the wall. In a perfectly
passive condition, the wall may relax in part or
as a whole;
but in a competent abdomen the
reflexes are also competent, and as soon as an of the Society of the Lying-in Hospital of New
York, 1903-04, by Agnes C. Vietor, M.D. (In
press.) of the Society of the Lying-in Hospital of New
York, 1903-04, by Agnes C. Vietor, M.D. (In
press.) decade and beyond there is marked tendency in
all individuals towards increased incompetence
of the abdomen, with increased visceral ptosis. press.)
(B) Postnatal. I
h press.)
(B) Postnatal. I
h ,
ptosis. Competence of the abdominal walls is, then,
evolutionary;
and while related to evolution of
the abdominal viscera, is a later stage of develop-
ment, and thus appears as to some extent inde-
pendent of this latter;
so that the abdominal
viscera may proceed to the attainment of their
normal position, although the walls are insuffi-
ciently developed towards competence; and it is,
at least, possible that competent walls might be
developed around viscera that had not entirely
attained their normal position. ( )
In the supine position of infancy, the postnatal
evolution of the abdomen proceeds. This evo-
lution differs from the prenatal in that there
are added the reactions of a new environment. ( )
In the supine position of infancy, the postnatal
evolution of the abdomen proceeds. This evo-
lution differs from the prenatal in that there
are added the reactions of a new environment. (1) Beginning of two
new forms of visceral
reflex activity with birth: (a) respiratory (abso-
lutely new), and (b) digestive; this latter is new
in regard to the presence in the stomach and
bowels of food derived from the outside world;
and the new activities set in motion by this factor
in the liver, pancreas, kidneys, etc., setting free
numerous new reflex stimuli to the muscles of
the abdominal wall. ( )
(B) Postnatal.
b E
l
i
f
ll
i STUDY
OF EVOLUTION
OF THE
HUMAN
ABDOMEN
LEADS BACK TO EARLIER VERTEBRATE SERIES,
AND
O POSTNATAL AND FORWARD TO POSTNATAL DEVELOPMENT. E
l
i
f
bd
i
l
ll
d
i Evolution of human abdominal walls and vis-
cera closely related to evolution of mammalian,
or even vertebrate, abdominal walls and viscera. ,
Prenatal human development
so closely
re-
sembles prenatal vertebrate development that
failures in evolution of the human abdominal
walls and viscera would most naturally be ex-
pected along lines suggesting the varying verte-
brate paths. a y subsequent
g
This instability of the abdomen exists at the
present day in the most primitive as well as the
most civilized peoples; and its existence can be
traced among all peoples as far back as pictorial
history extends. Illustrations. paths. Study of the evolution of the vertebrate ab-
dominal walls and viscera, taken in connection
with the fact that a progressive postnatal evolu-
tion of the human abdominal walls and viscera is
constantly present in every individual, show that
so-called
congenital
variations
in
either
the
abdominal walls or viscera should not necessarily
be regarded
as end results. These congenital
variations may be merely retarded steps of the
still ascending ladder; and, in such cases, their
normal evolution may be accelerated or assisted
by a knowledge of the normal postnatal develop-
ment. y
Incompetence
of the abdominal walls with
the consequent displacement of the abdominal
viscera, continually struggling towards compe-
tence of the abdominal walls with the
conse-
quent retention of the viscera in position is,
therefore,
a generic condition, or even a verte-
brate condition. Seeking the anatomical basis for this generic
condition, one is carried backward to the verte-
brate series, and one notes that competence of the
abdominal walls is correlated successively with successively
—
(1) The ascent of the trunk from a horizontal
to a vertical plane;
an ascent in which the dorsal
surface or back leads, the ventral surface lagging. This ascent begins at the cephalic extremity of
the body and proceeds caudad. OUTLINE
OF
EVOLUTION
OF
VERTEBRATE
ABDO-
MEN,
INCLUDING
MAN,
SHOWING
CONTINUITY
AND CORRELATIONS. OUTLINE
OF
EVOLUTION
OF
VERTEBRATE
ABDO-
MEN,
INCLUDING
MAN,
SHOWING
CONTINUITY
AND CORRELATIONS. body
proceeds
(2) The tendency of the ventral and dorsal
surfaces of this vertical trunk to approach each
other in approximately parallel planes. The Boston Medical and Surgical Journal as published by
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For personal use only. No other uses without permission. From the NEJM Archive. Copyright © 2010 Massachusetts Medical Society. ( )
(B) Postnatal.
b p
Nevertheless, bearing in mind the law that,
given a cavity which contains no free space, and
whose contents are movable, whenever the walls
of the cavity are displaced, in whole or part, its
contents are displaced in whole or part; and as
it can be demonstrated that the abdominal wall
is under the constant control of visceral and other
reflexes, and is competent
or incompetent
ac-
cording as these reflexes are active or in abey-
ance, it is rational to think that abdominal walls
which have not attained competence must always
affect the relative size and volume, and hence
the relative or actual position of the contained
viscera. And, conversely, that
viscera
which
have not attained their normal size and volume,
and hence their relative or actual position, must
affect the attainment of competency by the con-
taining walls. (2) Beginning of
new surface stimuli from
changed environment (thermal, tactile, etc.). (2) Beginning of
new surface stimuli from
changed environment (thermal, tactile, etc.). c a ged
(
, tactile,
)
(3) Increased and new activity of the muscles
of the lower extremities, spine, pelvis, thorax,
upper extremities, and even the neck and head. Activity of all these regions can be shown to have
direct connection with the activity and develop-
ment of the abdominal walls
— the lower extremi-
ties, pelvis,
lumbar spine
and thoracic
cage
being most immediately and powerfully related
though the upper extremities are also of great
importance importance. Such a large number of contributing factors
sufficiently
indicate
the
complexities
of
the
process of evolution of the abdominal walls. taining
Hence the study of visceral ptosis requires
the study of the development of both viscera and
walls. p
Clinical
observations
of
the
writer during
fourteen years show that this complex process is
still in progress;
that the human
race is still
struggling towards the attainment of a compe-
tent abdomen and the retention of the abdominal
viscera against gravity
and
other displacing
forces; that only a minority of the race attain
this higher development, and that though at-
tained, it is an unstable possession and may be
lost at any subsequent age. STUDY
OF EVOLUTION
OF THE
HUMAN
ABDOMEN STUDY
OF EVOLUTION
OF THE
HUMAN
ABDOMEN
LEADS BACK TO EARLIER VERTEBRATE SERIES,
AND FORWARD TO POSTNATAL DEVELOPMENT. The Boston Medical and Surgical Journal as published by
The New England Journal of Medicine. Downloaded from nejm.org at WEILL CORNELL MEDICAL COLLEGE
For personal use only. No other uses without permission. From the NEJM Archive. Copyright © 2010 Mass ( )
(B) Postnatal.
b body
proceeds
(2) The tendency of the ventral and dorsal
surfaces of this vertical trunk to approach each
other in approximately parallel planes. (A) Prenatal. (A') Status at term. For details, see Report of Research Fellowship medium of weight transference between the spine
and the acetabula. (3) The affiliation of the pelvis with the spine,
and its consequent association with the trunk
rather than with the lower extremities
— the
pelvis becoming virtually an exjDanded sacrum,
following this latter bone in all its positions;
a
transference of affinities from the position of
proximal division of the lower extremity to that
of distal division of the trunk. (3) The affiliation of the pelvis with the spine,
and its consequent association with the trunk
rather than with the lower extremities
— the
pelvis becoming virtually an exjDanded sacrum,
following this latter bone in all its positions;
a
transference of affinities from the position of
proximal division of the lower extremity to that
of distal division of the trunk. This bony support is supplemented by fibrous
and muscular tissues, but the weight of the trunk
walls and viscera is so great that until these walls
have adapted themselves to the
new position
by transitional changes, the lower extremities
are still called upon to assist in weight-bearing
in the older mammalian way. This is done by
the
femora continuing flexed,
abducted
and
rotated outward, with corresponding positions
of the leg and foot. (4) The gradual transference of the weight-
bearing function which, beginning with the body
wall itself, is transferred to four specialized out-
growths of this wall, the two fore and two hind
limbs. These limbs combine the function
of
weight-bearing (a more or less passive process)
with various specialized functions (active proc-
esses),
prehension,
locomotion,
etc.;
and
in
proportion as one of these classes of functions
predominates, the other is impaired, and
con-
versely. Thus,
exercise of the weight-bearing
function impairs
the
exercise
of
the higher
specializing functions; and exercise of the higher
specializing functions impairs the
exercise of
the weight-bearing function. eg
This is a frequent position of many mammals,
and these processes may be shown in develop-
ment in studying the change from the horizontal
to the vertical trank in one of the higher mam-
malia, as the dog or bear. ( )
(B) Postnatal.
b ,
g
As the trunk is erected and the weight-bearing
is transferred to the lower extremities, the pelvis
follows the sacrum, which, in turn, follows the
main spine in its vertical ascent. As
a result,
the pelvis is no longer erect, resting on the tuber
ischii or on the oblique plane posterior or anterior
to these processes, and assisting the abdominal
walls and back in the support of the viscera. In-
stead, the pelvis becomes inclined obliquely to
the vertical plane,
so that the crests of the ilii
tend to fall forward and downward, and the tuber
ischii rise backward and upward. Instead of a
containing basin, acting as a receptacle for the
viscera, the pelvis becomes a tilted basin, throw-
ing the viscera forward and downward against
the abdominal walls. g
g
(5) The gradual transference of the weight-
bearing function from the cephalic towards the
caudal extremity of the trunk, and the conse-
quent development of the cephalic division and
outgrowths
of
the trunk,
so set free, towards
independence and more highly specialized func-
tionating. tionating. These
processes may be seen epitomized
in
studying the ascent of the infant from the prone
to the creeping position. creeping position. (6) The assumption along this path of the
weight-bearing function by the pelvis, which is
now the lowest division of a more or less vertical
trunk. The pelvis is more or less erect, resting
on the tuber ischii or on the oblique plane pos-
terior to these processes,
more, rarely
on the
oblique plane anterior to these processes. It
acts
as a containing basin and assists the ab-
dominal walls and back in the support of the
viscera. In this position, the hind limbs
are
freed from weight-bearing, but the thighs remain
flexed,
abducted
and
rotated outward,
in
a
position well suited to that variation of their
weight-bearing
function which
leads
to their
supplementing the pelvis and abdominal walls
in supporting the weight
of the viscera, but
also
a
position
favoring
earlier
specialized
functions. This forward inclination of the pelvis follows
the extension of the lower extremities (thigh)
in the plane of the trunk. It may be studied in
the stillborn, in the supine position, by changing
the lower extremities (thigh) from the position
of spontaneous flexion, abduction and outward
rotation, present at term, to that of extension,
adduction and forward rotation. g
p
y
The New England Journal of Medicine. Downloaded from nejm.org at WEILL CORNELL MEDICAL COLLEGE LIBRARY on June 27, 2016.
For personal use only. No other uses without permission. From the NEJM Archive. Copyright © 2010 Massachusetts Medical Society. The Boston Medical and Surgical Journal as published by The Boston Medical and Surgical Journal as published by
The New England Journal of Medicine. Downloaded from nejm.org at WEILL CORNELL MEDICAL COLLEGE LIBRARY on June 27, 2016.
For personal use only. No other uses without permission. From the NEJM Archive. Copyright © 2010 Massachusetts Medical Society. The Boston Medical and Surgical Journal as published by The Boston Medical and Surgical Journal as published by WHAT SHOULD BE THE ATTITUDE OF PUBLIC
SANATORIA TOWARD CASES OF TUBERCU-
LAR LARYNGITIS; WITH SUGGESTIONS AS
TO THE GENERAL PLAN OF TREATMENT OF
SUCH
CASES
IN
SANATORIA. sion,
larynx s,
rule,
In 1901, the German government published and
distributed at the British Congress on Tubercu-
losis the results of the treatment of 6,273 patients
treated àt 31 sanatoria. I can find no reference
in this report to laryngeal tuberculosis at all, nor
does any mention of the throat occur in the copy
of the statistical card used by the Imperial health
board, though there is a column for complications
of all kinds. In the application blank of our own
Massachusetts State Sanatorium there is a line
for condition of the throat, but a search of the
reports and the publications emanating from the
sanatorium, so far as I have had access to them,
would seem to show that in the past at least, this
factor in tuberculosis has not had much promi-
nence given to it, as I find no reference whatever
to such a thing as laryngeal tuberculosis. b
i BY GEORGE L. RICHARDS, M.D., FALL RIVER, MASS.,
Otologist and Laryngologist Fall River Union Hospital, Fellow Ameri-
can Laryngological Association, American Otological Association, etc. Viewed from the standpoint of its literature
for the last few years, the workers in the field of
tuberculosis can be divided into two classes with
somewhat different points of view, though dealing
with the same general problem. general problem. First, that class of workers who treat tubercu-
losis purely from the standpoint of an affection of
the lungs, with the knowledge that it secondarily
involves other organs, but who in the main dis-
regard those organs either as etiological factors
or to be specially considered from the therapeutic
standpoint;
and,
secondly,
the
distinctively
special workers in the diseases of the upper air
tract, who treat and write of tuberculosis
as
they see it, namely,
as affecting the larynx, the
pharynx and the nose. Very little on the subject
of the laryngeal aspects of tuberculosis can be
found in the writings of the first class, and rela-
tively little as to the pulmonary side of it in the
writings of the second. It has seemed to me that
there should be more community of effort in the
ranks of the two workers, and that the laryngeal
aspect of these cases was being decidedly neglected
by phthisiologists in general. WHAT SHOULD BE THE ATTITUDE OF PUBLIC
SANATORIA TOWARD CASES OF TUBERCU-
LAR LARYNGITIS; WITH SUGGESTIONS AS
TO THE GENERAL PLAN OF TREATMENT OF
SUCH
CASES
IN
SANATORIA. I believe, there-
fore, that it is time for the public sanatorium and
the workers in behalf of it to take more into ac-
count the laryngeal side of these cases, not only
as regards treatment, but as regards early diag-
nosis as well, and that the phthisiologist and the
laryngologist should work together, or else that
the phthisiologist should be, from a diagnostic
standpoint at least, sufficiently trained in laryn-
gology to make a proper laryngological examina-
tion of every case on which he passes an opinion,
and be competent to properly treat the laryngeal
complication should it be present. U l
I
h
i t k
h thing
laryngeal
From the little which will be found in the publi-
cations of the general phthisiologist on the laryn-
geal side of this subject, it would seem as though
laryngeal tuberculosis could almost be ignored
as a prominent factor in the disease, yet when one
turns to the writings of the distinct specialists in
throat diseases, one finds a mass of literature
devoted to it. Statistics as to the absolute fre-
quency of laryngeal tuberculosis are frequently
unreliable because the average case of consump-
tion does not have an examination of the larynx
made unless the laryngeal symptoms are so mani-
fest that the patient's physician or his friends
send him, sooner, or later, to the laryngologist. Such statistics as I have been able to find would
seem to show that the relative proportion is at
least as high as 15% to 20% in which there are
positive laryngeal lesions, and some statistics give
it very much higher. Habershon examined the
records of post-mortem examinations at Bromp-
ton for ten years and found 1,255 cases of pul-
monary tuberculosis,
853 in
men and
402 in
women. Among them 595
cases of tubercular
laryngitis were recorded, about 48%, of which
116 only were in females. l
h lf
f h o y
For the last year and a half all of the patients
entering
the
Massachusetts
State
Sanatorium
have had a laryngeal examination made, and the
results of such examination recorded. No reports
have been published nor have they as yet been
tabulated, but I am informed that the proportion
of patients showing laryngeal lesions would not
be anywhere near as high as above stated. ( )
(B) Postnatal.
b These processes may be shown in studying the
young infant, in the supine position, as it spon-
taneously develops this extension of the lower
extremity, with forward rotation of the pelvis;
and in the child, as it progresses from the sitting
to the standing position. standing pos t o
(8) And, finally,
the
concentration
of
the
caudad
transference
of
weight-bearing
along
each lower extremity, independently,
so that,
in its highest form, each lower extremity is in
itself able to support the weight of the entire
body, leaving the other lower extremity free to
exercise its specialized function of locomotion,
etc., in the vertical plane. This is an occasional position of many mam-
mals, especially of the anthropoids and of the
sitting child. Its evolution may be shown in
the study of the ascent of the infant from the
supine to the sitting position. etc.,
plane. Illustrations of (1) to (7) in studying the changes
produced in passing from the horizontal to the
vertical trunk in one of the higher mammals, as
the dog or the bear. i supine
sitting position. (7) The further transference caudad of the
weight-bearing function to the two lower
ex-
tremities, which become, for the first time in the
animal series, fully extended in the plane of the
vertical trunk. dog
Similar changes observed in studying the new-
born and infant. b The expanded, weight-bearing
base
of
the
trunk, the pelvis, is suddenly contracted to the
small area of
contact
between
the
acetabula
and the heads of the femora, in connection with
the bony portions of the pelvis which act as
a Study of the newborn, the infant and the child
illustrates similar changes, and also the evolution
of (8). Study of the newborn, the infant and the child
illustrates similar changes, and also the evolution
of (8). ( )
Mechanism of this evolution as observed in the
study of attitudes and spontaneous movements ( )
Mechanism of this evolution as observed in the
study of attitudes and spontaneous movements this
purpose. The Boston Medical and Surgical Journal as published by
The New England Journal of Medicine. Downloaded from nejm.org at WEILL CORNELL MEDICAL COLLEGE LIBRARY on June 27, 2016.
For personal use only. No other uses without permission. From the NEJM Archive. Copyright © 2010 Massachusetts Medical Society. The Boston Medical and Surgical Journal as published by
The New England Journal of Medicine. Downloaded from nejm.org at WEILL CORNELL MEDICAL COLLEGE LIBRARY on June 27, 2016.
For personal use only. No other uses without permission. From the NEJM Archive. Copyright © 2010 Massachusetts Medical Society. The Boston Medical and Surgical Journal as published by The Boston Medical and Surgical Journal as published by
The New England Journal of Medicine Downloaded from nejm org at WEILL CORNELL MEDICAL COLLEGE LIBRARY on June 27 2016 ( )
(B) Postnatal.
b On looking
over
a number of
reports of public and private sanatoria, I find
almost no reference to the laryngeal complica-
tions, although now and then an announcement
of a private sanatorium will state that it has a
well-equipped throat room, and a picture of such
a room may be given, equipped with more or
less impressive looking apparatus, yet in the
statistics no mention will be made of the presence
or absence of tubercular laryngitis,
nor do I
think that in the routine examination for admis-
sion, the larynx is, as a rule, examined. of newborn and infants, and the creeping, sitting
and standing child. this
purpose. On looking
over
a number of
reports of public and private sanatoria, I find
almost no reference to the laryngeal complica-
tions, although now and then an announcement
of a private sanatorium will state that it has a
well-equipped throat room, and a picture of such
a room may be given, equipped with more or
less impressive looking apparatus, yet in the
statistics no mention will be made of the presence
or absence of tubercular laryngitis,
nor do I
think that in the routine examination for admis-
sion, the larynx is, as a rule, examined. standing
Illustration of this mechanism by diagrams,
measurements and photographs of stillborn, and
by photographs of infants and children. by photographs
Illustrations of continuity of this mechanism
in the adolescent and adult by diagrams, meas-
urements and photographs. (To be continued.) (To be continued.) WHAT SHOULD BE THE ATTITUDE OF PUBLIC
SANATORIA TOWARD CASES OF TUBERCU-
LAR LARYNGITIS; WITH SUGGESTIONS AS
TO THE GENERAL PLAN OF TREATMENT OF
SUCH
CASES
IN
SANATORIA. J b
H
f L
d co p cat o
p
Unless I am very much mistaken, the average
sanatorium physician and the average sanatorium
is inclined to discriminate against cases of laryn-
geal tuberculosis
as regards the admittance of
patients, and is very seldom properly equipped
to care for them if these lesions happen to affect
any of the patients under their care. Most sana-
toria either make no provision for the treatment
of the throat complications of tuberculosis, or,
if they do, do so only in a half-hearted manner,
and seldom have properly trained assistants for a y
e e
g
In a paper by W. Jobson Home, of London,
presented at the annual meeting of the British
Medical Association at Leicester, in 1905, he refers
to a clinical and pathological research which was
|
https://openalex.org/W3172617793
|
https://ojs.uph.edu/index.php/DIL/article/download/3564/1602
|
Indonesian
| null |
Perpektif Makna Cinta dalam Kumpulan Puisi Esai Atas Nama Cinta Karya Denny JA [The Perspective of Meaning of Love in the Compilation of Poetry Titled "Atas Nama Cinta" by Denny JA]
|
Diligentia : Journal of Theology and Christian Education
| 2,021
|
cc-by-sa
| 5,397
|
Keywords: Essay Poetry, The Meaning of Love, Reconstruction, Social Education Keywords: Essay Poetry, The Meaning of Love, Reconstruction, Social Education Keywords: Essay Poetry, The Meaning of Love, Reconstruction, Social Educatio E-ISSN: 2686-3707 E-ISSN: 2686-3707 Diligentia: Journal of Theology and Christian Education
ojs.uph.edu/index.php/DIL Correspondence email: pitaya.rahmadi@uph.edu Received: 13/05/2021 1 Burhan Nurgiyantoro, Teori Pengkajian Fiksi (Yogyakarta, Indonesia: Gajah Mada University
Press, 2010), 181. Accepted: 28/05/2021 Published: 31/05/2021 Perpektif Makna Cinta dalam Kumpulan Puisi Esai
Atas Nama Cinta Karya Denny JA Pitaya Rahmadi
Universitas Pelita Harapan, Indonesia Abstract The community plays a role in empowering the use of literary works, including, in this case, poetry
essays to express the realities that happened in society. Through the five essay poems written by Denny
JA, various facts are presented to be used as lessons for readers as part of society. This collection of
stories of different kinds of love offered by Denny JA opens the eyes and awakens the readers' minds
that love is no love is never free or natural. Love is still imprisoned by religion, discrimination, by the
moral norms of society. Is there true love that transcends religion, overcomes discrimination, and is
above the moral norms of society? This collection of five poetry essays Atas Nama Cinta by Denny JA
serves as a starting point for reflections to answer the restlessness expressed inf the series of questions
above. The meaning of love as described by Denny JA raises the perspective of both internal social
problems related to the psyche due to conflicting thoughts that exist in the person's character—and
externally associated with the character's interaction with the surrounding environment. The meaning
of love needs to be reconstructed to convey educational values to readers as part of society. Literary
works, including poetry, are not only psychological expressions of the author but also reflect recorded
records of various events in a community so that poetry can present a history of conflicts that exist in
the author's memory. 2 Herman J. Waluyo, Apresiasi Puisi (Jakarta, Indonesia: Gramedia Pustaka Utama, 2002), 93.
3 Denny J.A., Menjelaskan Puisi Esai: Visi Denny JA Dan Respon Sastrawan Kritikus Indonesia+ Manca
Negara. (Jakarta, Indonesia: Cerah Budaya Indonesia, 2019), 103.
4 Nurgiyantoro, Teori Pengkajian Fiksi, 181. Pendahuluan Karya sastra termasuk puisi, di samping merupakan ekspresi kejiwaan pengarang, juga
mencerminkan rekaman berbagai peristiwa dalam suatu masyarakat Dengan itu, puisi
memiliki potensi menghadirkan konflik baik internal maupun eksternal. Dalam kaitan itu,
Stanton1 menyatakan bahwa konflik eksternal dibagi menjadi dua yaitu konflik fisik dan
konflik sosial. Konflik fisik adalah konflik yang disebabkan oleh adanya pertentangan antara
tokoh dengan lingkungan alam; dan Konflik sosial adalah konflik yang disebabkan oleh
adanya kontak sosial atau masalah-masalah yang muncul akibat adanya hubungan antar
manusia. Kumpulan puisi esai “Atas Nama Cinta” karya Denny JA, mendeskripsikan adanya
konflik yang dialami oleh tokoh-tokoh terhadap lingkungannya. Bertemali dengan hal itu,
melalui peristiwa atau tragedi yang digambarkan dalam puisi, penyair berusaha meyakinkan
pembaca tentang tingginya martabat manusia. Karena itu, manusia harus dihargai, Diligentia Vol. 3, No. 2, May 2021 Page 173 Page 173 dihormati, diperhatikan hak-haknya, dan diperlakukan secara adil dan manusiawi.2
Menyimak dan menggarisbawahi apa yang disampaikan oleh Waluyo di atas menjadi sebuah
persetujuan bahwa Denny JA dalam puisi esai Atas Nama Cinta, telah memaparkan keresahan
hatinya terkait diskriminasi (konflik eksternal), baik terhadap kaum perempuan maupun
terhadap orang-orang yang terpinggirkan. Tampaknya masalah diskriminasi selalu bercokol
dalam pikiran seorang Denny JA, bahkan catatan pikiran itu mungkin tak kan terhapus
selama kehidupan Denny. Warna-warni perasaaan emosi yang ada dalam perasaan dan pikiran seorang Denny JA
bermula dari rekaman fakta dalam kehidupan yang ia amati, bermetamorfosa menjadi karya
fiktif yang mengharu biru emosi pembacanya. Atas Nama Cinta yang merupakan kumpulan puisi esai menghadirkan konflik para
tokoh yang menarik untuk dikaji karena berkaitan dengan permasalahan diskriminasi sosial. Oleh karena itu peneliti mengangkat judul penelitian Perspektif Makna Cinta terhadap konflik
reformasi sosial. analisis dalam penelitian ini memanfaatkan karakteristik prosa oleh karena
dalam puisi esai terdapat kesejajaran unsur intrinsik strukturnya dengan karya sastra prosa
yaitu adanya konflik. unsur konflik inilah yang akan menjadi pusat kajian penelitian ini. Puisi Esai Puisi esai yang ditawarkan Denny JA memiliki sejumlah karakteristik yang baru. Pertama, puisi esai mengeksplorasi sisi batin individu yang sedang berada dalam sebuah
konflik sosial. Kedua, puisi esai menggunakan bahasa yang mudah dipahami. Semua
perangkat bahasa seperti metafor, analogi, dan sebagainya justru bagus untuk dipilih. Prinsip
puisi esai, semakin sulit puisi itu dipahami publik luas, semakin buruk puisi itu sebagai
medium komunikasi penyair dan dunia di luarnya. Ketiga, puisi esai adalah fiksi. Boleh saja
puisi esai itu memotret tokoh asli yang hidup dalam sejarah. Namun, realitas itu diperkaya
dengan aneka tokoh fiktif dan dramatisasi. Hal penting yang dimuat dalam puisi esai adalah
renungan dan kandungan moral yang disampaikan lewat sebuah kisah, bukan semata potret
akurat sebuah sejarah. Keempat, puisi esai tidak hanya lahir dari imajinasi penyair tapi hasil
riset minimal realitas sosial. Puisi merespons isu sosial yang sedang bergetar di sebuah
komunitas, apa pun itu. Catatan kaki menjadi sentral dalam puisi esai. Catatan kaki itu
menunjukkan bahwa fiksi ini berangkat dari fakta sosial. Kelima, puisi esai berbabak dan
panjang. Pada dasarnya puisi esai itu adalah drama atau cerpen yang dipuisikan.3 Dari
paparan di atas jelaslah di sini bahwa kumpulan puisi “Atas Nama Cinta” karya Denny JA
merupakan kisah dengan karakteristik prosa yang sarat dengan konflik para tokoh di
dalamnya. Konflik Karya sastra tidak bisa dilepaskan dari konflik, bahkan konflik menjadi jantung utama
cerita. Tanpa adanya konflik, sebuah karya tidak dapat menjadi karya yang baik. Stanton4
menyatakan bahwa konflik dalam cerita dapat dibagi menjadi dua jenis yaitu pertama,
konflik internal dan eksternal. Pertama, konflik internal atau konflik kejiwaan, yaitu konflik Diligentia Vol. 3, No. 2, May 2021 Page 174 akibat adanya pertentangan antara manusia dengan dirinya sendiri misalnya pertentangan
antara dua keinginan, pilihan yang berbeda, atau masalah lainnya. Kedua, konflik eksternal
adalah konflik yang terjadi antara seseorang tokoh dengan sesuatu di luar dirinya, mungkin
dengan lingkungan alam atau lingkungan manusia. akibat adanya pertentangan antara manusia dengan dirinya sendiri misalnya pertentangan
antara dua keinginan, pilihan yang berbeda, atau masalah lainnya. Kedua, konflik eksternal
adalah konflik yang terjadi antara seseorang tokoh dengan sesuatu di luar dirinya, mungkin
dengan lingkungan alam atau lingkungan manusia. 6 John Cresswell, Riset Pendidikan (Yogyakarta, Indonesia: Pustaka Pelajar, 2005), 221.
7 M. Atar Semi, Metode Penelitian Sastra (Bandung, Indonesia: Angkasa, 2012), 89. Metode Penelitian Metode yang digunakan dalam penelitian ini adalah metode deskriptif kualitatif. Menurut Basrowi dan Suwandi, metode kualitatif adalah jenis penelitian yang menghasilkan
penemuan-penemuan yang tidak dicapai dengan prosedur-prosedur statistik atau dengan
cara kuantifikasi lainnya.5 Secara lebih operasional, menurut Creswell, penelitian kualitatif
adalah penelitian yang dilakukan dengan mengumpulkan basis data teks sehingga analisis
data teks itu terdiri atas permbagian menjadi kelompok kalimat yang disebut segmen teks,
dan menentukan makna tiap-tiap kelompok kalimat.6 Oleh karena itu, penelitian ini
berbentuk penguraian secara deskriptif. Metode deskriptif menurut Semi adalah penelitian
yang mendasarkan diri pada data yang berupa kata-kata atau gambar-gambar bukan angka-
angka.7 Dalam analisis kumpulan puisi Atas Nama Cinta karya Denny JA ini, peneliti
mengamati data berupa baris-baris kata puisi kemudian ditentukan jenis konflik yang terjadi
pada tokoh-tokoh yang dipaparkan. 5 Basrowi and Suwandi, Memahami Penelitian Kualitatif (Jakarta, Indonesia: Rineka Cipta, 2008 5 Basrowi and Suwandi, Memahami Penelitian Kualitatif (Jakarta, Indonesia: Rineka Cipta, 2008),
24.
6 John Cresswell, Riset Pendidikan (Yogyakarta, Indonesia: Pustaka Pelajar, 2005), 221.
7 M. Atar Semi, Metode Penelitian Sastra (Bandung, Indonesia: Angkasa, 2012), 89. Deskripsi Data Atas Nama Cinta secara keseluruhan memuat 5 puisi dengan setting berbeda. Sapu
Tangan Fang Yin sebagai puisi pertama, menyuguhkan keresahan hati, kemirisan, dan
kebiadaban moral yang mendera gadis Tionghoa korban perkosaan sadis dalam kerusuhan
Mei ’98, sehingga ia harus melarikan diri ke Amerika supaya kehidupannya bisa terus
berlanjut. Selama tiga belas tahun beban psikologis-trauma yang hebat mewarnai terapi yang
harus dijalaninya sebelum akhirnya muncul kerinduan Fang Yin akan Indonesia, tanah
kelahirannya, termasuk kenangan cintanya bersama kekasihnya Kho. Romi dan Yuli dari Cikeusik adalah puisi esai yang kedua. Pada puisi kedua ini Denny JA
menggoreskan mirisnya cinta yang terhalang karena ego orang tua. Karakter Yuli dan Romi
digambarkan sebagai pasangan yang saling mencintai. Perjalanan cintanya hampir menuju
puncak peresmian pernikahan, tetapi semuanya batal demi dan atas nama aliran agama. Denny dengan runtut menggambarkan kerasnya aliran agama yang dianut orang tua Yuli,
sampai-sampai ia mendiskreditkan aliran agama lain. Fanatisme yang tidak pada tempatnya
ini, akhirnya menjadi dasar untuk membangun benteng tinggi yang memisahkan cinta Yuli
dan Romi, sehingga pernikahan yang merupakan puncak cinta kedua sejoli harus BATAL. Tragis dan haru, Yuli pergi untuk selamanya dengan membawa cinta tulus kekasihnya yang
terpisahkan oleh ego orang tua. Puisi ketiga, Minah Tetap Dipancung. Puisi ini mengisahkan Minah, seorang TKW yang
bekerja di Arab Saudi yang harus menjalani hukuman pancung karena didakwa membunuh
majikannya. Pada puisi esai ketiga ini Denny JA menggambarkan dua dunia yang sangat Diligentia Vol. 3, No. 2, May 2021 Page 175 Page 175 berlawanan. Di Arab Saudi, Minah menjalani kehidupan pedih-perih dan ketidakberdayaan
pemberontakannya terhadap majikan. berlawanan. Di Arab Saudi, Minah menjalani kehidupan pedih-perih dan ketidakberdayaan
pemberontakannya terhadap majikan. Di aspek yang lain, Minah memiliki dunia tersendiri yang penuh ‘bunga’, yaitu
bayangan indahnya cinta kepada suami dan keluarga. Kontras yang ditampilkan oleh Denny,
menyeret pembaca pada kegeraman terhadap perilaku manusia laki-laki Arab Saudi yang
membawa Minah pada hukuman pancung. Harapan Minah pulang dari Arab Saudi
membawa devisa menjadi kenangan duka bagi suami, keluarga, dan bangsa Indonesia. Puisi esai keempat, Cinta Terlarang Batman dan Robin. Kali ini Denny JA sangat berani
menggambarkan cinta terlarang yang tidak semua orang mau memaparkannya. Kisah cinta
Batman dan Robin yang disuguhkan oleh Denny adalah sebuah kisah unik yang dirasakan
oleh pasangan sejenis. Penggambaran konflik yang tertuang mengenai terlarangnya menjalin
asmara dengan sesama jenis dilukiskan dengan cukup lembut, walaupun sebagian orang
yang sudah membacanya dapat saja berpikir bahwa yang dikemukakan penyair adalah hal
yang tabu. Pembahasan Melalui kumpulan puisi esai Atas Nama Cinta ini, Denny JA kuat sekali memaparkan
fakta yang dikemas dalam karya sastra fiktif. Benar-benar suatu ide cerdas, menurut penulis,
terkait apa yang dikreasikan oleh Denny JA, yaitu memadukan kenyataan hidup sehari-hari
dengan fiksi, sehingga tercipta bentuk puisi esai yang lugas, namun tetap mampu mengharu-
biru perasaan pembaca. Dengan puisi esai, Denny JA sebenarnya berdiri di antara 2 bentuk
karya sastra –--cerpen dan puisi–-- tanpa harus memikirkan kompleksitas unsur intrinsik
cerpen; namun juga tidak harus ‘bermeditasi’ untuk memilih dan menggunakan diksi dalam
puisinya seperti format puisi pada umumnya. Deskripsi Data Pandangan demikian karena masyarakat pada umumnya masih menganggap
cinta sejenis sebagai sebuah aib dalam keluarga, sehingga kalau bisa disembunyikan. Itulah
gambaran cinta sejenis dalam puisi esai keempat Denny JA. Puisi kelima atau yang terakhir, Bunga Kering Perpisahan. Tema puisi esai ini sebenarnya
masih dekat dengan puisi kedua Romi dan Yuli dari Cikeusik. Albert seorang anak pendeta,
memiliki perasaan cinta pada Dewi, seorang Islam. Kembali masalah agama menjadi paparan
inspirasi Denny untuk mengilhami tokoh Dewi. Dikisahkan, Dewi dipaksa orang tuanya untuk menikah dengan Joko karena alasan
seagama. Dalam puisi ini ditunjukkan bagimana di Indonesia, jodoh masih merupakan area
kekuasaan orang tua. Dalam kasus Romi dan Yuli yang dipaparkan, Denny JA
menggambarkan bagaimana orang tua Dewi mengatasnamakan agama sebagai tameng yang
kuat dan tidak bergeming menghadapi lembutnya perasaan cinta Dewi kepada Albert. Romansa modern yang dipisahkan dengan alasan klasik! Cinta Berselimut Diskriminasi Denny JA melalui kumpulan puisi esai Atas Nama Cinta mengajak pembaca untuk
membuka empati dan simpati pada korban diskriminasi, baik di Indonesia maupun di dunia
luar yang masih sangat terkait erat dengan Indonesia. Hal ini tercermin dari dua puisi esainya
yang berjudul Atas Nama Cinta dan Minah Tetap Dipancung. Dari kedua puisi esai tersebut
tercermin pertanyaan bagaimana peran pemerintah saat paparan konflik yang ada di kedua
puisi tersebut terbuka? ; di mana kehadiranmu “Indonesia”, saat rakyatmu menangis histeris,
melolong-lolong, menengadah pasrah minta pertolongan. Tragisnya, “Indonesia” ternyata Diligentia Vol. 3, No. 2, May 2021 Page 176 Page 176 tidak hadir. Diawali dengan Atas Nama Cinta seorang korban perkosaan dalam sejarah kelam
Mei 1998, tidak hadir. Diawali dengan Atas Nama Cinta seorang korban perkosaan dalam sejarah kelam
Mei 1998, (Sapu Tangan Fang Yin: 20) Tanpa menjelaskan unsur intrinsik sebuah cerita, entah setting maupun karakter tokoh,
pembaca dibawa oleh penyair pada pengalaman kehidupan pahit seorang gadis Tionghoa,
Fang Yin, yang mewakili keresahan dan ketakutan yang mencekam etnis Tionghoa pada
kerusuhan Mei 1998. “Indonesia” negeri yang selama ini dicintainya, harus ditinggalkan
karena keberingasan sekelompok orang yang membabi buta atas nama nasionalisme semu. Kebiadaban yang dipertontonkan itu menjadikan Fang Yin dan “sebangsanya” dengan dada
gemetar diselimuti ketakutan sangat, lari terbirit menyingkir dari tanah air, dengan
membawa trauma ketakutan yang dihujamkan dalam kehinaan moral. Ingin ia bakar selembar kenangan itu
Saksi satu-satunya. Sisa trauma masa lalu
Selama ini disimpannya diam-diam setangan itu
Tak ingin ada orang lain mengganggu. Ingin ia bakar selembar kenangan itu Tak ingin ada orang lain mengganggu. (Sapu Tangan Fang Yin: 23) Garam yang kasar mengoyak dan melukai kulit; terasa perih-pedih, menganga luka hati
yang ingin ditinggalkan Fang Yin sebagai kenangan pahit kehidupan. Trauma mendalam
yang ingin disimpannya tanpa ada orang lain tahu. Denny JA mendeskripsikan betapa luka
Fang Yin menjadi sebuah rahasia yang tersimpan. Dari kutipan teks tersebut mencerminkan
betapa Denny tahu dan sangat memahami situasi saat kerusuhan, bahkan sampai pada detail
perasaan korbannya. Menggelisahkan bercampur aduk dengan kebencian yang sulit untuk
terbalaskan. Kini ia ingin pulang, rindunya membara Ia ingin Indonesia seperti dirinya: menang melawan masa lalu Musibah dan bencana datang tak terduga Yang penting tetap harus punya mimpi. Ini Indonesia baru, kata mereka. Ya, ya… niatnya pun teguh: aku segera kembali ke sana! Page 177 Diligentia Vol. 3, No. 2, May 2021 Aku segera pulang ke sana! Aku segera hidup di sana! Cinta Berselimut Diskriminasi (Sapu Tangan Fang Yin: 48) Proses penyembuhan yang terekam di ingatan Fang Yin seolah seperti “garam” yang
berkali kali melukai dan membuat pedih, “garam” menjadi sumber luka, sumber rasa pedih. Kini Fang Yin kembali melihat “garam” itu, tapi dengan mata yang berbeda. Bukan karena ia
rindu merasakan luka, namun karena ia perlahan-lahan meyakini, bahwa “garam” yang sama
juga dapat menjadi penyembuh luka, “garam” adalah penyelamat penutup trauma
kebiadaban moral sekelompok manusia atas nama cinta. Senada dengan puisi esai Sapu Tangan Fang Yin penggalan teks puisi esai berikut ini
seperti catatan penjelas yang menunjukkan pemberontakan dan pergolakan batin seorang
Minah, TKW polos di Saudi Arabia. (Sebagai ibu muda yang lugu dari desa (Sebagai ibu muda yang lugu dari desa
Minah tak mengerti pernah ada sebuah zaman
Ketika budak boleh diperkosa majikan….) Minah tak mengerti pernah ada sebuah zaman Minah tak mengerti pernah ada sebuah zaman Ketika budak boleh diperkosa majikan….) Ketika budak boleh diperkosa majikan….) (Minah Tetap Dipancung: 103) Dengan segala kesederhanaan pemikiran dan tujuan yang tidak pernah terpikirkannya
sama sekali, ternyata zaman berubah. Budak dapat diperkosa majikan adalah realita yang
sama sekali tak pernah muncul dalam hati Minah saat berangkat ke Saudi. Gusti Allah, Sudah kulakukan semua ajaran baik
Tetapi mengapa tetap saja kena celaka? Kau berjanji melindungi
Kaum tertindas, kaum yang lemah Sudah kulakukan semua ajaran baik (Minah Tetap Dipancung: 106) (Minah Tetap Dipancung: 106) Aminah, betapa bangga aku padamu: Kau berjuang untuk keluarga Membela kehormatan diri. (Minah Tetap Dipancung: 115) (Minah Tetap Dipancung: 115) Minah merasa sebagai TKW yang sudah melakukan hal yang baik di mata Tuhannya,
tetapi kenapa masih dirundung prahara yang mengakhiri jalan hidupnya? Di manakah
engkau berada “Indonesia”, ketika rakyatmu dipancung tak berdaya? Diligentia Vol. 3, No. 2, May 2021 Page 178 Page 178 Kedua puisi di atas menjadi representasi pertanyaan Denny JA kepada penguasa yang
harusnya memiliki andil yang besar dalam prahara yang cukup genting. Nasib warga negara
yang mengalami trauma seperti yang dialami oleh Fang Yin perlu mendapat perhatian oleh
penguasa. Tidak hanya itu, hal kritis mengenai penanganan tenaga kerja Indonesia (TKI) pun
perlu menjadi sorotan. Pahlawan devisa seharusnya memiliki hak perlindungan kerja dari
negara. Dalam hal ini, puisi esai bergerak sebagai jangkar yang melabuhkan kapal berbentuk
kritikan kepada negara. Hal ini berjalan beriringan dengan sifat sastra yang sejatinya
memihak kepada masyarakat. Cinta Berselimut Diskriminasi Sarjono menerangkan bahwa sastrawan perlu menulis dan
berpihak kepada rakyat yang teraniaya, pada orang-orang yang tidak berpihak pada koalisi
apapun, pada orang-orang yang tidak setuju dengan gaya pemerintahan yang sewenang-
wenang.8 Lebih lanjut Sarjono menerangkan bahwa sudah selayaknya sastra memberikan
ruang kepada masyarakat yang tersingkirkan oleh hiruk-pikuk budaya politik serta
pembesaran media. Dalam hal ini, Denny JA menunjukkan kebolehannya sebagai seorang
sastrawan yang memberikan ruang bagi rakyat-rakyat yang dikerdilkan secara asasi. 8 Agus R Sarjono, “Sastra Indonesia Di Tengah Huru Hara,” in Mozaik Sastra Indonesia: Dimensi
Sastra Dari Berbagai Perspektif (Bandung, Indonesia: Nuansa Cendekia, 2005), 37. Cinta Berbalut Agama Agama, agama, dan agama! Dalam area berbeda agama menjadi oase yang
menyejukkan hati, namun Denny JA hadir ketika agama menjadi kendala dan penghalang
dalam area cinta. Bahkan paparan puisi esainya dengan jelas mengungkapkan agama menjadi
senjata ampuh yang dipaksakan untuk ditaati, yang tak perlu meminta alasan ketika
persoalan datang. Atas nama agama semua jadi tersudahi walau dengan gelimang hati yang
tersakiti. Adalah nama Romi dan Yuli dalam puisi Romi dan Yuli dari Cikeusik, yang menjadi
korban agama mematahkan ketulusan cinta mereka yang hampir menuju puncak. Harapan
hidup bahagia bersama orang yang dicintai luruh terkoyak atas nama agama. Hal yang sama
terjadi pada Albert dan Dewi dalam puisi Bunga Kering Perpisahan. Keempat tokoh dalam dua
puisi esai tersebut adalah inspirasi Denny JA untuk memaparkan bagaimana kakunya
tembok tebal yang bernama agama membentengi keterpisahan cinta mereka. Pedas, keras ucapan ibunya Kita di Indonesia, tidak di Amerika. Di sini agama di atas segala
Tak terkecuali cinta remaja. (Romi dan Yuli dari Cikeusik: 54) (Romi dan Yuli dari Cikeusik: 54) (Romi dan Yuli dari Cikeusik: 54) Orang Tua Yuli bagai kena setrum Bagai tersambar halilintar: Dan dalam kegeramanmereka berkata, Demi nama baik keluarga Demi nama baik keluarga 8 Agus R Sarjono, “Sastra Indonesia Di Tengah Huru Hara,” in Mozaik Sastra Indonesia: Dimensi
Sastra Dari Berbagai Perspektif (Bandung, Indonesia: Nuansa Cendekia, 2005), 37. Diligentia Vol. 3, No. 2, May 2021 Page 179 Pernikahan harus dibatalkan! (Romi dan Yuli dari Cikeusik: 69) Agama di atas segalanya adalah sebuah kata penutup permasalahan dan senjata ampuh
ketika alasan lain tidak ditemukan. Gelora cinta berakhir luka dan petaka bagi yang
memilikinya. Aliran Ahmadiyah adalah sebuah kontroversi bagi aliran non-Ahmadiyah. Permasalahan aliran garis keras melawan “garis tidak keras”, bahkan kalau ada sambungan
cinta di antara keduanya seolah menjadi sebuah kemustahilan untuk bersatu, tak terkecuali
Romi dan Yuli. Pasangan kekasih ini menerima tamparan telak karena kisah cintanya. Apa
pun yang sudah dicapai dan dijalani, bahkan sesuatu yang sudah direncanakan matang oleh
keempat tokoh di atas akan dengan sangat mudah dan cepat dipatahkan dengan senjata
“agama”. Sepuluh tahun ia hidup
Bersama Joko, suami pilihan Ayah
Perkawinannya selalu redup
Karena Albert pilihan hatinya… Sepuluh tahun ia hidup (Bunga Kering Perpisahan: 155) (Bunga Kering Perpisahan: 155) Dewi sejak awal merasa
Albert lelaki istimewa. Tapi Dewi seorang Muslimah
Sedangkan Albert anak pendeta. Dewi sejak awal merasa
Albert lelaki istimewa. Tapi Dewi seorang Muslimah
Sedangkan Albert anak pendeta. Dewi sejak awal merasa Albert lelaki istimewa. Tapi Dewi seorang Muslimah Sedangkan Albert anak pendeta. (Bunga Kering Perpisahan: 157) Dalam penggalan teks puisi tersebut, Denny JA menggambarkan bagaimana
kegelisahan tokoh Dewi menjalani pernikahannya bersama pria yang tidak dicintainya. Cintanya bukan untuk Jono, lelaki pilihan orang tuanya. Cinta matinya hanyalah untuk
Albert, anak Pendeta. Atas nama agama Dewi dan Albert harus terpisah dengan membawa
masing-masing luka batin dan hati yang teriris. Itu artinya mereka bukan jodoh, arti yang
harus diterima Dewi tanpa alasan apa pun. Itu artinya
Ia bukan jodoh
Yang dikirim Allah
Untukmu! Itu artinya Untukmu! (Bunga Kering Perpisahan: 163) Melalui kedua puisi di atas, keprihatinan Denny menghadapi fanatisme agama lebih
tampak seperti gejolak pemberontakan batin yang diujudkan dalam kalimat tanya dengan Diligentia Vol. 3, No. 2, May 2021 Page 180 Page 180 tanda baca yang keras. Mengapa masih terdapat cinta yang kandas-terkoyak disebabkan
egoisme orang tua dengan mengatasnamakan agama? Kedua konteks ini secara nyata jamak
terjadi di masyarakat. Mengapa masih saja terjadi topik ini di masyarakat? Bahkan sejak Siti
Nurbaya pun hal ini tidak muncul sebagai topik cerita, apalagi sebagai problema realita
kehidupan. tanda baca yang keras. Mengapa masih terdapat cinta yang kandas-terkoyak disebabkan
egoisme orang tua dengan mengatasnamakan agama? Kedua konteks ini secara nyata jamak
terjadi di masyarakat. Mengapa masih saja terjadi topik ini di masyarakat? Bahkan sejak Siti
Nurbaya pun hal ini tidak muncul sebagai topik cerita, apalagi sebagai problema realita
kehidupan. Cinta di Tengah Penjara Sesuatu yang bertentangan dengan norma-norma yang berlaku di masyarakat adalah
sebuah ketidaklaziman. Dalam konteks tertentu dan terasa lebih ironis, apabila ‘yang
berbeda’ selalu dianggap sebagai musuh masyarakat. Denny JA cukup berani memaparkan
puisi esai Cinta Terlarang Batman dan Robin, suatu gambaran cinta yang jelas bertentangan
dengan strata norma yang berlaku di masyarakat. Bambang disebut Batman, Bambang disebut Batman,
Karena ia jagoan
Berani berbeda
Keras prinsip hidupnya. Sedangkan Amir seorang peragu
Goyah pendirian selalu;
Karena akrab dengan Bambang Sang Batman
Ia kebagian sebutan Robin. Karena ia jagoan Karena ia jagoan Berani berbeda Keras prinsip hidupnya. 9 Muslimin, “Modernisasi Dalam Novel Belenggu Karya Armijn Pane,” Jurnal Sastra, Bahasa, Dan
Budaya 1, no. 1 (2011): 126.
10 Ikhsanniah et al., “Nilai-Nilai Edukasi Puisi Dalam Kolom Apresiasi Harian Pontianak Post,”
Jurnal Pendidikan Dan Pembelajaran Khatulistiwa 3, no. 7 (2014), 2. Eros telah menyatukan keduanya Eros telah menyatukan keduanya Di pesantren. Siapakah yang mesti disalahkan? Siapakah yang mesti disalahkan? Semua terjadi begitu saja, tanpa rencana. Semua terjadi begitu saja, tanpa rencana. (Cinta Terlarang Batman dan Robin:124) Dua manusia sejenis telah dipersatukan oleh eros justru di pesantren! Dengan
ketertarikan dan rasa saling melengkapi, rasa saling mengayomi, saling menerima, dan masih
banyak “saling” lainnya, menjadi alasan kedua insan sejens ini ingin bersatu dalam eros. Kedua insan ini pasti ingin cinta mereka dimaklumi dan diterima masyarakat tanpa dianggap
menabrak norma yang ada di dalamnya. Denny JA cukup berani terbuka memaparkan percintaan terlarang ini dengan latar
pesantren, yang dipercaya sebagai tempat berlangsungnya pendidikan agama yang sangat
kuat. Ironis memang, masalah yang seharusnya jauh dari setting pesantren, tetapi justru
sebaliknya. Mata masyarakat dibuat terbelalak oleh paparan Denny dengan puisi esai yang
satu ini. Masyarakat prihatin, namun juga mencibir. Dalam hal ini, Denny JA sedang
menuangkan konflik berbalut tradisi yang revolusinya terendus tajam oleh masyarakat. Kebudayaan sebagai satu aspek paradigma kultural yang sangat menentukan cara pandang Diligentia Vol. 3, No. 2, May 2021 Page 181 Page 181 sekelompok orang terhadap suatu isu.9 Oleh karena itu, tidak heran jika satu hal yang memicu
satu transformasi budaya dapat menimbulkan pergolakan atau kontroversi yang intens. Kisah Bambang (Batman) dan Robin tidaklah semata-mata sebuah kisah cinta biasa. Justru
konflik kisah yang dihadapi oleh kedua tokoh merupakan satu tanda arus perkembangan
budaya terjadi. Hanya saja, keberpihakan sebagian besar masyarakat timur pada norma
agama dan adat istiadat menelurkan sebuah polemik terhadap perasaan yang dialami
Bambang dan Robin. Sekali lagi, sastra menjadi cerminan akan kenyataan sosial yang muncul
di permukaan. Pemilihan judul dan latar puisi ini sangat menggelitik, tak hanya secara
individu bagi tokoh agama, namun juga masyarakat dengan segala aturan norma moralnya. Keterbukaan Denny menjadi satir dan tamparan keras bagi suatu golongan yang mungkin
tidak menyangka bahwa topik ini benar terjadi dalam institusi yang bernama pesantren. Homoseksual yang biasanya menjadi isu sensitif dan orang segan atau enggan
membicarakannya, namun Denny dengan gamblang membuka tabir sensitivitas tersebut
dengan tujuan membukakan mata pembaca agar peka dan mesti bersikap seperti apa yang
terkait dengan cinta terlarang tersebut. 10 Ikhsanniah et al., “Nilai-Nilai Edukasi Puisi Dalam Kolom Apresiasi Harian Pontianak Post
l Pendidikan Dan Pembelajaran Khatulistiwa 3, no. 7 (2014), 2. Diligentia Vol. 3, No. 2, May 2021 Diligentia Vol. 3, No. 2, May 2021 Page 182 Page 182 Melalui puisi Romi dan Yuli dari Cikeusik yang mencuplik kisah tragis penyerangan
Ahmadiyah tahun 2011, masyarakat dapat berefleksi mengenai perbedaan yang seharusnya
menjadi kekuatan, bukan justru menjadi motif sengketa. Perihal keyakinan yang dijunjung
oleh kedua pihak yang bertentangan semestinya menjadi hal yang dihormati. Selain kisah
cinta yang dipampangkan oleh Denny JA, toleransi yang disuguhkan pun perlu menjadi
sorotan dan perenungan. Sastra (dalam hal ini mewujud bentuk puisi esai) digunakan sebagai
senjata yang dapat meletuskan toleransi. Kisah lainnya, dari puisi Minah Tetap Dipancung pun mengemas nilai-nilai pembelajaran
yang dapat dipetik. Tekad dan keberanian Minah untuk mengabdikan tenaga di negeri jiran
pantas untuk selalu dikenang oleh masyarakat. Dalam lingkup yang lebih kecil, semangat
juang yang dipertontonkan oleh Denny JA melalui tokoh Minah dapat menjadi satu asupan
reflektif bagi setiap anggota keluarga untuk menjadi berdampak bagi satu sama lain, saling
berjuang dan mengabdikan diri terhadap keluarga adalah satu nilai berharga yang tidak
boleh lepas. Tidak hanya itu, puisi ini mempertontonkan kiprah perempuan yang tidak jarang
menjadi korban di dalam dunia kerja. Padahal, tekad bulat yang dimiliki oleh Minah, pertama
kali dipicu oleh kondisi suaminya yang tidak lagi bekerja. Malam itu aku di samping suami tercinta Menyusun rencana. Sudah sekian lama suamiku nganggur Sudah sekian lama suamiku nganggur Anak perempuanku, delapan tahun, Belum juga ia bersekolah Anak perempuanku, delapan tahun, Belum juga ia bersekolah Aku belum bisa bayar uang iurannya. Aku belum bisa bayar uang iurannya. (Minah Tetap Dipancung: 91) Ketidakadilan bagi tenaga kerja Indonesia tidak sekali dua kali terjadi. Penindasan yang
berujung kematian ini paling sering terjadi bagi kawula wanita. Sastra dapat digunakan
sebagai senjata yang memerangi lambatnya penanganan serta bisunya pemerintah menindak
kesewenang-wenangan yang dialami oleh pahlawan devisa. Realitas objektif yang tercermin dalam dua puisi terakhir dapat menampilkan sastra
sebagai satu dimensi yang dinamis. Norma, nilai, adat-istiadat, institusi, dan berbagai hal
lainnya membentuk satu realitas objektif yang dapat memberikan proses belajar dan
pengalaman (Prasetyawan, 2005). Puisi Cinta Terlarang Batman dan Robin menjadi satu
perlambangan norma dan tradisi masyarakat timur yang cenderung masih mengikat, tidak
sebebas norma atau kebudayaan barat. Belajar dari hal ini, pembaca seharusnya mengerti
bahwa adat istiadat yang mengikat masyarakat senantiasa memiliki andil di dalam setiap
perilaku. Hal serupa juga dikemas dalam puisi Bunga Kering Perpisahan. Agama sebagai
noktah atau kiblat perilaku sangat jelas tergambar dalam puisi ini. 11 Syahrudin Y.S., “Sastra, Imajinasi, Dan Empirisme Relijius,” in Mozaik Sastra Indonesia: Dimensi
Sastra Dari Berbagai Perspektif (Bandung, Indonesia: Nuansa Cendekia, 2005), 56. Muara Puisi Esai Semua hal adalah guru, semua tempat adalah sekolah. Melalui karya sastra, sudah
seharusnya masyarakat semakin mampu memilah dan memahami setiap konteks yang ada,
tidak mendiskriminasi seseorang perkara perbedaan cara pandang atau perilaku, juga tidak
menjadi sosok yang paling benar dalam menyikapi segala isu sosial yang mencuat di
masyarakat. Sasaran akhir dari sebuah karya sastra tidak lain dan tidak bukan adalah untuk
memanusiakan manusia10 Masyarakat sebagai pemegang peran pemberdayaan paling
penting dapat memanfaatkan karya sastra (dalam hal ini puisi esai) guna menyingkap realita
dan sebagai bahan pembelajaran sepanjang hayat. Melalui kelima puisi esai yang dituliskan
oleh Denny JA, tercuplik berbagai kenyataan yang dapat dijadikan sebagai pembelajaran. Kisah Fang Yin yang mencuplik tragedi 98 dapat ditelisik menggunakan kaca mata historis
dan dijadikan sebagai satu bahan refleksi mendalam bagi masyarakat Indonesia. Kesewenang-wenangan aparat dan berbagai golongan yang berandil di dalam tragedi kelam
Indonesia berimbas bukan saja pada timpangnya politik dan ekonomi negara. Lebih spesifik,
hal ini berimbas pada mental dan psikis orang-orang yang terdampak, dan dalam hal ini
berimbas pada Fang Yin. Minggu-minggu pertama di Amerika Minggu-minggu pertama di Amerika
Fang Yin belum sadar apa sebenarnya yang terjadi
Raga dan jiwanya lemah, perlu pemulihan dari dahsyatnya trauma
Ke manapun ia pergi, orang tuanya dan seorang psikolog mendampingi
(Sapu Tangan Fang Yin: 21) Fang Yin belum sadar apa sebenarnya yang terjadi Raga dan jiwanya lemah, perlu pemulihan dari dahsyatnya trauma Raga dan jiwanya lemah, perlu pemulihan dari dahsyatnya trauma Ke manapun ia pergi, orang tuanya dan seorang psikolog mendampingi
(Sapu Tangan Fang Yin: 21) 12 Rencan Carisma Marbun, “Kasih Dan Kuasa Ditinjau Dari Perspektif Etika Kristen,” Jurnal
Teologi Cultivation 3, no. 1 (2019), 664. https://doi.org/10.46965/jtc.v3i1.259.
13 Marbun, “Kasih Dan Kuasa Ditinjau Dari Perspektif Etika Kristen,” 666.
14 Ayub Sugiharto, “Keselamatan Ekslusif Dalam Yesus Di Tengah Kemajemukan Beragama,”
Angelion: Jurnal Teologi Dan Pendidikan Kristen 1, no. 2 (2020),99. https://doi.org/10.38189/jan.v1i2.66.
15 Sugiharto, “Keselamatan Ekslusif Dalam Yesus Di Tengah Kemajemukan Beragama,” 107. Diligentia Vol. 3, No. 2, May 2021 Apabila karya sastra
dihubungkan dengan agama, maka nilai-nilai religius sastra memiliki nafas, karena ia hadir
dalam konsep yang riil11. Melalui hal ini, kita belajar bahwa nilai benar dan salah yang hidup
di masyarakat bergantung pada perspektif yang mereka yakini. Satu hal menjadi benar hanya
karena agama membenarkannya. Satu hal menjadi benar hanya karena tradisi
mendukungnya, begitu pula sebaliknya. Oleh karena itu, dalam hal ini karya sastra berperan
mengedukasi masyarakat untuk dapat memandang segala sesuatu dari berbagai sudut
pandang. Diligentia Vol. 3, No. 2, May 2021 Page 183 Page 183 Kasih menjadi peta selubung paling jelas yang menggambarkan lima puisi esai ciptaan
Denny JA. Hanya saja, dapat terlihat bahwa kasih pula yang menjadi pemantik konflik dalam
setiap kisah yang disuarakan dalam bentuk puisi esai. Di sisi lain, konflik tersebut juga dipicu
oleh berbagai keberagaman yang ada. Keberagaman kepercayaan, sudut pandang, pemikiran,
turut mengambil andil yang besar dalam mengolah kisah kasih yang terjalin dari masing-
masing tokoh. Karena itu, diperlukan satu kaca mata/perspektif yang terbaik dalam
mengupas lima kisah tragis yang disiapkan oleh Denny JA ini. Perspektif yang baik tentu saja dilandasi oleh Alkitab. Muara dari kasih yang dilakukan
manusia sepatutnya adalah respons dari kasih Allah yang terlebih dahulu datang dalam
pribadi manusia12. Lebih lanjut, kasih yang adalah landasan filsafat etika Kristen tidak
ditunjukkan dengan memperhatikan jasa, kelas sosial, suku, bangsa, atau keluarga orang
yang dikasihi13. Hal ini tentu akan bersinggungan dengan perspektif lain yang diyakini oleh
masyarakat. Konteks kemajemukan kepercayaan yang hadir di Indonesia dapat dijadikan
sebagai satu ujian penting dalam menelisik kasih yang diimani oleh orang percaya. Hal ini
berkenaan dengan Sugiharto yang mengatakan orang percaya perlu menanamkan toleransi
(menghargai dan menghormati pemeluk kepercayaan lain)14. Analogi toleransi yang dipaparkan di dalam Alkitab dapat menjadi satu landasan yang
baik bagi orang percaya dalam menjalankan keseharian bertoleransi. Karena kasih Allah,
Allah toleran terhadap manusia, namun tidak terhadap dosa manusia15. Oleh sebab itu, sikap
menghargai dan mengasihi orang lain yang memiliki perbedaan cara pandang tidak serta-
merta menjadikan kita mengambil sikap membenarkan perilaku atau cara pandang mereka. Dalam hal mengasihi, kita dapat mengambil peran menyuarakan pendapat, memberikan
masukan yang membangun, dan tidak menghakimi. Niscaya, cita-cita perdamaian atas nama
cinta yang dikumandangkan dalam puisi esai Denny JA dapat terjadi, tanpa konflik yang
berarti. Penutup Kumpulan kisah berbagai macam percintaan yang dipaparkan oleh Denny JA ini
membukakan mata dan menyadarkan pikiran pembacanya bahwa tidak ada cinta yang
sebebas-bebasnya, cinta masih saja terpenjara oleh agama, oleh diskriminasi, oleh norma
moral masyarakat. Adakah cinta yang sejati yang mengatasi agama, mengatasi diskriminasi, dan di atas
norma moral masyarakat? Kumpulan lima puisi esai Atas Nama Cinta karya Denny JA ini
adalah titik awal permenungan untuk menjawab kegelisahan serangkaian pertanyaan di atas. Sekali lagi perlu digarisbawahi bahwa masyarakat sebagai pemegang peran
pemberdayaan paling penting dapat memanfaatkan karya sastra (dalam hal ini puisi esai)
guna menyingkap realita dan sebagai bahan pembelajaran sepanjang hayat. Melalui kelima
puisi esai yang dituliskan oleh Denny JA, tercuplik berbagai kenyataan yang dapat dijadikan
sebagai pembelajaran. Diligentia Vol. 3, No. 2, May 2021 Page 184 Page 184 Daftar Pustaka Basrowi, and Suwandi. Memahami Penelitian Kualitatif. Jakarta, Indonesia: Rineka Cipt Basrowi, and Suwandi. Memahami Penelitian Kualitatif. Jakarta, Indonesia: Rineka Cipta, 2008. Cresswell, John. Riset Pendidikan. Yogyakarta, Indonesia: Pustaka Pelajar, 2005. Basrowi, and Suwandi. Memahami Penelitian Kualitatif. Jakarta, Indonesia: Rineka Cipta, 2008. Cresswell, John. Riset Pendidikan. Yogyakarta, Indonesia: Pustaka Pelajar, 2005. Cresswell, John. Riset Pendidikan. Yogyakarta, Indonesia: Pustaka Pelajar, 2005. Ikhsanniah, Lidya, Martono, and Nanang Heryana. “Nilai-Nilai Edukasi Puisi Dalam Kolom
Apresiasi Harian Pontianak Post.” Jurnal Pendidikan Dan Pembelajaran Khatulistiwa 3, no. 7 (2014): 1-10. JA, Denny. Menjelaskan Puisi Esai: Visi Denny JA Dan Respon Sastrawan Kritikus Indonesia+
Manca Negara. Jakarta, Indonesia: Cerah Budaya Indonesia, 2019. Marbun, Rencan Carisma. “Kasih Dan Kuasa Ditinjau Dari Perspektif Etika Kristen.” Jurnal
Teologi Cultivation 3, no. 1 (2019): 663-672. https://doi.org/10.46965/jtc.v3i1.259. Muslimin. “Modernisasi Dalam Novel Belenggu Karya Armijn Pane.” Jurnal Sastra, Bahasa,
Dan Budaya 1, no. 1 (2011): 126. Nurgiyantoro, Burhan. Teori Pengkajian Fiksi. Yogyakarta, Indonesia: Gajah Mada University
Press, 2010. Sarjono, Agus R. “Sastra Indonesia Di Tengah Huru Hara.” In Mozaik Sastra Indonesia:
Dimensi Sastra Dari Berbagai Perspektif. Bandung, Indonesia: Nuansa Cendekia, 2005. j
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Dimensi Sastra Dari Berbagai Perspektif. Bandung, Indonesia: Nuansa Cendekia, 2005. g
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Semi, M. Atar. Metode Penelitian Sastra. Bandung, Indonesia: Angkasa, 2012. Sugiharto, Ayub. “Keselamatan Ekslusif Dalam Yesus Di Tengah Kemajemukan Beragama.”
Angelion: Jurnal Teologi Dan Pendidikan Kristen 1, no. 2 (2020): 98-112. https://doi.org/10.38189/jan.v1i2.66. p
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Waluyo, Herman J. Apresiasi Puisi. Jakarta, Indonesia: Gramedia Pustaka Utama, 2002 Y.S, Syahrudin. “Sastra, Imajinasi, Dan Empirisme Relijius.” In Mozaik Sastra Indonesia:
Dimensi Sastra Dari Berbagai Perspektif, 56. Bandung, Indonesia: Nuansa Cendekia, 2005. y
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Dimensi Sastra Dari Berbagai Perspektif, 56. Bandung, Indonesia: Nuansa Cendekia, 2005.
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Effects of supplementation of tropical fruit processing by-products on lipid profile, retinol levels and intestinal function in Wistar rats
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Anais da Academia Brasileira de Ciências
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Effects of supplementation of tropical fruit
processing by-products on lipid profile, retinol
levels and intestinal function in Wistar rats KAMILA S. BATISTA, HASSLER CLEMENTINO CAVALCANTE, JÉSSYCA A. DE SOUSA
GOMES, LAIANE A. DA SILVA, NATÁLIA S. DE HOLANDA CAVALCANTI, ESTEFÂNIA F. GARCIA, FRANCISCA NAYARA D.D. MENEZES, TAMIRES A.S. DE LIMA, EVANDRO L. DE SOUZA, MARCIANE MAGNANI & JAILANE DE SOUZA AQUINO Abstract: Fruits agro-industrial by-products may have a great variety of bioactive
compounds that promote health. Thus, the effects of supplementation with acerola,
cashew and guava processing by-products for 28 days on retinol level, lipid profile and
on some aspects related to intestinal function in rats were investigated. The animals
supplemented with different fruit by-products presented similar weight gain, faecal pH
values and intestinal epithelial structures; however, they showed higher moisture and
Lactobacillus spp. and Bifidobacterium spp. counts in faeces compared to the control
group. Supplementation with the cashew by-product decreased the blood glucose,
acerola and guava by-products reduced serum lipid levels and all fruit by-products tested
increased serum and hepatic retinol. The results indicated that acerola and guava by-
products possess a potential hypolipidemic effect. The three fruit by-products increase
the hepatic retinol deposition and the faecal populations of beneficial bacterial groups
and modulated aspects of intestinal function. The findings of this study can contribute
to sustainable fruticulture and support future clinical studies with the supplementation
of by-products. Key words: blood glucose, by-products, gut microbiota, lipid profile, retinol status. An Acad Bras Cienc (2023) 95(2): e20201684 DOI 10.1590/0001-3765202320201684
Anais da Academia Brasileira de Ciências | Annals of the Brazilian Academy of Sciences
Printed ISSN 0001-3765 I Online ISSN 1678-2690
www.scielo.br/aabc | www.fb.com/aabcjournal An Acad Bras Cienc (2023) 95(2): e20201684 DOI 10.1590/0001-3765202320201684
Anais da Academia Brasileira de Ciências | Annals of the Brazilian Academy of Sciences
Printed ISSN 0001-3765 I Online ISSN 1678-2690
www.scielo.br/aabc | www.fb.com/aabcjournal An Acad Bras Cienc (2023) 95(2) INTRODUCTION and lycopene contents. Cashew is considered
a source of fibres, carotenoids, vitamin C and
polyphenols, while guava is an important source
of vitamins A and C, fibre, pectin and potassium
(Ellong et al. 2015, Vargas-Murga et al. 2016). Brazil produces approximately 40 million tons
of tropical, subtropical and temperate fruits per
year, providing a great variety of fruits throughout
the year (FAO 2018). Among the more popular
and most frequently processed native or exotic
fruits in Brazil are acerola (Malpighia emarginata
D.C.), cashew (Anacardium occidentale L.)
and guava (Psidium guajava L.), which are
greatly appreciated because of their sensory
characteristics (flavour and colour), nutritional
quality and bioactive compound content. Acerola is recognised as one of the greatest
natural vitamin C sources, with high carotenoid Frozen tropical fruit pulps have become
popular worldwide due to their practical
consumption (Dantas et al. 2019). However, fruit
pulp processing generates a large volume of
by-products such as skins, seeds and bagasse,
which are often inadequately disposed into the
environment (Medeiros et al. 2019). The fruit
processing by-products may contain dietary
fibres and different antioxidant compounds An Acad Bras Cienc (2023) 95(2) An Acad Bras Cienc (2023) 95(2) EFFECTS OF TROPICAL FRUIT PROCESSING BY-PRODUCTS KAMILA S. BATISTA et al. PB, Brazil (latitude 07º 26’ 19” S, longitude 34º
54’ 52” W; altitude 49 m) during September and
October 2018. By-products were donated by
the Polpa Ideal Indústria Ltda. (João Pessoa,
PB, Brazil). The total sample contained 20 kg
of each by-product (skin, seeds and bagasse)
homogenised from different batches. Each by-
product was subjected to lyophilisation (L-101
lyophilizer, LIOTOP, São Carlos, SP, Brazil) at - 47
°C, with a vacuum pressure below 150 µHg and
a lyophilisation rate of 1 m/h, for approximately
12 h. The freeze-dried by-products were ground
in a domestic blender (average particle size < 1.0
mm) and stored under refrigeration (-10°C) and
protected from light. such as phenolic compounds, which are
considered dietary components associated with
the modulation of bowel transit time, decreased
gastric emptying, delayed glucose absorption,
decreased postprandial glycaemia and reduced
blood cholesterol and triacylglycerol (TAG) levels
due to their physical properties that provide
viscosity to the luminal content (Ayala-Zavala
et al. 2011, Batista et al. 2018). INTRODUCTION In addition, by-
products contain carotenoids that can exert
vitamin A activity in various physiological
processes such as regulation of glucose and
lipid metabolisms, cell proliferation and
differentiation, and the immune system (Saeed
et al. 2017, García-Cayuela et al. 2018). The intake of different fruit by-products
seems to influence on maintaining the balance
of the gut microbiota. Fruit by-products contain
one or more components capable of selective
fermentation, which promote changes in the
gut microbiota composition and activity, that is,
they have a potential prebiotic effect (Batista
et al. 2018). These effects provide health
benefits, given that the balance of commensal
and pathogenic bacteria of the gut microbiota
has been associated with decreased risk of
developing metabolic diseases (Belizário et al. 2018). For total carotenoids quantification, initially
18 mL of acetone P.A. was added to 0.5 g of by-
products samples and after homogenization,
the samples were read on a spectrophotometer
(Genesys 10S UV-Vis Spectrophotometer
model, Madison, USA) at wavelengths of 470
nm, 645 nm, and 662 nm in the absence of
light at a temperature of 25°C (Lichtenthaler &
Buschmann 2001). The results were expressed in
mg/100 g of sample dry weight. The ascorbic acid concentration was
determined using the Tillmans titrimetric
method (2,6-Dichlorophenolindophenol
sodium) (method 967.21) (AOAC 2016). A
solution of 2,6-dichlorophenol-indophenol
was discoloured by ascorbic acid in 0.5 g of
by-product sample using a standard ascorbic
acid solution. The spectrophotometer (Genesys
10S UV-Vis Spectrophotometer model, Madison,
USA) was calibrated at 100% transmittance
using 5 mL 2% HPO3 blank solution and 10 mL of
water. Samples were read at a wavelength of 518
nm and results were expressed as mg/100g of
sample dry weight. Considering these aspects and the need for
a sustainable destination of fruit processing by-
products, the present study aimed to evaluate
the effects of supplementation with acerola,
cashew and guava by-products on the retinol
levels, lipid profile and some parameters
associated with intestinal function in rats. MATERIALS AND METHODS Pulp fruit processing by-products EFFECTS OF TROPICAL FRUIT PROCESSING BY-PRODUCTS KAMILA S. BATISTA et al. Figure 1. Flowchart of the
experimental study with
rats supplemented or
not with acerola, cashew,
or guava by-products.
CG = control group; AEG=
acerola by-product
experimental group; CEG
= cashew by-product
experimental group;
GEG= guava by-product
experimental group. Figure 1. Flowchart of the
experimental study with
rats supplemented or
not with acerola, cashew,
or guava by-products.
CG = control group; AEG=
acerola by-product
experimental group; CEG
= cashew by-product
experimental group;
GEG= guava by-product
experimental group. Pulp fruit processing by-products Acerola (Malpighia emarginata D.C.), cashew
(Anacardium occidentale L.) and guava (Psidium
guajava L.) by-products were grown in Alhandra, An Acad Bras Cienc (2023) 95(2) e20201684 2 | 14 EFFECTS OF TROPICAL FRUIT PROCESSING BY-PRODUCTS Animals and diets relative humidity 50-55% and 12 hr light-dark
cycles, with water and standard diet (Presence,
Paulínea, SP, Brazil) provided ad libitum. The experimental method was approved under
protocol number no. 050514 by the Animal
Experimentation Ethics Committee (Comissão
de Ética no Uso de Animais – CEUA), Federal
University of Paraíba (Universidade Federal da
Paraíba – UFPB) (João Pessoa, PB, Brazil), and
all experiments followed the standards of
the Brazilian Society of Science in Laboratory
Animals (Sociedade Brasileira de Ciência em
Animais de laboratório - SBCAL). The rats were
acclimatized for one week at the Experimental
Nutrition Laboratory - LANEX (UFPB) and were
maintained in cages (4 animals/cage) at 21 ± 1°C, The biological assay was performed
during 28 days using 32 adult Wistar male rats
(average initial weight 290.00 ± 10.00 g) at ±80
days old randomised into four groups which
received saline solution (control group; CG,
n=8), acerola processing by-product (acerola
by-product experimental group; AEG, n=8),
cashew processing by-product (cashew by-
product experimental group; CEG, n=8) and
guava processing by-product (guava by-product
experimental group; GEG, n=8) (Figure 1). The An Acad Bras Cienc (2023) 95(2) e20201684 3 | 14
Figure 1. Flowchart of the
experimental study with
rats supplemented or
not with acerola, cashew,
or guava by-products. CG = control group; AEG=
acerola by-product
experimental group; CEG
= cashew by-product
experimental group;
GEG= guava by-product
experimental group. Figure 1. Flowchart of the
experimental study with
rats supplemented or
not with acerola, cashew,
or guava by-products. CG = control group; AEG=
acerola by-product
experimental group; CEG
= cashew by-product
experimental group;
GEG= guava by-product
experimental group. Figure 1. Flowchart of the
experimental study with
rats supplemented or
not with acerola, cashew,
or guava by-products. CG = control group; AEG=
acerola by-product
experimental group; CEG
= cashew by-product
experimental group;
GEG= guava by-product
experimental group. An Acad Bras Cienc (2023) 95(2) e20201684 3 | 14 EFFECTS OF TROPICAL FRUIT PROCESSING BY-PRODUCTS KAMILA S. BATISTA et al. freeze-dried by-products were administered to
the AEG, CEG, and GEG groups at the dose of 400
mg/kg of animal weight. The fruit by-products
were diluted in saline solution (1.6%, w/v) and
gavage was performed twice a day at 4 h interval
(Batista et al. 2018). The body weight and the
dietary intake measurements were performed
weekly using a digital electronic scale (Prix III,
Toledo, São Bernardo do Campo, SP, Brazil). Serum and liver retinol determination Retinol was extracted from serum and liver
homogenate (Aquino et al. 2016). The retinol
levels were determined by high-performance
liquid chromatography (UltiMate 3000, Thermo
Scientific Dionex, Waltham, MA, USA) using a
Dionex chromatograph containing a C18 column
measuring 4.60 x 2.50 mm x 5.00 µm, a pre-
column, a detector set to 325 nm, and a mobile
phase flow rate (methanol) of 1.50 mL for min. Animals and diets fat samples were collected, weighed to calculate
the percentage of this fat in relation to the body
weight of the animal. The carcass was used to
determine total fat in the rats’ body (Folch et
al. 1957). The colon was removed for histological
analysis, and the liver was removed to quantify
hepatic retinol. Somatic parameters, lipid profile and blood
glucose After 28 days, the animals were fasted for 8 h
and anaesthetised intraperitoneally using 75 mg
of ketamine hydrochloride per kg of body weight
and 5 mg of xylazine hydrochloride per kg of body
weight. Somatic parameters were evaluated with
anaesthetized animals, as follows: body weight;
chest circumference measured immediately
before the hind paw, abdominal circumference
measured immediately behind the front leg,
and naso-anal length measured using metric
tape. The body mass index (BMI) and Lee index
(Novelli et al. 2007) were calculated through the
equations: BMI = body weight (g)/length (cm)
squared; Lee index = cube root of body weight
(g)/length (cm). An Acad Bras Cienc (2023) 95(2) e20201684 4 | 14 Faecal analyses Animal faeces were collected on the 26th to 28th
days of the experiment to obtain samples in
representative quantities for analysis (Batista
et al. 2018, Tavares et al. 2021). Part of the
faecal samples was collected fresh for bacterial
count analysis and part was stored in a freezer
-20°C for pH and moisture analysis. The pH
was determined using a digital potentiometer
(Q400AS, Quimis, São Paulo, SP, Brazil) and the
moisture was determined following the 934.01
method (AOAC 2016). The anaesthetized animals were then
euthanised via cardiac puncture. Blood samples
were collected to perform glycaemia level using
a glucometer (model Performa, Accu-check,
Jaguaré, SP, Brazil) and to obtain serum by
centrifugation (MPW-351R centrifuge, MPW-Med. Instrument, Warsaw, Poland) at 1,000 x g for 10
min at 4°C for determining lipid profile using
commercial kits (Labtest, Lagoa Santa, MG, Brazil)
and LabMax 240 Premium automatic analyzer
(Labtest). Sample analyses were performed
at 505 nm for TAG level; 500 nm for total
cholesterol (TC); 600 nm for very low-density
lipoprotein (VLDL), low-density lipoprotein (LDL)
and high-density lipoprotein (HDL). The visceral For analysis of bacteria count in faeces,
the fresh faecal samples were diluted in sterile
peptone (1:9, w/v). Then, 20 µL aliquots were
inoculated using the micro drop technique (Miles
et al. 1938) on selective agar for Lactobacillus
spp. (de Man, Rogosa and Sharpe – MRS,
HiMedia, Mumbai, MH, India), Bifidobacterium
spp. (Bifidobacterium agar, HiMedia) and
Enterobacteriaceae (MacConkey, HiMedia). Plates were incubated under anaerobiosis
(Lactobacillus spp. and Bifidobacterium spp.; An Acad Bras Cienc (2023) 95(2) e20201684 4 | 14 EFFECTS OF TROPICAL FRUIT PROCESSING BY-PRODUCTS KAMILA S. BATISTA et al. Anaerobic System Anaerogen, Oxoid Ltd.,
Basingstoke, Hampshire, United Kingdom) or
aerobiosis (Enterobacteriaceae) at 37°C for 48
hr. Results are expressed as log10 colony-forming
units (CFU)/g (APHA 2001). Software Inc., San Jose, CA, USA) statistical
software. Software Inc., San Jose, CA, USA) statistical
software. Histological colon evaluation Colon samples collected for histological
evaluation were processed using routine
histological techniques and stained with
Haematoxylin and Eosin (H&E). Stasis, leukocyte
migration, haemorrhage, vasodilation, necrosis,
epithelial preservation, hypertrophy and
hyperplasia of the outer muscular layer were
evaluated at a total 40x magnification using an
optical microscope (Motic BA 200, Kowloon Bay,
Kowloon, Hong Kong) (Batista et al. 2018). RESULTS AND DISCUSSION The by-products of acerola and cashew
presented the highest amount of carotenoids,
however, the by-product of acerola stood out
from the others by containing almost one
hundred times more ascorbic acid (Table I). The
by-products of acerola, cashew and guava have
high amounts of ascorbic acid and carotenoids
compared to other fruit by-products such as
melon, orange, mandarin, and grapefruit (Rico et
al. 2020, Reynoso-Camacho et al. 2021). However,
not only the amounts of antioxidant compounds
in each by-products should be considered to
assess the benefits of their consumption. The
carotenoid fractions and the interfering factors
in the bioavailability of ascorbic acid present in
each by-product also impacts the bioactivity in
body, demonstrating the importance of carrying
out animal model interventions to study the
fruit by-products (Gómez-García et al. 2020,
Mieszczakowska-Frąc et al. 2021). Statistical analysis The sample size (32 animals randomized into
four groups, n = 8) was calculated to meet
a minimum statistical power of 80%, with a
minimally detectable effect size of 1.0 and
a significance level of 0.05 (a = 0.05). Results
were expressed as the mean and standard
deviation. The data showed normal distribution
by the Shapiro-Wilk test and were therefore,
subjected to parametric one-way Analysis of
Variance (ANOVA) and the Tukey’s post hoc test
at a 5% significance level (P ≤ 0.05), when there
was a difference between the obtained data. The analysis and graphic design were carried
out using SigmaPlot 12.5 for Windows (Systat The cashew by-product experimental
group (CEG) presented higher dietary intake in
comparison to the control group (CG), acerola
by-product experimental group (AEG) and guava
by-product experimental group (GEG) (P ≤ 0.05;
Figure 2a). Although all experimental groups
(AEG, CEG and GEG) consumed a greater amount
of diet, they presented similar weight gain to
CG (P > 0.05) (Figure 2b). These results can be Table I. Total carotenoids and ascorbic acid contents in acerola, cashew and guava processing by-products. Bioactive compounds
(mg/100 g of dry weight)
By-products
Acerola
Cashew
Guava
Total carotenoids
4.66±0.11a
4.82±0.14a
4.29±0.06b
Ascorbic acid
149.40±5.30a
1.69±0.20b
1.41±0.20b
Note: a,b Different lowercase letters in the same row indicate a significant difference between the mean ± standard deviation of
each by-product (one-way ANOVA, P ≤ 0.05, Tukey’s post hoc test). carotenoids and ascorbic acid contents in acerola, cashew and guava processing by-products. Table I. Total carotenoids and ascorbic acid contents in acerola, cashew and guava processing b Note: a,b Different lowercase letters in the same row indicate a significant difference between the mean ± standard deviation of
each by-product (one-way ANOVA, P ≤ 0.05, Tukey’s post hoc test). An Acad Bras Cienc (2023) 95(2) e20201684 5 | 14 EFFECTS OF TROPICAL FRUIT PROCESSING BY-PRODUCTS KAMILA S. BATISTA et al. (P > 0.05) (Table II). The acerola and guava by-
products have also been shown to significantly
reduce the final body weight of the AEG and
GEG groups (P ≤ 0.05). Statistical analysis The beneficial effects
of these fruit by-products on body weight
and body composition are suggested when it
is noted that the body length, abdominal and
chest circumferences, visceral and body fat
were similar between experimental and control explained by an increase in gastrointestinal
motility with consequent reduction in the
amount of absorbed and metabolized energy,
promoted by supplementation with the fruit by-
products (Batista et al. 2018). Despite the higher dietary intake of the CEG
group, the cashew by-product maintained the
body weight of the supplemented rats, providing
similar final body weight to the other groups Figure 2. Food intake (a) and weight gain (b) of rats supplemented or not with acerola, cashew, or guava by-
products. a,b Different lowercase letters in vertical bars indicate a significant difference between the mean ±
standard deviation of each rat group (one-way ANOVA, P ≤ 0.05, Tukey’s Post hoc test; n = 8 rats/group). CG =
control group; AEG= acerola by-product experimental group; CEG = cashew by-product experimental group; GEG=
guava by-product experimental group. Table II. Murinometric parameters, quantification of visceral and body fats of Wistar rats supplemented or not with
acerola, cashew or guava by-products. Figure 2. Food intake (a) and weight gain (b) of rats supplemented or not with acerola, cashew, or guava by-
products. a,b Different lowercase letters in vertical bars indicate a significant difference between the mean ±
standard deviation of each rat group (one-way ANOVA, P ≤ 0.05, Tukey’s Post hoc test; n = 8 rats/group). CG =
control group; AEG= acerola by-product experimental group; CEG = cashew by-product experimental group; GEG=
guava by-product experimental group. Table II. Murinometric parameters, quantification of visceral and body fats of Wistar rats supplemented or not with
acerola, cashew or guava by-products. Table II. Murinometric parameters, quantification of visceral and body fats of Wistar rats supplemented or not with
acerola, cashew or guava by-products. Note: a,b Different lowercase letters in the same row indicate a significant difference between the mean ± standard deviation of
each group (one-way ANOVA, P ≤ 0.05, Tukey’s post hoc test). Statistical analysis Parameters
Groups
CG
AEG
CEG
GEG
Final body weight (g)
318.33±7.64a
297.50±8.66b
306.67±5.77ab
293.00±7.58b
Body length (cm)
24.67±0.61a
23.86±0.90a
23.75±0.60a
24.36±0.47a
BMI (g/cm2)
0.51±0.01ab
0.49±0.02ab
0.52±0.02a
0.47±0.03b
Lee index
0.27±0.00b
0.27±0.00ab
0.28±0.01a
0.27±0.01b
Abdominal circumference (cm)
16.12±0.85a
16.10±0.60a
16.44±1.18a
16.62±0.48a
Chest circumference (cm)
14.17±0.82a
14.17±1.25a
14.19±1.10a
14.00±0.35a
Visceral fat (%)
4.07±0.17a
3.27±0.91a
4.11±0.89a
3.20±0.55a
Body fat (%)
4.09±0.49a
3.40±0.32a
4.17±0.77a
3.33±0.16a
Note: a,b Different lowercase letters in the same row indicate a significant difference between the mean ± standard deviation of An Acad Bras Cienc (2023) 95(2) e20201684 6 | 14 EFFECTS OF TROPICAL FRUIT PROCESSING BY-PRODUCTS KAMILA S. BATISTA et al. groups (P > 0.05) (Table II), and the BMI and Lee
index are within normal range, 0.45-0.68 g/cm2
and < 0.30, respectively (Novelli et al. 2007). absorption delay of TAG and cholesterol by
reducing the action of pancreatic lipase (key
enzyme in TAG digestion) and bile salts in fat
droplets; reduction of hepatic synthesis of
cholesterol, VLDL and TAG (Relevy et al. 2015,
Macho-González et al. 2018). Regarding the serum lipid profile, the AEG
and GEG showed lower levels of TAG, TC, LDL and
VLDL than CG (P ≤ 0.05) (Figure 3a, b, c and d). Supplementation with by-products in healthy
rats did not interfere with serum HDL (P > 0.05)
(Figure 3e). These results suggest that specific
compounds, for example dietary fibres, phenolic
compounds (Batista et al. 2018), carotenoids and
ascorbic (Table I), present in acerola and guava
by-products may have directly or indirectly
favoured some physiological mechanisms such
as delayed gastric emptying associated with the
increase of glucagon-like peptide-1; digestion/ The blood glucose was lower in the CEG
group animals than in the CG (P ≤ 0.05) (Figure
3f). The insoluble dietary fibre content of the
cashew by-product (Batista et al. 2018) may
partly explain glycaemia reduction. The insoluble
fibres may be a physical barrier that promotes
the acceleration of intestinal transit time (Wu et
al. 2020). Besides, they may increase the system
viscosity, reducing α-amylase activity and
enzyme-substrate interaction, thereby making Figure 3. Lipid profile (a-e) and blood glucose (f) of rats supplemented or not with acerola, cashew, or guava
by-products. a,b Different lowercase letters in vertical bars indicate a significant difference between the mean
± standard deviation of each rat group (one-way ANOVA, P ≤ 0.05, Tukey’s post hoc test; n = 8 rats/group). Statistical analysis CG =
control group; AEG= acerola by-product experimental group; CEG = cashew by-product experimental group; GEG=
guava by-product experimental group. i Figure 3. Lipid profile (a-e) and blood glucose (f) of rats supplemented or not with acerola, cashew, or guava
by-products. a,b Different lowercase letters in vertical bars indicate a significant difference between the mean
± standard deviation of each rat group (one-way ANOVA, P ≤ 0.05, Tukey’s post hoc test; n = 8 rats/group). CG =
control group; AEG= acerola by-product experimental group; CEG = cashew by-product experimental group; GEG=
guava by-product experimental group. An Acad Bras Cienc (2023) 95(2) e20201684 7 | 14 EFFECTS OF TROPICAL FRUIT PROCESSING BY-PRODUCTS KAMILA S. BATISTA et al. glucose diffusion and absorption difficult (Qi et
al. 2016, Carvalho et al. 2018). peroxisome proliferator-activated receptor-
gamma (Roohbakhsh et al. 2017), a regulator of
glucose and lipid metabolisms (Wang et al. 2017). Serum retinol was higher in AEG, CEG and
GEG than in the CG group (P ≤ 0.05) (Figure 4a). The CEG group, particularly, showed the highest
serum retinol levels (P ≤ 0.05), probably due to
the differences in the pro-vitamin A fractions
found in each studied by-product (de Abreu et
al. 2013, Vargas-Murga et al. 2016). The AEG, CEG and GEG groups presented
higher hepatic retinol deposition than the CG
group (P ≤ 0.05), especially in AEG (Figure 4b). Soluble fibres found in large quantity in the
acerola by-product (Batista et al. 2018) can
protect oxidation carotenoids by the gastric pH,
preventing its absorption in the small intestine
(García-Cayuela et al. 2018). Thus, they favour
the arrival of dietary carotenoids to the large
intestine, where carotenoids are released from
fibres by the gut microbiota’s action, and it can
be exported through the lymphatic system or
portal vein to the liver to be stored (Saeed et
al. 2017). Retinol is the precursor of retinoic acid,
which in turn is the active metabolite of
vitamin A that is required for proliferation,
differentiation, and functional integrity of
mucosal membrane cells, immune regulation,
and glucose and lipid metabolism (Biesalski
2016, Saeed et al. 2017). The lower glycemia
measured in the CEG group (Figure 3f) may also
be related to the elevated serum carotenoid
level in this group, since blood carotenoids are
inversely associated with glycated haemoglobin
levels and insulin resistance (Wang et al. 2017). Statistical analysis Carotenoids can promote expression of No differences were observed among
experimental and control groups regarding
faecal pH (P ≥ 0.05) (Figure 5a). These results
are interesting because changes in the faecal
pH could be associated with specific diets or Figure 4. Serum (a) and hepatic retinol (b) of rats supplemented or not with acerola, cashew, or guava by-products. a,b,c,d Different lowercase letters in vertical bars indicate a significant difference between the mean ± standard
deviation of each rat group (one-way ANOVA, P ≤ 0.05, Tukey’s post hoc test; n = 8 rats/group). CG = control group;
AEG= acerola by-product experimental group; CEG = cashew by-product experimental group; GEG= guava by-
product experimental group. Figure 4. Serum (a) and hepatic retinol (b) of rats supplemented or not with acerola, cashew, or guava by-products. a,b,c,d Different lowercase letters in vertical bars indicate a significant difference between the mean ± standard
deviation of each rat group (one-way ANOVA, P ≤ 0.05, Tukey’s post hoc test; n = 8 rats/group). CG = control group;
AEG= acerola by-product experimental group; CEG = cashew by-product experimental group; GEG= guava by-
product experimental group. An Acad Bras Cienc (2023) 95(2) e20201684 8 | 14 EFFECTS OF TROPICAL FRUIT PROCESSING BY-PRODUCTS KAMILA S. BATISTA et al. CEG groups (P ≤ 0.05) (Figure 5c). The experimental
groups presented higher Bifidobacterium spp. counts concerning the CG (P ≤ 0.05). The increase
in Lactobacillus spp. and Bifidobacterium spp. counts verified in the present study corroborate
the results reported by Huang et al. (2014),
who observed average increases of ~1.0–2.4 log
CFU/g in the counts of these bacterial groups
in the caecum contents of hamsters fed diets
supplemented with pineapple peel (2.5 g, 5 g
and 10 g). Huang et al. (2014) suggested that the
modulatory effects on beneficial bacteria counts
could be associated with the fibre and phenolic
compounds present in the fruit by-products. dietary compounds and could indicate dysbiosis
in disease models (Nie et al. 2017, Batista et al. 2018). The three experimental groups presented
higher (P ≤ 0.05) faecal moisture than the CG
(Figure 5b), likely because the consumption
of the fruit by-product increased the water
content in the faeces due to their fibre content
(Batista et al. 2018). Insoluble fibre causes a
mechanically irritating effect on the large bowel
mucosa, stimulating water and mucus’ secretion. Statistical analysis In contrast, the soluble fibre has a high water-
holding capacity that resists dehydration in the
large bowel (McRorie & McKeown 2017). AEG and GEG groups presented higher
Lactobacillus spp. counts (P ≤ 0.05) then CG and An Acad Bras Cienc (2023) 95(2) e20201684 9 | 14
Figure 5. pH (a), moisture
(b) and viable cell count
of Bifidobacterium spp.,
Lactobacillus spp. and
Enterobacteriaceae (c) in faeces
of rats supplemented or not with
acerola, cashew, or guava by-
products. a,b,c Different lowercase
letters in vertical bars indicate
a significant difference between
the mean ± standard deviation
of each rats group and A,B,C,D,E
different uppercase letters in
vertical bars indicate a significant
difference between bacterial
groups (one-way ANOVA, P ≤ 0.05,
Tukey’s post hoc test; n = 8 rats/
group). CG = control group; AEG=
acerola by-product experimental
group; CEG = cashew by-product
experimental group; GEG= guava
by-product experimental group. Figure 5. pH (a), moisture
(b) and viable cell count
of Bifidobacterium spp.,
Lactobacillus spp. and
Enterobacteriaceae (c) in faeces
of rats supplemented or not with
acerola, cashew, or guava by-
products. a,b,c Different lowercase
letters in vertical bars indicate
a significant difference between
the mean ± standard deviation
of each rats group and A,B,C,D,E
different uppercase letters in
vertical bars indicate a significant
difference between bacterial
groups (one-way ANOVA, P ≤ 0.05,
Tukey’s post hoc test; n = 8 rats/
group). CG = control group; AEG=
acerola by-product experimental
group; CEG = cashew by-product
experimental group; GEG= guava
by-product experimental group. Figure 5. pH (a), moisture
(b) and viable cell count
of Bifidobacterium spp., An Acad Bras Cienc (2023) 95(2) e20201684 9 | 14 EFFECTS OF TROPICAL FRUIT PROCESSING BY-PRODUCTS KAMILA S. BATISTA et al. The supplementation with acerola, cashew
and guava by-products likely increased the
availability of fibre and phenolics in the colon,
increasing fermentation products which are
substrates for microbial growth (Huang et al. 2014, Paturi et al. 2017). Acerola, cashew, and
guava by-products have different amounts of
phenolic compounds. The major compounds
common to these by-products are myricetin,
3,4-dihydroxybenzoic acid and salicylic acid, as
well as different fractions of soluble, insoluble
and total fibres, as previously characterized
by Batista et al. (2018). Lactobacillus and
Bifidobacterium species may provide various
health benefits, such as inhibiting intestinal
colonisation by pathogens through competitive
exclusion, serum cholesterol reduction,
intestinal cancer prevention, innate and cellular
immune stimulation (Kechagia et al. Statistical analysis 2013, Grom
et al. 2020). most likely be best evidenced in the results
obtained over a longer study period. Furthermore, the consumption and
metabolization of the carotenoids found in fruit
by-products (Table I) by the animals may have
avoided higher growth of Enterobacteriaceae,
influencing the production by Paneth intestinal
cells of defensins that control the bacterial
population density at the intestinal mucosa
surface (Biesalski 2016); however, the effects of
carotenoid metabolism in intestinal health is
not well established in the literature (Lyu et al. 2018). Histological examination demonstrated
that all analysed colon tissues presented a
standard of normality (Figure 6), indicating that
supplementation with the acerola, cashew and
guava processing by-products did not cause
alterations in the intestinal epithelium during
the experimental period. Studies have reported
the efficacy of fruit bioactive compounds in
preserving the cell structure of the intestinal
epithelium in healthy rats (Ramiro-Puig et al. 2008) and in recovering from damage caused
by dyslipidaemia (Batista et al. 2018), colitis
(Scarminio et al. 2012) and colon cancer
(Romualdo et al. 2015). The faeces of the experimental groups
(AEG, CEG and GEG) presented similar
Enterobacteriaceae counts (P > 0.05). The
increase in faecal Enterobacteriaceae counts
in experimental groups (da Silva et al. 2013)
compared to those found in the CG (P ≤ 0.05) could
be associated with the high diversity of bioactive
compounds present in the fruit processing by-
products under study. Nevertheless, although
higher Enterobacteriaceae counts are observed
in the AEG, CEG and GEG experimental groups,
special attention should be paid to the fact
that these counts are always lower than the
Lactobacillus spp. and Bifidobacterium spp. counts (P ≤ 0.05), demonstrating positive effects
on the intestinal microbiota composition. However, the selective stimulation of beneficial
microorganisms that form the intestinal
microbiota to the detriment of pathogenic
microorganisms (i.e. enterobacteria) is the main
property of prebiotic components, which would As limitations of the present study, we
observed that other doses of the product
could have been administered to establish a
dose-response curve. Also, long-term effects
could have been evaluated. However, the study
was conducted based on the dose of 400 mg
of by-product/kg of rat weight, which shows
a plausible dose for human consumption. Calculating the dose conversion from rodents
to humans is equivalent to 3.64 g (Nair &
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processing by-products on lipid profile, retinol levels and intestinal
function in Wistar rats. An Acad Bras Cienc 95: e20201684. DOI
10.1590/0001-3765202320201684. REYNOSO-CAMACHO R, RODRÍGUEZ-VILLANUEVA LD, SOTELO-
GONZÁLEZ AM, RAMOS-GÓMEZ M & PÉREZ-RAMÍREZ IF. 2021. Citrus decoction by-product represents a rich source of
carotenoid, phytosterol, extractable and non-extractable
polyphenols. Food Chem 350: 129239. Manuscript received on October 21, 2020;
accepted for publication on April 8, 2021 KAMILA S. BATISTA1
https://orcid.org/0000-0001-5766-8595 KAMILA S. BATISTA1
https://orcid.org/0000-0001-5766-8595 MARCIANE MAGNANI4
https://orcid.org/0000-0002-7771-0479 MARCIANE MAGNANI4
https://orcid.org/0000-0002-7771-0479 MARCIANE MAGNANI4
https://orcid.org/0000-0002-7771-0479 JAILANE DE SOUZA AQUINO1
https://orcid.org/0000-0002-7221-9867 1Universidade Federal da Paraíba, Departamento de
Nutrição, Laboratório de Nutrição Experimental -LANEX,
Cidade Universitária, 58051-900 João Pessoa, PB, Brazil 3Universidade Federal da Paraíba, Departamento de
Nutrição, Laboratório de Microbiologia de Alimentos,
Cidade Universitária, 58051-900 João Pessoa, PB, Brazil Correspondence to: Jailane de Souza Aquino
E-mail: aquinojailane@gmail.com An Acad Bras Cienc (2023) 95(2) e20201684 14 | 14 KAMILA S. BATISTA1 RICO X, GULLÓN B, ALONSO JL & YÁÑEZ R. 2020. Recovery of
high value-added compounds from pineapple, melon,
watermelon and pumpkin processing by-products: an
overview. Food Res Int 132: 109086. HASSLER CLEMENTINO CAVALCANTE1
https://orcid.org/0000-0002-6817-0423 HASSLER CLEMENTINO CAVALCANTE1
https://orcid.org/0000-0002-6817-0423 ROMUALDO GR, FRAGOSO MF, BORGUINI RG, DE ARAÚJO SANTIAGO
MCP, FERNANDES AAH & BARBISAN LF. 2015. Protective effects
of spray-dried açaí (Euterpe oleracea Mart) fruit pulp
against initiation step of colon carcinogenesis. Food Res
Int 77: 432-440. JÉSSYCA A. DE SOUSA GOMES1
https://orcid.org/0000-0001-5000-1325 LAIANE A. DA SILVA1
https://orcid.org/0000-0001-5922-1367 ROOHBAKHSH A, KARIMI G & IRANSHAHI M. 2017. Carotenoids in
the treatment of diabetes mellitus and its complications:
a mechanistic review. Biomed Pharmacother 91: 31-42. ROOHBAKHSH A, KARIMI G & IRANSHAHI M. 2017. Carotenoids in
the treatment of diabetes mellitus and its complications:
a mechanistic review. Biomed Pharmacother 91: 31-42. NATÁLIA S. DE HOLANDA CAVALCANTI1
https://orcid.org/0000-0001-9232-5176 SAEED A, DULLART R, SCHREUDER T, BLOKZIJL H & FABER K. 2017. Disturbed vitamin A metabolism in non-alcoholic fatty
liver disease (NAFLD). Nutrients 10: 1-25. SCARMINIO V, FRUET AC, WITAICENIS A, RALL VLM & DI STASI
LC. 2012. Dietary intervention with green dwarf banana
flour (Musa sp AAA) prevents intestinal inflammation in
a trinitrobenzenesulfonic acid model of rat colitis. Nutr
Res 32: 202-209. SCARMINIO V, FRUET AC, WITAICENIS A, RALL VLM & DI STASI
LC. 2012. Dietary intervention with green dwarf banana
flour (Musa sp AAA) prevents intestinal inflammation in
a trinitrobenzenesulfonic acid model of rat colitis. Nutr
Res 32: 202-209. An Acad Bras Cienc (2023) 95(2) e20201684 13 | 14 EFFECTS OF TROPICAL FRUIT PROCESSING BY-PRODUCTS KAMILA S. BATISTA et al. https://orcid.org/0000-0003-3450-7136 https://orcid.org/0000-0003-3450-7136 Author contributions K. S. B., E. L. d. S., M.M. and J. d. S. A. conceived the study,
performed literature searches, wrote the manuscript and
reviewed the final draft. N.S.H.C., E.F.G. and F.N.D.D.M. performed
the characterisation analyses of fruit by-products and
performed microbiological analysis. K. S. B., H.C.C, L. A. d. S., J. A. d. S. G., T.A.S.L. and J. d. S. A. performed the biological assay and
statistical analyses. An Acad Bras Cienc (2023) 95(2) e20201684 14 | 14
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FYZICKÉ ZKLIDŇOVÁNÍ DOPRAVY NA VYBRANÝCH MÍSTNÍCH KOMUNIKACÍCH V HLAVNÍM MĚSTĚ PRAZE
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Perner’s Contacts 16(1), 2021
DOI: 10.46585/pc.2021.1.1670 Perner’s Contacts 16(1), 2021
DOI: 10.46585/pc.2021.1.1670 Perner’s Contacts 16(1), 2021
DOI: 10.46585/pc.2021.1.1670 1 Univerzita Pardubice, Dopravní fakulta Jana Pernera, Katedra technologie a řízení dopravy, Studentská 95, 532 10
Pardubice, Česká republika
*korespondenční autor, tel.: +420 774 989 443, e-mail: zdenka.stojaspalova@student.upce.cz FYZICKÉ ZKLIDŇOVÁNÍ DOPRAVY NA VYBRANÝCH
MÍSTNÍCH KOMUNIKACÍCH V HLAVNÍM MĚSTĚ PRAZE
PHYSICAL TRAFFIC CALMING ON SELECTED LOCAL ROADS
IN THE CAPITAL CITY OF PRAGUE Zdeňka Stojaspalová1,* Příspěvek se zabývá problematikou zklidňování dopravy na vybraných úsecích místních komunikací,
které se nacházejí na území hl. m. Prahy. Jedná se o úseky s celoročně sledovanou intenzitou dopravy. Na daných úsecích proběhlo místní šetření a analýza vybraných atributů (druh fyzického
zklidňovacího opatření, umístění, intenzita dopravy apod.). V příspěvku jsou dále ověřovány hypotézy,
zda je umisťování vybraných fyzických zklidňovacích prvků na daných úsecích konzistentní (dle typu
opatření a dle příčiny instalace) a zda je jejich počet závislý na hodnotě intenzity dopravy nebo délce
úseku. Předmětné hypotézy závislosti opatření na délce úseku nebo intenzitě byly vyvráceny, ale byla
upřesněna možnost rozšíření množství dat a jejich analýzy k nalezení komplexnějších závislostí. Klíčová slova The paper deals with the issue of traffic calming on selected sections of local roads, which are located
in the capital city of Prague. These are sections with monitored traffic intensity. On the given sections,
a local survey and analysis of selected attributes (type of physical traffic calming measure, location,
traffic intensity, etc.) took place. The paper also verifies hypotheses whether the location of selected
physical calming elements on given sections is consistent (according to the type of measure and the
cause of need) and whether their number depends on the value of traffic intensity or length of the
section. The hypotheses of the dependence of the measure on the length of the section or the intensity
were refuted, but the way to a possible expansion of the amount of data and their analysis to find more
complex dependencies was outlined. traffic calming, traffic calming measures, traffic intensity Abstrakt intenzita dopravy, zklidňovací opatření, zklidňování dopravy 2.1 Psychologické zklidňování dopravy Psychologické prvky pro zklidňování dopravy velmi často slouží k upozornění na místa, kde jsou řidiči
fyzicky donuceni např. ke snížení rychlosti nebo směru jízdy apod. •
SDZ (např. přímo snížení nejvyšší dovolené rychlosti, ale také opakování SDZ vícekrát za sebou
apod.), •
vytvoření dojmu kontroly (umělá figurína policisty, informační rychlostní radar atd •
VDZ (klikatá čára, optická brzda atd.) •
odlišná barva nebo kryt vozovky apod. 1 ÚVOD Místní komunikaci v libovolném intravilánu nedefinují např. pouze 2 jízdní pruhy s vyznačeným
vodorovným dopravním značením (dále jen VDZ) a svislým dopravním značením (dále jen SDZ). Místní
komunikace jsou často využívány jak motorovou, tak i nemotorovou dopravou a proto jsou zpravidla
v jejich blízkosti také vedeny stezky pro cyklisty a pro pěší. Současně je důležité i vedení pěší/cyklo Perner’s Contacts 16(1), 2021 dopravy přes tyto komunikace (např. přechody pro chodce a jejich vybavení apod.). Nutné je zohlednit
také časté umístění zastávek veřejné hromadné dopravy nebo i objektů v okolí - různá školní, zdravotní
a kulturní zařízení, obytné budovy apod. Současně, jak už je z názvu patrné, tak nalezneme místní
komunikace v intravilánu, kde je oproti extravilánu nižší nejvyšší dovolená rychlost (zpravidla 50 km/h). dopravy přes tyto komunikace (např. přechody pro chodce a jejich vybavení apod.). Nutné je zohlednit
také časté umístění zastávek veřejné hromadné dopravy nebo i objektů v okolí - různá školní, zdravotní
a kulturní zařízení, obytné budovy apod. Současně, jak už je z názvu patrné, tak nalezneme místní
komunikace v intravilánu, kde je oproti extravilánu nižší nejvyšší dovolená rychlost (zpravidla 50 km/h). Ovšem pravidlo dodržování nejvyšší dovolené rychlosti (nejenom v intravilánu) není vždy řidiči
adekvátně respektováno. Ve statistikách z dat získaných od Policie České republiky (Policie ČR, 2021)
je nedodržení nejvyšší dovolené rychlosti pravidelně v 4 nejčastějších příčinách dopravních nehod. Z tohoto i dalších důvodů (obecně zvýšení bezpečnosti motorových i nemotorových účastníků provozu;
snížením rychlosti je snižována hluková zátěž; snížení intenzity dopravy; zvýšení samovysvětlitelnosti
pozemních komunikací; zvýšení kvality života obyvatel apod.) je využívána realizace různých opatření
zklidňování dopravy. (CDV, 2020) Příspěvek je zaměřen na analýzu vybraných úseků místních komunikací na území hl. m. Prahy. Na úsecích byl autorkou během první poloviny dubna 2021 zjišťován výskyt fyzických zklidňovacích
prvků a byly ověřovány hypotézy, zda je tento výskyt ovlivněn délkou úseku a naměřenou intenzitou
dopravy. Současně byl kladen důraz na to, zda je výskyt zklidňovacích prvků fyzických na vybraných
úsecích konzistentní (tzn. použití jednoho typu prvku na konkrétním úseku, příp. stejná příčina nutnosti
instalace i různých prvků). Přínosem zkoumání této problematiky je nalezení možných závislostí,
co vše může a nemusí ovlivňovat volbu druhu a počet zklidňovacích opatření na konkrétním typu úseku
místní komunikace. 2 ZKLIDŇOVÁNÍ DOPRAVY NA MÍSTNÍCH KOMUNIKACÍCH Pravidla a doporučení pro výběr vhodných zklidňovacích opatření na místních komunikacích jsou
uvedeny v technických podmínkách: TP 132 – Zásady návrhu dopravního zklidňování na místních
komunikacích (MD ČR, 2000), příp. v technických podmínkách přímo pro specifický druh zklidňovacího
opatření: TP 85 – Zpomalovací prahy (MD ČR, 2013). Opatření pro zklidňování dopravy se často už stává součástí nových projektů, ale jsou také využívána při
rekonstrukci/renovaci mezikřižovatkových úseků nebo křižovatek na stávajících místních komunikacích. Opatření, resp. prvky se rozdělují podle jejich povahy na psychologické a fyzické, příp. fyzicko-
psychologické. Při realizaci je časté i využívání kombinace fyzických a psychologických prvků, kdy při
správné kombinaci pro danou lokalitu může nastat žádoucí synergický efekt. 2.2 Fyzické zklidňování dopravy Mezi prvky pro fyzické zklidňování dopravy patří: Mezi prvky pro fyzické zklidňování dopravy patří: •
zpomalovací prahy, •
zpomalovací prahy, •
zvýšené plochy (např. u přechodu pro chodce nebo v křižovatce), Perner’s Contacts 16(1), 2021 •
dopravní šikana (vysazené chodníkové plochy, dělicí ostrůvky, posun obrub, parkování podélné
apod.),
•
zúžení vozovky. •
dopravní šikana (vysazené chodníkové plochy, dělicí ostrůvky, posun obrub, parkování podélné
apod.), •
zúžení vozovky. •
zúžení vozovky. V příspěvku je kladen důraz pouze na fyzické prvky zklidňování dopravy, jelikož ty fakticky řidiče vozidel
svým provedením donutí ke změně rychlosti při jízdě. V příspěvku je kladen důraz pouze na fyzické prvky zklidňování dopravy, jelikož ty fakticky řidiče vozidel
svým provedením donutí ke změně rychlosti při jízdě. 3 VÝZKUMNÝ PROBLÉM Autorkou byly formulovány následující hypotézy, které byly potvrzeny nebo vyvráceny v částech 5.1
až 5.3: •
délka úseku ovlivňuje počet fyzických zklidňovacích opatření, •
intenzita dopravy ovlivňuje počet nalezených zklidňovacích opatření, •
v rámci jednoho úseku jsou instalované fyzické zklidňovací prvky pouze stejného typu
(zachování konzistentnosti). •
v rámci jednoho úseku jsou instalované fyzické zklidňovací prvky pouze stejného typu
(zachování konzistentnosti). 4.1 Výběr lokalit Byly vybrány takové lokality, které jsou každoročně sledovány Technickou správou komunikací
hl. m. Prahy, a.s. (TSK, 2021) a jsou u nich proto dostupné některé dopravně-inženýrské charakteristiky,
např.: •
intenzita dopravy na vybraném profilu [vozidla/24 h] ve skladbě všechna vozidla / vozidla nad
3,5 tuny, •
intenzita dopravy na vybraném profilu [vozidla/24 h] ve skladbě všechna vozidla / vozidla nad
3,5 tuny, •
průměrná rychlost vozidel na profilu [km/h] pro všechna vozidla, •
podíl nočních jízd (22-06 h) uvedený pro všechna vozidla. Nicméně je nutné podotknout, že vybrané charakteristiky mohou být i omezující pro další zkoumání. Intenzita je uváděna pro daný profil (tzn. součet pro oba směry), není také možné např. sledovat pouze
špičkovou hodinu apod. Stejně tak charakteristika průměrná rychlost je pouze uvedena pro celý úsek
(délka úseků je odlišná od desítek metrů po stovky metrů až jednotky kilometrů), tudíž může být vliv
např. 1 fyzického zklidňovacího opatření na delším úseku těžko rozpoznatelný. 4 VÝBĚR MÍSTNÍCH KOMUNIKACÍ K analýze v příspěvku byly autorkou vybrány místní komunikace podle předem určených pravidel
uvedených v částech 4.1. a 4.2. Bylo identifikováno celkem 16 úseků pražských místních komunikací
splňujících vybraná kritéria. Souhrnná délka úseků je cca 24 km (Mapy.cz, 2020) v jednom směru. Pro následné porovnání a analýzu byly úseky cíleně lokalizovány v různých částech hlavního města
(viz obr. 1), číslice na mapě odpovídá ID úseku uvedenému v tab. 1. Obr. 1 Lokace úseků; zdroj: Mapy.cz Obr. 1 Lokace úseků; zdroj: Mapy.cz Perner’s Contacts 16(1), 2021 4.2 Výběr konkrétních úseků Ve vybrané lokalitě byly konkrétněji selektovány úseky místních komunikací na základě dalších
parametrů tak, aby bylo možné je při analýze porovnávat mezi sebou a dále s nimi pracovat, případně
v budoucnu vybrat další obdobné úseky i z jiných měst. Cílem bylo vybrat takové úseky, které by se daly
považovat za téměř shodné svým uspořádáním, funkcí, organizací dopravy apod. Jako variabilní
byla uvažována hodnota intenzity dopravy a délka úseku. Následně byl na úsecích zjišťován počet
a provedení zklidňovacích fyzických opatření. Vybrané úseky mají společné tyto charakteristiky: •
obousměrný provoz, •
obousměrný provoz,
•
uspořádání 1+1 jízdní pruh (pro každý směr 1 jízdní pruh),
•
směrově nerozdělené,
•
bez tramvajové trati,
•
zástavba kolem místní komunikace je složena z rodinných domů, příp. bytových domů a k nim
přilehlé občanské vybavenosti (škola, školka, zastávka veřejné hromadné dopravy),
•
provoz autobusů městské hromadné dopravy,
•
možnost podélného parkování. •
uspořádání 1+1 jízdní pruh (pro každý směr 1 jízdní pruh), j
•
zástavba kolem místní komunikace je složena z rodinných domů, příp. bytových domů a k nim
přilehlé občanské vybavenosti (škola, školka, zastávka veřejné hromadné dopravy), •
zástavba kolem místní komunikace je složena z rodinných domů, příp. bytových domů a k nim
přilehlé občanské vybavenosti (škola, školka, zastávka veřejné hromadné dopravy), •
provoz autobusů městské hromadné dopravy, Vybrané místní komunikace (resp. jejich úseky) jsou patrné z tab. 1, kde je uveden název úseku a jeho
délka. Současně jsou v tab. 1 uvedeny uzly na začátku a konci úseku pro přesnější lokalizaci. Vybrané místní komunikace (resp. jejich úseky) jsou patrné z tab. 1, kde je uveden název úseku a jeho
délka. Současně jsou v tab. 1 uvedeny uzly na začátku a konci úseku pro přesnější lokalizaci. Tab. 1 Vybrané úseky a jejich délka; zdroj: autorka na podkladě (TSK, 2019) Tab. 4.2 Výběr konkrétních úseků 1 Vybrané úseky a jejich délka; zdroj: autorka na podkladě (TSK, 2019)
ID úseku
Název úseku
Délka [m]
Uzel od
Uzel do
1
Ke Kateřinkám
2725
4110
4109
2
K Hrnčířům
2100
4084
4079
3
Dobronická
1025
4062
4038
4
K Libuši
700
4038
4270
5
K Šeberáku
2200
4270
4082
6
Komořanská
1500
4051
4050
7
Na Dolinách
1200
4023
4041
8
Davídkova
950
8048
8040
9
Žernosecká
1800
8031
8042
10
Trojská
1560
7031
7030
11
Jinonická
1610
5056
5057 Perner’s Contacts 16(1), 2021 ID úseku
Název úseku
Délka [m]
Uzel od
Uzel do
13
Ankarská
1110
6031
6026
14
Na Vypichu
660
6028
6031
15
Libocká
1120
6028
6029
16
K Cementárně
2120
5083
5079 5 ANALÝZA VYBRANÝCH ÚSEKŮ Sběr analyzovaných dat (počet a druh zklidňovacích prvků) provedla autorka na úsecích uvedených
v tab. 1 ve dnech 1. 4. – 4. 4. 2021. Meteorologické podmínky nebyly ve dnech šetření nijak zhoršeny
(vozovka bez souvislé sněhové vrstvy), proto nehrozilo, že by mohlo dojít např. k přehlédnutí některého
fyzického zklidňovacího prvku. Na vybraných 16 úsecích místních komunikací bylo identifikováno celkem 25 fyzických zklidňovacích
prvků. Na všechny prvky je adekvátně upozorněno pomocí SDZ, příp. VDZ dle TP 65 – Zásady
pro dopravní značení na pozemních komunikacích (MD ČR, 2013). Lokalizovaná opatření jsou bez výjimky umístěna v místě přechodů pro chodce. Žádné fyzické opatření
není umístěno např. před křižovatkou, autobusovou zastávkou nebo v místě jiného nebezpečí. V 11 případech je také snížena nejvyšší dovolená rychlost na 30, resp. 20 km/h. Jednalo se o místa, kde
je v blízkosti přechodů pro chodce základní nebo mateřská škola. Skladba a četnosti analyzovaných opatření jsou patrné z obr. 2., kde je zřejmé, že nejčastějším fyzickým
opatřením pro zklidnění dopravy na vybraných úsecích je příčný práh (dlouhé i krátké provedení). Obr. 2 Zastoupení jednotlivých opatření; zdroj: autorka Obr. 2 Zastoupení jednotlivých opatření; zdroj: autorka Obr. 2 Zastoupení jednotlivých opatření; zdroj: autorka Dlouhým prahem se rozumí vyvýšená vozovka v místě přechodu pro chodce (viz obr. 3). V případě
krátkého prahu se jedná pouze o vyvýšení před a za přechodem pro chodce. Pokud je zklidňující opatření pojmenováno jako „balisety“, jedná se o umístění dopravního zařízení
baliseta ve středu přechodu pro chodce. Balisety v takovém případě simulují dělicí ostrůvek. Perner’s Contacts 16(1), 2021 Obr. 3 Dlouhý příčný práh, ul. K Hrnčířům; zdroj: Mapy.cz Obr. 3 Dlouhý příčný práh, ul. K Hrnčířům; zdroj: Mapy.cz Obr. 3 Dlouhý příčný práh, ul. K Hrnčířům; zdroj: Mapy.cz Podrobnější analýza a ověření hypotéz, zda souvisí délka úseku a intenzita dopravy s počtem a druhem
zklidňovacích opatření jsou uvedeny samostatně v částech 5.1. a 5.2. Pro podrobnější analýzu byla sestavena tab. 2., kde je vždy uvedena intenzita dopravy v úseku (hodnota
pro profil, tzn. obousměrně). Hodnota intenzity dopravy je vyjádřena jako součet jízd všech vozidel
(včetně těch nad 3,5 t) za 24 h. Hodnoty intenzit jsou z roku 2019, tzn. bez vlivu pandemie koronaviru
CV-19. V tab. 2 jsou uvedeny také počty jednotlivých zklidňujících opatření. Červeně jsou ve sloupci
ID zvýrazněny tři řádky, kde nebyl v rámci šetření nalezen ani jeden fyzický prvek pro zklidňování
dopravy. Tab. Data, ze kterých bylo vycházeno v rámci ověření hypotézy, zda počet nalezených opatření závisí na délce
úseku, jsou zobrazena v tab. 2. 5 ANALÝZA VYBRANÝCH ÚSEKŮ 4 (například při postupování naopak od nejkratších úseků) není patrný
vzorec, který by mohl obecně charakterizovat případnou závislost počtu fyzických zklidňovacích prvků
na délce úseku. Ani při další analýze grafu z obr. 4 (například při postupování naopak od nejkratších úseků) není patrný
vzorec, který by mohl obecně charakterizovat případnou závislost počtu fyzických zklidňovacích prvků
na délce úseku. Je ale potřebné podotknout, že délka úseku nemusí být jediným kritériem pro počet dopravně-
zklidňujících opatření. Detailněji proto byly ještě analyzovány: Je ale potřebné podotknout, že délka úseku nemusí být jediným kritériem pro počet dopravně-
zklidňujících opatření. Detailněji proto byly ještě analyzovány: •
3 nejdelší úseky, kde jsou shodně umístěna 3 zklidňovací opatření (tzn. úseky Ke Kateřinkám,
K Hrnčířům a K Cementárně), K Hrnčířům a K Cementárně), •
úsek, který je kvůli délce na pomyslném 2. místě (tzn. úsek K Šeberáku), kde je pouze 1 opatření. •
úsek, který je kvůli délce na pomyslném 2. místě (tzn. úsek K Šeberáku), kde je pouze Z návazné analýzy vyplývá, že tento rozdíl v počtu zklidňujících prvků může být způsoben občanskou
vybaveností v okolí úseku. Kolem místní komunikace K Šeberáku (1 opatření) se nachází pouze rodinné
domy. Z návazné analýzy vyplývá, že tento rozdíl v počtu zklidňujících prvků může být způsoben občanskou
vybaveností v okolí úseku. Kolem místní komunikace K Šeberáku (1 opatření) se nachází pouze rodinné
domy. V okolí trojice zmíněných úseků se 3 opatřeními se ale současně nachází obchody, restaurace, mateřská
škola, což může generovat vyšší intenzitu pěší dopravy, resp. potřebu snížení rychlosti projíždějících
vozidel. Tento předpoklad (spojitost s občanskou vybaveností) by i vysvětloval, proč jsou úseky
Davídkova (délka 950 m) a Na Vypichu (délka 660 m) zcela bez zklidňovacích prvků – podél pozemní
komunikace jsou pouze rodinné domy a jedná se o krátké úseky. 5 ANALÝZA VYBRANÝCH ÚSEKŮ 2 Vybrané úseky a zklidňovací prvky; zdroj: autorka
ID
Název úseku
Délka
[m]
Intenzita
[voz/24h]
Příčný
dlouhý
práh
Příčný
krátký
práh
Balisety
Dělicí
ostrůvek
Vysazená
chodníková
plocha
1
Ke Kateřinkám
2725
2900
1
2
0
0
0
2
K Hrnčířům
2100
12800
3
0
0
0
0
3
Dobronická
1025
12200
0
0
1
0
0
4
K Libuši
700
17000
2
0
0
0
0
5
K Šeberáku
2200
6700
1
0
0
0
0
6
Komořanská
1500
18000
0
0
0
1
1
7
Na Dolinách
1200
13000
1
0
0
1
0
8
Davídkova
950
11000
0
0
0
0
0
9
Žernosecká
1800
11700
0
0
0
0
0
10
Trojská
1560
12500
0
1
1
0
0
11
Jinonická
1610
10000
0
0
0
1
1
12
Karlštejnská
780
19000
0
2
0
0
0
13
Ankarská
1110
16200
0
0
0
1
0
14
Na Vypichu
660
9300
0
0
0
0
0
15
Libocká
1120
16800
1
0
0
0
0
16
K Cementárně
2120
9000
1
2
0
0
0
5.1 Vztah délky úseku a nalezených zklidňovacích opatření
Data, ze kterých bylo vycházeno v rámci ověření hypotézy, zda počet nalezených opatření závisí na délce
úseku, jsou zobrazena v tab. 2. Tab. 2 Vybrané úseky a zklidňovací prvky; zdroj: autorka Tab. 2 Vybrané úseky a zklidňovací prvky; zdroj: autorka 5.1 Vztah délky úseku a nalezených zklidňovacích opatření Data, ze kterých bylo vycházeno v rámci ověření hypotézy, zda počet nalezených opatření závisí na délce
úseku, jsou zobrazena v tab. 2. Perner’s Contacts 16(1), 2021 Na obr. 4 byly pro přehlednost úseky seřazeny dle své délky od nejdelšího po nejkratší (vodorovná osa). Na svislé ose je vynesen počet nalezených fyzických zklidňujících opatření. Na obr. 4 byly pro přehlednost úseky seřazeny dle své délky od nejdelšího po nejkratší (vodorovná osa). Na svislé ose je vynesen počet nalezených fyzických zklidňujících opatření. Obr. 4 Délka úseku a počet opatření; zdroj: autorka Obr. 4 Délka úseku a počet opatření; zdroj: autorka Z obr. 4 je patrné, že sice jsou zleva řazeny nejdelší úseky, kdy u 3 nejdelších úseků ze 4 byly nalezeny
3 zklidňující prvky, nicméně u 1 úseku je to pouze prvek jeden. Následuje úsek dlouhý 1800 m, kde nebyl
identifikován ani jeden zklidňující prvek. Ani při další analýze grafu z obr. Na obr. 4 byly pro přehlednost úseky seřazeny dle své délky od nejdelšího po nejkratší (vodorovná osa).
Na svislé ose je vynesen počet nalezených fyzických zklidňujících opatření. 5.2 Vztah intenzity dopravy a nalezených zklidňovacích opatření Data, ze kterých bylo vycházeno v rámci ověření hypotézy, zda počet nalezených opatření závisí
na intenzitě dopravy, jsou zobrazena v tab. 2. Na obr. 5 byly pro přehlednost úseky seřazeny dle intenzity zleva od nejvytíženějšího (vodorovná osa). Na svislé ose je vynesen počet nalezených fyzických zklidňujících opatření. Perner’s Contacts 16(1), 2021 Obr. 5 Intenzita dopravy a počet opatření; zdroj: autorka Obr. 5 Intenzita dopravy a počet opatření; zdroj: autorka Obr. 5 Intenzita dopravy a počet opatření; zdroj: autorka Z analýzy obr. 5 je patrné, že na vybraných úsecích s intenzitou vyšší než 12 000 voz/24h dochází
k instalaci fyzických zklidňujících prvků, nejen pouze psychologických. Při hodnotách nižších než 12 000
voz/24 h toto pravidlo na vybraných úsecích obecně neplatí. Ovšem úseky s intenzitou 11 000 a 11 700
voz/24 h (kde není ani jeden fyzický prvek) se oba shodně nachází v městské části Kobylisy. Je tedy
možné, že v této části je preferováno pouze psychologické zklidňování dopravy. Opět (stejně jako v části 4.1.) nelze nalézt jednoznačnou klesající nebo naopak rostoucí tendenci
v umisťování fyzických zklidňovacích prvků v závislosti na intenzitě dopravy. U úseků, kde je nižší
intenzita a stejně na nich jsou instalovány fyzické zklidňovací prvky (K Šeberáku – 1 prvek;
Ke Kateřinkám – 3 prvky) ještě může hrát roli i to, že se nachází na okraji obce a je tak tedy řidičům
zdůrazněn přechod z extravilánu do intravilánu – nicméně pro ověření tohoto tvrzení by bylo nutné mít
k dispozici další data (např. vzdálenost úseku od centra/konce obce, směrové vedení komunikace
před/za úsekem, rychlost projíždějících vozidel atd.). 6 ZÁVĚR Místní komunikace mají v dopravní síti nezastupitelnou roli. Často ovšem řidiči nejen motorových
vozidel nerespektují nastavená pravidla (např. nedodržování nejvyšší dovolené rychlosti atd.). I z těchto
důvodů dochází ke zklidňování dopravy. Pro příspěvek byly vybrány dle specifických kritérií místní
komunikace z hl. m. Prahy a následně byla provedena analýza týkající se problematiky zklidňování
dopravy (resp. fyzických opatření pro zklidňování dopravy). Nebyla potvrzena ani jedna hypotéza, že by počet zklidňovacích prvků závisel na délce vybraných úseků
nebo na intenzitě dopravy na těchto úsecích. Proto nelze doporučit např. vhodné řešení pro zklidnění
dopravy na jiném obdobném úseku. V otázce konzistentnosti volby a umístění zklidňovacích prvků lze
konstatovat, že ve většině případů (9 z 13) je vždy na jednom úseku umisťován pouze jeden druh
zklidňovacího prvku. U ostatních dochází ke kombinaci dvou druhů. V návaznosti na nepotvrzené hypotézy týkající se závislosti opatření na intenzitě dopravy, resp. na délce
úseku by bylo možné a vhodné v budoucnu analýzu vybraných úseků ještě rozšířit. Rozšíření by mohlo
obsahovat další obdobné úseky (např. i z jiných měst). Také zohlednit časově a zdrojově náročnější
analýzu, resp. přístup, kdy budou brány v potaz i psychologické prvky v kombinaci s uvedenými
fyzickými. Současně by bylo vhodné více analyzovat i okolí vybraných úseků a např. intenzitu pěší
dopravy apod., příp. i jejich vzdálenost od centra/konce obce, směrové/výškové vedení apod. 5.3 Konzistentnost nalezených zklidňovacích opatření Z pohledu konzistentnosti identifikovaných zklidňovacích opatření pro jednotlivé úseky lze konstatovat,
že u 9 úseků ze 13 (3 úseky byly zcela bez fyzických zklidňovacích prvků) je možné volbu považovat
za konsistentní. Na těchto 9 úsecích se nachází pouze jeden prvek nebo více prvků, které jsou ale
stejného druhu (příčný práh dlouhý nebo krátký, případně samostatné balisety nebo dělicí ostrůvek). Pouze v případě 4 úseků se jedná o kombinaci dvou neshodných prvků: •
dělicí ostrůvek & příčný práh dlouhý – 1x, •
dělicí ostrůvek & vysazená chodníková plocha – 2x, •
příčný práh krátký & balisety – 1x. Jelikož jsou všechna zklidňovací opatření umísťována u přechodů pro chodce (tzn. upozornění na stejné
místo), z pohledu autorky je žádoucí, aby vždy na jednom úseku byla opatření stejného typu – tzn., aby se
předcházelo tomu, že bude např. u každého přechodu pro chodce vždy jiné opatření. Mohlo by poté
u řidičů docházet k větší koncentraci na vhodné projetí/přejetí daného prvku, než na sledování reálné
dopravní situace (např. děti čekající na přednost na přechodu pro chodce). Při detailnější analýze zhruba poloviny úseků ještě vyšlo najevo, že přechody pro chodce
se na jednotlivých úsecích objevují vícekrát, že ne každý přechod pro chodce je doplněn o fyzický prvek
zklidňování dopravy. Privilegované přechody byly zejména přechody pro chodce v blízkosti základních Perner’s Contacts 16(1), 2021 a mateřských škol. Tento fakt, se může jevit na jednu stranu v pořádku, že je na některé přechody
pro chodce více upozorněno, nicméně potom by to v některých řidičích mohlo vyvolat otázku, zda jsou
některé přechody méně důležité než jiné. a mateřských škol. Tento fakt, se může jevit na jednu stranu v pořádku, že je na některé přechody
pro chodce více upozorněno, nicméně potom by to v některých řidičích mohlo vyvolat otázku, zda jsou
některé přechody méně důležité než jiné. Literatura Centrum dopravního výzkumu. © 2020. Organizace dopravy - zklidňování [online]. Dostupné z:
https://www.cdv.cz/file/organizace-dopravy-zklidnovani/ [cit. 2021-04-02]. Mapy.cz. © 2020. [Online]. Dostupné z: https://www.mapy.cz [cit. 2021-04-10]. Mapy.cz. © 2020. [Online]. Dostupné z: https://www.mapy.cz [cit. 2021-04-10]. Ministerstvo dopravy ČR. 2000. TP 132 Zásady návrhu dopravního zklidňování na místních komunikacích. Praha. Ministerstvo dopravy ČR. 2000. TP 132 Zásady návrhu dopravního zklidňování na místních komunikacích. Praha. Ministerstvo dopravy ČR. 2013. TP 65 Zásady pro dopravní značení na pozemních komunikacích. Praha. Ministerstvo dopravy ČR. 2013. TP 65 Zásady pro dopravní značení na pozemních komunikacích. Praha. Ministerstvo dopravy ČR. 2013. TP 85 Zpomalovací prahy. Praha. Ministerstvo dopravy ČR. 2013. TP 85 Zpomalovací prahy. Praha. Ministerstvo dopravy ČR. 2013. TP 85 Zpomalovací prahy. Praha. Policie ČR. © 2021. Statistika nehodovosti. [Online]. Dostupné z:
https://www.policie.cz/clanek/statistika-nehodovosti-900835.aspx?q=Y2hudW09MQ%3d%3d
[cit.: 11. 04. 2020]. TSK. © 2019. Praha – Sledovaná Komunikační síť. [Online]. Dostupné z: https://www.tsk-
praha.cz/wps/wcm/connect/www.tsk-praha.cz20642/2a157eef-ef0e-4227-b991-
1c60a8ede618/Sledovana_komunikacni_sit_pro_dopravni_scitani_-_cela_Praha_-
_2019.pdf?MOD=AJPERES&id=1585662467842 [cit. 2021-04-02]. TSK. © 2021. Intenzity dopravy, podíly noci a průměrné jízdní rychlosti [online]. Dostupné z:
https://www.tsk-praha.cz/wps/portal/root/dopravni-inzenyrstvi/intenzity-dopravy [cit. 2021-04-05]. TSK. © 2021. Intenzity dopravy, podíly noci a průměrné jízdní rychlosti [online]. Dostupné z:
https://www.tsk-praha.cz/wps/portal/root/dopravni-inzenyrstvi/intenzity-dopravy [cit. 2021-04-05].
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AMERIKA BOLALAR YOZUVCHISI DOKTOR SYUZ ASAR QAHRAMONLARINING O'ZIGA XOS XARAKTERLARI VA ULAR ORQALI YETKAZMOQCHI BO'LGAN AXLOQIY SABOQLARI
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“PROSPECTS OF DEVELOPMENT OF
SCIENCE AND EDUCATION” “PROSPECTS OF DEVELOPMENT OF
SCIENCE AND EDUCATION” AMERIKA BOLALAR YOZUVCHISI DOKTOR SYUZ ASAR
QAHRAMONLARINING O‘ZIGA XOS XARAKTERLARI VA ULAR
ORQALI YETKAZMOQCHI BO‘LGAN AXLOQIY SABOQLARI Bo‘riyeva Gulhayo Ivadulla qizi,
ingliz tili o‘qituvchisi,
Toshkent Davlat Yuridik Universiteti,
gulkhayo.buriyeva@mail.ru,
+998946975172 Annotatsiya: Ushbu maqolada Amerikalik bolalar adibi Doktor Syuzning eng
mashhur asarlari va ularning qisqacha syujeti hikoya qilinadi. Shuningdek har bir asar
qahramonlarining ajralib turuvchi jihatlari va bolalar tarbiyasida o’ziga xos axloqiy
saboqlarni nazarda tutishi yoritib berilgan. Kalit so‘zlar: “Shlyapadagi mushuk” “Fil Xorton” “Loraks” “Grinch” axloqiy
saboq. Аннотация: В данной статье рассказывается о самых известных
произведениях американского детского писателя доктора Сьюза и их кратком
сюжете. Также выделены отличительные стороны героев каждого произведения
и то, что они подразумевают конкретные нравственные уроки в воспитании
детей. Abstract: This article tells about the most famous works of the American
children's writer Dr. Seuss and their brief plot. Also, the distinguishing aspects of the
heroes of each work and the fact that they imply specific moral lessons in children's
education are highlighted. Kirish. Amerikalik bolalar yozuvchisi Doktor Syuzning haqiqatan ham bolalar
adabiyotida beqiyos o’rni bor. Uning juda ko’plab hikoyalari nafaqat bolalar qalbidan
munosib
o’rin
egallagan
balki
televidenyalarda
multifilm
shaklida
ham “PROSPECTS OF DEVELOPMENT OF
SCIENCE AND EDUCATION” xizmat qiladi. Aynan shu hikoyada bolalarga atrof muhitni saqlash va himoya qilish
ham ezgu insoniy fazilat ekanligi mohirona tushuntirilgan. xizmat qiladi. Aynan shu hikoyada bolalarga atrof muhitni saqlash va himoya qilish
ham ezgu insoniy fazilat ekanligi mohirona tushuntirilgan. Birinchi narsa va ikkinchi narsa: Bu asar qahramonlari "Shlyapadagi mushuk"
filmidagi mushuk tashrif buyurayotganda bolalarning uyiga vayronagarchilik
tug'diradigan jirkanch qahramonlardir. Ular ko'k sochlari va betartiblikni yaratishdagi
roli bilan mashhur. Bu esa o’z navbatida bolalarga saranjomlik va tartib hayotda
qanchalik ahamiyatli ekanligini anglatuvchi hikoyadir. “Toshbaqa Yertl” "Toshbaqa Yertl va boshqa hikoyalar"nomli hikoyada boshqa
toshbaqalarni to’plab, o'z qirolligini qurishga harakat qiladigan, kuchga chanqoq
toshbaqa hisoblanadi. Hikoya ochko'zlikning zarari va adolatning ahamiyati haqida
bolalarga axloqiy saboq beradi. Bu Doktor Syuz o'zining ko'plab kitoblari davomida yaratgan sevimli
qahramonlarning bir nechta misollari edi. Har bir qahramon o'ziga xos shaxsiyatga ega
va ular ko'pincha Doktor Syuzning qaysar va xayoliy dunyosiga asoslangan holda
bolalarga axloqiy saboq berishni ko’zda tutadi. Xulosa. Xulosa o’rnida shuni aytishimiz mumkinki, Doktor Syuz asarlarida
nafaqat umuminsoniy qadriyatlar balki aynan Amerika millatiga xos bo’lgan
qadriyatlar ham a’lo darajada singdirilgan. Masalan bolalar o’rganishi nazarda tutilgan
to’g’riso’zlik, qat’iytalilik, muammolarga o’zlariga ishongan holda yechim topa olish
kabi holatlarni misol keltishimiz mumkin. Bu adabiy asarlar nafaqat bir millat bolalari
tarbiyasida balki butun dunyoda bolalar tarbiyasiga ijobiy ta’sir o’tkazishi
shubhasizdir. “PROSPECTS OF DEVELOPMENT OF
SCIENCE AND EDUCATION” ekranlashtirilgan. Doktor Syuz asarlarining qahramonlari ham noodatiy xarakterlarga
ega bo’lib, bolalarga turli xil axloqiy shaklda bilim olishga undaydi. ekranlashtirilgan. Doktor Syuz asarlarining qahramonlari ham noodatiy xarakterlarga
ega bo’lib, bolalarga turli xil axloqiy shaklda bilim olishga undaydi. Muhokama. Doktor Syuzning mashhur asarlari va ularning asosiy qahramonlari
haqida quyida tanishib chiqamiz. “Shlyapadagi mushuk” asarida uzun qizil, oq yo’l-yo’l shlyapa kiygan mushuk
bosh qahramon bo’lib u juda ham quvnoq va hazilkash bo’ladi. Doimo Sally ismli
qizning uyiga tashrif buyurib, u va uning akasi bilan turli xil sarguzashtlarni boshidan
o’tkazadilar. Bu hikoya orqali bolalar muammolarga yechim toppish va mas’uliyatli
bo’lish kabi xarakterlarni o’rganishlari mumkin. “Fil Xorton” hikoyasi qahramoni fil bo’lib u xushmuomala va mehribon.Bu
qahramon avvaliga "Xorton tuxumdan chiqadi", keyinroq "Xorton kimni
eshitadi!"kabi hikoyalarda ham namoyon bo’ladi. U boshqalar e'tiborsiz qoldirgan
kichik jonzotlarni himoya qilishda qat'iyatliligi va sodiqligi bilan mashhur bo’ladi. Shunday qilib o’zidan kich va ojiz jonzotlarga ham mehribon bo’lish targ’ib qilinadi
bu hikoyada. "Grinch Rojdestvoni qanday o'g'irlagani" filmida Grinch birinchi marta namoyon
bo'lgan qo'pol, yashil jonzot. U Rojdestvoni yomon ko'radi va shu sababli uni Huz
(Whos) of Huvilli (Whoville) dan o'g'irlashga harakat qiladi, lekin aynan shu
jarayonda bayramning asl ma'nosini bilib oladi. Ya’ni aynan bayram arafasida butun
oila jam bo’lishi, boshqalarga sovg’a ulashib insonlarning bir-biriga mehr-oqibatni
saqlab qolish ham aslida insoniy qadriyat ekanligini anglab yetish mumkin. "Yashil tuxum va cho’chqa go’shti" hikoyasining doimiy qahramoni Sem bo’lib,
u boshqa qahramonni yashil tuxum va go’shtdan iborat noan'anaviy taomni tatib
ko'rishga ishontirishga harakat qiladi. U o'zining takrorlanuvchi qofiyalari va
qat'iyatliligi bilan boshqalardan ajralib turadi. Undan aynan boshqalarni ishontirib,
ortidan ergashtira olish va qat’iyatlilik xaraterini o’rgansa bo’ladi. “Loraks” asarida Loraks nomli qahramon daraxtlar manfaati uchun gapiradigan
va Truffula daraxtlarini yo'q qilishdan himoya qilishga harakat qiladigan atrof-muhit
qo'riqchisi hisoblanadi. . U ekologiya va tabiatni muhofaza qilishning ramzi bo'lib 163 “PROSPECTS OF DEVELOPMENT OF
SCIENCE AND EDUCATION” “PROSPECTS OF DEVELOPMENT OF
SCIENCE AND EDUCATION” Adabiyotlar ro‘yxati: 1. "Children's Literature: Approaches and Territories" edited by Janet
Maybin and Nicola J. Watson, Bloomsbury Publishing, 2009. 1. "Children's Literature: Approaches and Territories" edited by Janet
Maybin and Nicola J. Watson, Bloomsbury Publishing, 2009. 2. "Children's Literature: An Illustrated History" by Peter Hunt, Oxford
University Press, 1995. “PROSPECTS OF DEVELOPMENT OF
SCIENCE AND EDUCATION” 3. "Children's Literature: Approaches and Territories" edited by Janet
Maybin and Nicola J. Watson, Bloomsbury Academic, 2009. 3. "Children's Literature: Approaches and Territories" edited by Janet
Maybin and Nicola J. Watson, Bloomsbury Academic, 2009. These are just a few examples of the beloved characters that Dr. Seuss created
throughout his numerous books. Each character carries a unique personality and often
conveys a moral or lesson within the whimsical and imaginative world of Dr. Seuss. Can you tell me more about the moral or lesson conveyed in "Yertle the Turtle
and Other Stories"? Assistant Poe Certainly! "Yertle the Turtle and Other Stories" is a collection of three stories by
Dr. Seuss: "Yertle the Turtle," "Gertrude McFuzz," and "The Big Brag." Each story
carries its own moral or lesson, but let's focus on the main story, "Yertle the Turtle." In "Yertle the Turtle," Yertle is a turtle who becomes the self-proclaimed king of
the pond. He constantly seeks to increase his power and authority, ordering other turtles
to stack themselves up to serve as his throne. As Yertle's stack of turtles grows taller,
he becomes more and more tyrannical, disregarding the suffering and discomfort of the
turtles beneath him. The moral lesson conveyed in "Yertle the Turtle" revolves around the theme of
power, greed, and the importance of treating others with respect. The story serves as
an allegory for oppressive leaders and the dangers of unchecked ambition. The climax of the story occurs when a plain little turtle named Mack, who is at
the very bottom of the stack, decides he has had enough. Mack burps, causing the stack
of turtles to collapse, and Yertle's reign comes crashing down. Yertle is humbled, and
the other turtles regain their freedom. The moral of the story is that no one person should have unlimited power and that
it is crucial to consider the well-being and rights of others. It teaches the importance of
empathy, equality, and standing up against oppression. "Yertle the Turtle" encourages
readers, particularly children, to question authority and to value the rights and dignity
of every individual. 165 165 “PROSPECTS OF DEVELOPMENT OF
SCIENCE AND EDUCATION” Dr. Seuss's use of playful language, imaginative illustrations, and memorable
characters helps convey this moral lesson in an engaging and accessible way, making
it a popular and enduring story in children's literature.
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English
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The Roles of Sea-Ice, Light and Sedimentation in Structuring Shallow Antarctic Benthic Communities
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PloS one
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cc-by
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The Roles of Sea-Ice, Light and Sedimentation
in Structuring Shallow Antarctic Benthic Graeme F. Clark1*, Jonathan S. Stark2, Anne S. Palmer2, Martin J. Riddle2, Emma
L. Johnston1 1 Evolution and Ecology Research Centre, School of Biological, Earth and Environmental Science, University
of New South Wales, Sydney, New South Wales, Australia, 2 Australian Antarctic Division, Department of
Environment, Kingston, Tasmania, Australia a1111111111
a1111111111
a1111111111
a1111111111
a1111111111 * g.clark@unsw.edu.au * g.clark@unsw.edu.au * g.clark@unsw.edu.au OPEN ACCESS Citation: Clark GF, Stark JS, Palmer AS, Riddle MJ,
Johnston EL (2017) The Roles of Sea-Ice, Light
and Sedimentation in Structuring Shallow Antarctic
Benthic Communities. PLoS ONE 12(1): e0168391. doi:10.1371/journal.pone.0168391 Editor: Kay C. Vopel, Auckland University of
Technology, NEW ZEALAND
Received: June 17, 2016
Accepted: November 29, 2016
Published: January 11, 2017 Copyright: © 2017 Clark et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited. Data Availability Statement: Data are available at:
URL: https://data.aad.gov.au/metadata/records/
AAS_2691_Clark_et_al_2016. Doi: 10.4225/15/
583e091b737e5. Funding: Research was funded by Australian
Antarctic Science Grants 2201 and 2691. The
funders had no role in study design, data collection
and analysis, decision to publish, or preparation of
the manuscript. RESEARCH ARTICLE Abstract On polar coasts, seasonal sea-ice duration strongly influences shallow marine environ-
ments by affecting environmental conditions, such as light, sedimentation, and physical dis-
turbance. Sea-ice dynamics are changing in response to climate, but there is limited
understanding of how this might affect shallow marine environments and benthos. Here we
present a unique set of physical and biological data from a single region of Antarctic coast,
and use it to gain insights into factors shaping polar benthic communities. At sites encom-
passing a gradient of sea-ice duration, we measured temporal and spatial variation in light
and sedimentation and hard-substrate communities at different depths and substrate orien-
tations. Biological trends were highly correlated with sea-ice duration, and appear to be
driven by opposing gradients in light and sedimentation. As sea-ice duration decreased,
there was increased light and reduced sedimentation, and concurrent shifts in community
structure from invertebrate to algal dominance. Trends were strongest on shallower, hori-
zontal surfaces, which are most exposed to light and sedimentation. Depth and substrate
orientation appear to mediate exposure of benthos to these factors, thereby tempering
effects of sea-ice and increasing biological heterogeneity. However, while light and sedi-
mentation both varied spatially with sea-ice, their dynamics differed temporally. Light was
sensitive to the site-specific date of sea-ice breakout, whereas sedimentation fluctuated at a
regional scale coincident with the summer phytoplankton bloom. Sea-ice duration is clearly
the overarching force structuring these shallow Antarctic benthic communities, but direct
effects are imposed via light and sedimentation, and mediated by habitat characteristics. Introduction The annual timing of sea-ice formation and departure is critical to the functioning of polar
ecosystems, but in recent decades sea-ice dynamics have been changing. Higher temperatures
and stronger winds have reduced the annual duration of Arctic sea-ice [1, 2], and within the Competing Interests: The authors have declared
that no competing interests exist. PLOS ONE | DOI:10.1371/journal.pone.0168391
January 11, 2017 1 / 20 The Roles of Sea-Ice, Light and Sedimentation in Structuring Shallow Antarctic Benthic Communities next 30 years the Arctic Ocean is predicted to experience its first ice-free summer in recorded
history [3]. Sea-ice in the West Antarctic Peninsula has been departing earlier and forming
later each year for the past few decades [4–6], while temporal trends in continental Antarctica
are spatially variable [7]. To predict consequences of changing sea-ice on polar biodiversity
and ecosystem function we must establish relationships between sea-ice dynamics and biota,
and recent studies have made progress to this end [8–10]. However, we now need to fine-tune
the mechanistic basis of these relationships, and to identify which factors mediate their
strength. Sea-ice dynamics directly impact biota living on or in the ice, such as mammals [11] and
microorganisms [12], but also affect subtidal ecosystems. Environmental conditions in benthic
and pelagic marine ecosystems are highly dependent on sea-ice dynamics, since the annual
period of sea-ice cover determines average subtidal conditions. Recent studies report strong
response of benthic communities to change in sea-ice cover [10, 13, 14], although surveys are
generally at a coarse taxonomic scale and there is little information on benthos/sea-ice associa-
tions for small or cryptic taxa. Given the high diversity and productivity of polar benthic com-
munities [5], better understanding of their relationship to sea-ice is critical for predicting
future polar biodiversity. Of the ways in which sea-ice affects benthic communities, the most obvious is reduced solar
irradiance [8, 15]. Decreased photosynthetically active radiation (PAR) inhibits photosynthesis
in algae [16] and diatoms [17], and protects organisms from potentially harmful ultraviolet
wavelengths [18, 19]. Light is pivotal in mediating competitive interactions between algae and
invertebrates, and in shallow waters can dictate overall community structure [20]. The amount
of light an area of seabed receives annually depends on the timing of sea-ice breakout relative
to the annual solar cycle, since sunlight is strongly seasonal at high latitudes [8]. Sea-ice deter-
mines both the type and quantity of primary productivity fuelling shallow benthic ecosystems. PLOS ONE | DOI:10.1371/journal.pone.0168391
January 11, 2017 The Roles of Sea-Ice, Light and Sedimentation in Structuring Shallow Antarctic Benthic Communities it reaches that location. For seabed of a given depth, susceptibility to ice-scour is therefore
related to the size distribution of ice-bergs in the local area. Evidence of links between sea-ice duration and shallow benthic community structure is grow-
ing, although patterns vary between study regions. A long-term study from the Arctic showed
that macroalgae increased in abundance as sea-ice duration shortened [10, 29]–a process also
described in Clark et al. (2013). Two study areas in the West Antarctic Peninsula have experi-
enced reduced sea-ice duration over the past decades, but different processes of ecological change
have been identified at each location. At Potter Cove (King George Island, 62˚ S) retraction of
sea-ice over 25-years led to macroalgal colonization [13], while at Rothera Station (Adelaide
Island, 68˚ S) reduced sea-ice increased the frequency of iceberg-scour and resulted in more dis-
turbed communities [5, 9]. In addition to variation in effects of sea-ice between locations, there
is also likely to be within-site variation that is not yet understood. Small-scale local factors, such
as habitat orientation and depth, might ameliorate or augment the effects of sea-ice on benthic
communities, and there is a need to understand how habitat features mediate sea-ice effects. Light, sedimentation, and disturbance to the benthos are likely to be mediated by habitat ori-
entation and depth. Both light and sedimentation are vertically oriented processes, so their
effects on benthic organisms should depend on the orientation of habitat relative to overhead
inputs [30, 31]. Effects of sea-ice might also change with depth due to light attenuation [32]. PAR decreases at an exponential rate through the water column [33], so effects of sea-ice on pri-
mary production and UV exposure should also decrease with depth. The frequency of ice-scour
is highest in the shallows and decreases with depth, since fewer icebergs are large enough to
reach deeper seabed [9, 28]. Knowledge of the interactions between sea-ice and habitat charac-
teristics in affecting benthos is important to improve predictions about how benthos may be
affected by changing climate. In this paper we present a rare multidisciplinary dataset, combin-
ing environmental and biological data collected from a seasonally ice-covered region of Antarc-
tic coast. Study area This study was conducted around Casey Station (66˚ 17’ S, 110˚ 32’ E), on the coast of Wilkes
Land, East Antarctica (Fig 1). The subtidal landscape consists of a mosaic of granitic gneiss
bedrock, boulder fields, and sediments [34]. Ambient sea temperature is approximately -1.8˚C
throughout the year. Access to these sites was permitted by the Australian Antarctic Division,
and the work did not involve endangered or protected species. We report a detailed survey of biota on subtidal boulders at 11 sites across a gradient of
sea-ice duration, and consider biological trends in the context of local light and sedimentation. Introduction Algae growing on the underside of sea-ice provide the benthos with small amounts of food
[21], supplemented by particles advected from ice-free areas [22]. When sea-ice breaks out,
however, pelagic phytoplankton bloom and dramatically increase total primary production
[23, 24]. Sea-ice also mediates physical disturbance to the benthos by influencing both sedimenta-
tion and ice-scour. Sea-ice forms a barrier between the water column and the atmosphere,
preventing wind-induced turbulence and water-column turnover to create relatively still,
depositional environments, where flow is generally limited to tidal currents [25, 26]. In such
conditions, particulate matter settles out of the water column and sediments accumulate on
the seafloor, potentially imposing sedimentation stress on benthic taxa. Sediment may be from
glacial or terrestrial sources, resuspended marine sediments, or organic matter produced in
the water column. A major source of organic matter is the summer phytoplankton bloom,
which typically occurs immediately after sea-ice breakout in response to the sudden increase
in light [23]. The role of sea-ice in phytoplankton deposition is two-fold: the timing of sea-ice
breakout determines when the phytoplankton bloom happens, and sea-ice cover then affects
the rate of deposition. Sea-ice attached to coast (‘fast-ice’) also prevents drifting ice-bergs from scouring the sea-
bed and inflicting catastrophic physical disturbance [27]. Studies in West Antarctica have
found that the frequency of ice-scour to be directly related to seasonal sea-ice duration [9], and
that this process strongly influences the composition and structure of benthic communities
[28]. Frequently scoured seabeds are in perpetual states of early colonisation [5], and late suc-
cessional communities only occur at depth or in areas protected by continuous ice cover. Depth of seabed can affect the rate of ice-scour, since the draught of an iceberg may be too
shallow to reach the seabed at a particular location, or too deep to avoid being grounded before 2 / 20 PLOS ONE | DOI:10.1371/journal.pone.0168391
January 11, 2017 Light meters Light meters were deployed on the seabed at eight sites around Casey Station (Fig 1, Table 1)
between 1998 and 2004. These were Dataflow™cosine corrected photosynthetic irradiance sen-
sors (400–700 nm) [35], with data logged to Dataflow™292 data loggers or Odyssey™integrated
data loggers. Light meters were mounted upright on stainless-steel tripods or star pickets, and
were positioned and secured on the seabed by divers at depths of 7 to 10 m. They were cali-
brated against three Li-Cor LI-190SA sensors (Lincoln, Nebraska, USA) for 1 w prior to
deployment, and set to record ambient light levels every 20 min. Due to the deterioration in
performance of light meters through time as a result of physical disturbance, shading or foul-
ing, only the first 12 months of light data are presented. Raw data from light meters was con-
verted into mol photons PAR m-2 yr-1. PLOS ONE | DOI:10.1371/journal.pone.0168391
January 11, 2017 3 / 20 The Roles of Sea-Ice, Light and Sedimentation in Structuring Shallow Antarctic Benthic Communities The Roles of Sea-Ice, Light and Sedimentation in Structuring Shallow Antarctic Benthic Communities Fig 1. Map of study region in the Windmill Islands, located on the coast of Wilkes Land, East Antarctica. Casey Station is marked with a star. Abbreviated site names correspond to those in Table 1. doi:10.1371/journal.pone.0168391.g001 Windmill Islands, located on the coast of Wilkes Land, East Antarctica. Casey Station is marked with a star. d to those in Table 1. Fig 1. Map of study region in the Windmill Islands, located on the coast of Wilkes Land, East Antarctica. Casey Station is marked with a star. Abbreviated site names correspond to those in Table 1. Fig 1. Map of study region in the Windmill Islands, located on the coast of Wilkes Land, East Antarctica. Casey Station is marked with a star. Abbreviated site names correspond to those in Table 1. doi:10.1371/journal.pone.0168391.g001 Fig 1. Map of study region in the Windmill Islands, located on the coast of Wilkes Land, East Antarctica. Casey Station is marked with a star. Abbreviated site names correspond to those in Table 1. doi:10.1371/journal.pone.0168391.g001 doi:10.1371/journal.pone.0168391.g001 Sedimentation Sediment flux was quantified by deploying sediment traps at six sites (Fig 1, Table 1 and S1
Table) for a range of intervals between December 2002 and December 2006. A total of 211
traps were deployed: 187 for periods of 9–89 days, 20 for periods of 260–375 days, and 4 for
periods of 640–724 days. At each site, traps were deployed in inner and outer zones, where PLOS ONE | DOI:10.1371/journal.pone.0168391
January 11, 2017 PLOS ONE | DOI:10.1371/journal.pone.0168391
January 11, 2017 4 / 20 Filters were then placed on individual
petri dishes, oven dried at 60˚C, cooled in a desiccator and their dry weights recorded. The Roles of Sea-Ice, Light and Sedimentation in Structuring Shallow Antarctic Benthic Communities Table 1. Summary of sea-ice conditions and types of data collected at each site (denoted by x). Site codes are used in Fig 1. Ice-rank is an ordinal
number given to sites where biota were sampled, based on light measurements and long-term visual observations (NA refers sites where biota were not sam-
pled). Lower numbers indicate later sea-ice breakout. Ice-zone is a classification is given to all sites, for categorical analyses and to describe unranked sites. Site
Code
Ice-rank
Ice-zone
Biota
Light
Sedimentation
Brown Bay
BB
NA
Closed
x
Sparkes Bay 1
SP1
NA
Closed
x
Sparkes Bay 2
SP2
NA
Closed
x
O’Brien Bay 1
OB1
1
Closed
x
x
x
O’Brien Bay 5
OB5
2
Closed
x
x
x
McGrady Cove
MC
3
Closed
x
x
x
O’Brien Bay 2
OB2
4
Intermediate
x
x
Newcomb Corner
NC
5
Intermediate
x
x
Shannon Bay
SB
6
Intermediate
x
x
O’Brien Bay 3
OB3
7
Intermediate
x
x
Honkala Island
HI
8
Intermediate
x
Beall Island
BI
9
Open
x
Powell Cove
PC
10
Open
x
Shirley Island
SI
11
Open
x
x Table 1. Summary of sea-ice conditions and types of data collected at each site (denoted by x). Site codes are used in Fig 1. Ice-rank is an ordinal
number given to sites where biota were sampled, based on light measurements and long-term visual observations (NA refers sites where biota were not sam-
pled). Lower numbers indicate later sea-ice breakout. Ice-zone is a classification is given to all sites, for categorical analyses and to describe unranked sites. inner zones where closer to the shore and generally experienced later annual sea-ice breakout. Prior to deployment, traps were scrubbed with water and detergent, rinsed with Milli-Q water
and placed in a 10% nitric acid bath for 24 hours, rinsed again with Milli-Q and sealed ready
for deployment. Traps had an internal diameter of 5.4 cm and an internal height of 13.7 cm. No preservatives were used to keep the settling particles. Collected sediment material was
transported to the laboratory at Casey Station and stored at 4˚C until processed (within 24
hours of collection). Sediment material was twice filtered through pre-weighed 0.8 micron
glass fiber filters and 0.45 micron cellulose acetate filters. PLOS ONE | DOI:10.1371/journal.pone.0168391
January 11, 2017 doi:10.1371/journal.pone.0168391.t001 Biological survey Points represent ALB recorded by light meters during the
first year of deployment, and dashed lines are estimated minimum annual light requirements of four species of
local algae (from Clark et al. 2013). Algae species are expected to be viable at sites where the ALB is above
their minimum requirement. Light measurements are not available for three sites marked with *, but from long-
term visual observation we know their annual timing of sea-ice departure and therefore approximate light
budgets. doi:10.1371/journal.pone.0168391.g002 preserved. Sponges and calcified bryozoans were preserved in 80% ethanol with Milli-Q water,
and other taxa were preserved in 7% formalin in seawater. Biological survey Divers collected boulders from 11 sites across a gradient of seasonal sea-ice cover (Fig 1, S2
Table). At each site divers collected eight boulders (mean surface area per boulder ± SE =
344 ± 19 cm2) from two depths, 6 and 12 m, 100–200 m apart. There was no significant
difference in the size of boulders collected from different sites or depths (2-factor ANOVA,
P > 0.05). Surveys were conducted across two austral summers (January–February 2006 and
October–December 2006; Table 1) but given the large temporal scales over which these com-
munities develop [36] we did not expect strong differences between summers. To test this we
sampled one site (O’Brien Bay 1) twice, once in each summer, and did not detect significant
differences in community structure (ANOSIM, R = 0.029, P = 0.32). Boulders were kept in aerated local seawater at -2˚C, and communities were censused live
under a dissecting microscope. Quadrats (2 x 2 cm) were randomly placed on a boulder, and
all taxa occurring within a quadrat were recorded. On each boulder we sampled six quadrats
on horizontal (up-facing) surfaces and six on vertical (side-facing) surfaces, giving a total of 48
cm2. Down-facing surfaces were not included because this surface was bare on boulders at silty
sites. The placement of quadrats was constrained to be > 1 cm from any edge and at least 2 cm
from another quadrat. Counts were made of serpulid and spirorbid polychaetes, and percent
cover was estimated for all other taxa. All cheilostomate bryozoans were identified to species. Other taxa were categorised into morphospecies. Voucher specimens were collected and PLOS ONE | DOI:10.1371/journal.pone.0168391
January 11, 2017 5 / 20 The Roles of Sea-Ice, Light and Sedimentation in Structuring Shallow Antarctic Benthic Communities Fig 2. Annual light budgets (ALB) at the seabed. Points represent ALB recorded by light meters during the
first year of deployment, and dashed lines are estimated minimum annual light requirements of four species of
local algae (from Clark et al. 2013). Algae species are expected to be viable at sites where the ALB is above
their minimum requirement. Light measurements are not available for three sites marked with *, but from long-
term visual observation we know their annual timing of sea-ice departure and therefore approximate light
budgets. doi:10 1371/journal pone 0168391 g002 Fig 2. Annual light budgets (ALB) at the seabed. PLOS ONE | DOI:10.1371/journal.pone.0168391
January 11, 2017 Light at the seabed Annual light budgets on the seabed ranged from 54 to 1248 mol photons m-2 yr-1 (Fig 2), and
were positively related to the duration of the annual ice-free period. Based on published mini-
mum annual light budgets for four species of local algae (Clark et al. 2013), algae should be
capable of surviving at eight of the eleven sites where we sampled biota (Fig 2). Light measure-
ments are not available for three sites, but from visual observations since 1997 we know their
annual timing of sea-ice departure, and therefore we assume that their light budgets were simi-
lar to those from Shirley Island. Sedimentation Sediment trap data show peaks in deposition around mid-summer (Fig 3A), which did not
appear to be related to site-specific sea-ice dynamics (Fig 3B). Sites where sediment flux was
measured are typically ice-covered until late summer, 1–2 months after peak sediment deposi-
tion was observed. However, sedimentation rates were higher in the inner area of bays, relative
to outer area (Table 2, Fig 3C), suggesting a spatial relationship between sediment flux and
sea-ice cover. For summer deployments, there was a positive relationship between the length
of trap deployment duration and average daily sediment flux (Table 2, Fig 3C). Supplementary
Table 1 shows minimum, mean, and maximum, sediment fluxes for each site and position in
bay. Statistical analyses Sites were ranked according to their sea-ice duration (‘Ice-rank’, Table 1), as determined by
annual light budgets (Fig 2) and visual observations by Riddle and Stark since 1997. For uni-
variate analysis, ice-rank was used as a continuous variable to represent the gradient in sea-ice
cover, because light measurements (and therefore date of sea-ice breakout) were not available
for three of the eleven sites. For multivariate analysis (PERMANOVA), sites were classified
into one of three ice-cover zones (‘Ice-zone’, Table 1), since this analysis requires categorical
predictors. Categories were open, intermediate, and closed, where closed sites are generally
ice-covered >11 mo yr-1, intermediate sites 7–11 mo yr-1, and open sites <7 mo yr-1. For boulder communities, multivariate community-level effects of sea-ice cover (open,
intermediate and closed), depth (6 and 12 m) and substrate orientation (horizontal and verti-
cal) were analysed with PERMANOVA [37] using PRIMER v.6 [38]. The design included the
above fixed factors, and two nested random effects of site (nested within sea-ice cover) and
boulder (nested within site). Data were fourth-root transformed to reduce the influence of
abundant taxa, and a resemblance matrix constructed using Bray-Curtis similarity. P-values
were obtained with 999 permutations. Effects of sea-ice cover (ice-rank), depth and substrate orientation on the abundance and
diversity of individual taxa on boulders were analysed with generalized linear mixed models
(GLMM) [39]. Spatial autocorrelation between surfaces within boulders, and boulders within PLOS ONE | DOI:10.1371/journal.pone.0168391
January 11, 2017 6 / 20 The Roles of Sea-Ice, Light and Sedimentation in Structuring Shallow Antarctic Benthic Communities sites, was modelled with random intercepts [40]. Percentage cover was modelled as a binomial
response, and species richness and density were modelled as Poisson responses. Sediment trap data collected in summer were analysed with a GLMM, testing the relation-
ship between deployment duration (number of days), position in a bay (inner or outer), and
sediment flux (g m-2 d-1). The response was modeled with a Gaussian distribution with log
link function, and Site (bay) was modeled as a random intercept to account for autocorrelation
within sites. Only data from traps deployed during summer were statistically analysed. All GLMMs were conducted using the ‘lme4’ package v.1.1–7 [41] in R v.3.1.1 (R Develop-
ment Team 2014). Parameters were estimated with Laplace approximations [42], and P-values
obtained with Z-tests. Plots of residuals were inspected for homoscedasticity. PLOS ONE | DOI:10.1371/journal.pone.0168391
January 11, 2017 Source
df
MS
Pseudo-F
P
Between Sites(Ice)
Ice
2
53630
5.8866
0.001
Depth
1
10699
3.4144
0.009
Orientation
1
25307
12.795
0.001
Depth x Orientation
1
679.18
0.58571
0.762
Ice x Depth
2
4410.6
1.4075
0.197
Ice x Orientation
2
5159.3
2.6085
0.015
Ice x Depth x Orientation
2
888.7
0.7664
0.673
Within Sites(Ice)
Site(Ice)
6
9110 5
10 71
0 001 Table 2. Estimates, standard errors (SE), t-values and P-values for fixed effects in GLMM testing the effects of deployment duration (number of
days) and position (inner or outer area of a bay) on daily sediment flux (g m-2 d-2). Random intercepts were allowed per site (bay). Estimates are in log
units. Significant differences (P < 0.05) are denoted in bold. Estimate
SE
t
P
(Intercept)
-0.460
0.009
-52.560
<0.001
Duration
0.031
0.003
11.380
<0.001
Position
-0.411
0.009
-47.680
<0.001
Duration: Position
-0.003
0.002
-1.400
0.163
doi:10.1371/journal.pone.0168391.t002 and P-values for fixed effects in GLMM testing the effects of deployment duration (number of
n daily sediment flux (g m-2 d-2). Random intercepts were allowed per site (bay). Estimates are in log
b ld Table 2. Estimates, standard errors (SE), t-values and P-values for fixed effects in GLMM testing the effects
days) and position (inner or outer area of a bay) on daily sediment flux (g m-2 d-2). Random intercepts were all
units. Significant differences (P < 0.05) are denoted in bold. encrusting algae in areas of shorter sea-ice duration. Community structure also varied with
depth (Table 3), but in the same direction as sea-ice relative to nMDS axes (Fig 4C). encrusting algae in areas of shorter sea-ice duration. Community structure also varied with
depth (Table 3), but in the same direction as sea-ice relative to nMDS axes (Fig 4C). There were very strong trends in the total cover of algae and invertebrates relative to sea-ice
duration, with the exception of two highly abundant invertebrates–Inversiula nutrix and spir-
orbid polychaetes. Algal cover was higher on horizontal surfaces within sites classified as hav-
ing shorter sea-ice duration (Table 4, Fig 5), where the incidence of light is greatest. Invertebrate cover (excluding I. nutrix and spirorbids) showed the opposite pattern, particu-
larly on vertical surfaces at 12 m depth (Table 4, Fig 5). There was less bare space in areas of
shorter sea-ice duration, with an apparent step-change to low bare space at sites in the open
zone (Table 4, Fig 5). The Roles of Sea-Ice, Light and Sedimentation in Structuring Shallow Antarctic Benthic Communities encrusting algae in areas of shorter sea-ice duration. Community structure also varied with
depth (Table 3), but in the same direction as sea-ice relative to nMDS axes (Fig 4C). There were very strong trends in the total cover of algae and invertebrates relative to sea-ice
duration, with the exception of two highly abundant invertebrates–Inversiula nutrix and spir-
orbid polychaetes. Algal cover was higher on horizontal surfaces within sites classified as hav-
ing shorter sea-ice duration (Table 4, Fig 5), where the incidence of light is greatest. Invertebrate cover (excluding I. nutrix and spirorbids) showed the opposite pattern, particu-
larly on vertical surfaces at 12 m depth (Table 4, Fig 5). There was less bare space in areas of
shorter sea-ice duration, with an apparent step-change to low bare space at sites in the open
zone (Table 4, Fig 5). Species richness was higher on vertical than horizontal substrates,
although there were no significant trends in relation to sea-ice duration (Table 4, Fig 5). Inver-
tebrate and algal richness did, however, increase and decrease with sea-ice duration, respec-
tively (Table 4, Fig 5). The bryozoan Inversiula nutrix was by far the most abundant taxon, and together with spir-
orbid polychaetes showed different trends than most other invertebrates. I. nutrix was abun-
dant at almost all sites (Fig 6), although the type of habitat it dominated varied with sea-ice
duration. In areas of extended sea-ice duration it dominated horizontal surfaces, but domi-
nance shifted to vertical surfaces in areas with shorter sea-ice duration (Table 4, Fig 6). Table 2. Estimates, standard errors (SE), t-values and P-values for fixed effects in GLMM testing the effects of deployment duration (number of
days) and position (inner or outer area of a bay) on daily sediment flux (g m-2 d-2). Random intercepts were allowed per site (bay). Estimates are in log
units. Significant differences (P < 0.05) are denoted in bold. Estimate
SE
t
P
(Intercept)
-0.460
0.009
-52.560
<0.001
Duration
0.031
0.003
11.380
<0.001
Position
-0.411
0.009
-47.680
<0.001
Duration: Position
-0.003
0.002
-1.400
0.163
doi:10.1371/journal.pone.0168391.t002
Table 3. Permutational multivariate analysis of variance (PERMANOVA) testing for differences in community structure with respect to ice cover,
depth and orientation. Significant differences (P < 0.05) are denoted in bold. Data are fourth-root transformed, and distances were based on the Bray-Curtis
similarity index. Boulder communities In total we recorded 42 taxa, including algae, polychaetes, bryozoans (mostly cheilostomate),
hydroids, sponges and ascidians (S3 Table). Multivariate analyses found that communities dif-
fered between ice-cover zones, but differences depended on substrate orientation (Table 3, Fig
4). Vertical substrates (lower-left quadrat of nMDS, Fig 4A) were characterized by a diversity
of bryozoa and sponges, while horizontal substrates (upper-right quadrat of nMDS, Fig 4A)
contained algae, sand tubes, and a different suite of bryozoa (Fig 4B). There was a strong gradi-
ent in community structure correlated with sea-ice duration (Fig 4C), but a surface plot shows
that change with sea-ice is not linear in relation to nMDS axes (Fig 4D). Change is relatively
linear in the lower-left quadrat of the nMDS, the region correlated with vertical substrates, but
diverges in the upper-right quadrat, the region correlated with horizontal substrates (Fig 4D). This represents a strong gradient in community structure relative to sea-ice on horizontal sur-
faces, compared to comparatively steady change with sea-ice on vertical surfaces. Horizontal
surfaces are correlated with sand tubes in areas of greater sea-ice duration, but brown PLOS ONE | DOI:10.1371/journal.pone.0168391
January 11, 2017 7 / 20 The Roles of Sea-Ice, Light and Sedimentation in Structuring Shallow Antarctic Benthic Communities Fig 3. Sedimentation flux through time. (A) Polygons are individual sediment traps, with height showing
average daily sediment flux and width showing deployment duration. Colour represents position in bay:
red = inner, green = outer. (B) As above, but data are partitioned into sites and position within bay. (C) Plots of
average daily sediment flux vs. deployment duration. Left-hand plot shows all traps, and right-hand plot shows
traps deployed during summer only. In right-hand plot, fitted lines are predicted values of GLMM for each
position in bay. doi:10.1371/journal.pone.0168391.g003 Fig 3. Sedimentation flux through time. (A) Polygons are individual sediment traps, with height showing
average daily sediment flux and width showing deployment duration. Colour represents position in bay:
red = inner, green = outer. (B) As above, but data are partitioned into sites and position within bay. (C) Plots of
average daily sediment flux vs. deployment duration. Left-hand plot shows all traps, and right-hand plot shows
traps deployed during summer only. In right-hand plot, fitted lines are predicted values of GLMM for each
position in bay. doi:10.1371/journal.pone.0168391.g003 8 / 20 PLOS ONE | DOI:10.1371/journal.pone.0168391
January 11, 2017 Species richness was higher on vertical than horizontal substrates,
although there were no significant trends in relation to sea-ice duration (Table 4, Fig 5). Inver-
tebrate and algal richness did, however, increase and decrease with sea-ice duration, respec-
tively (Table 4, Fig 5). The bryozoan Inversiula nutrix was by far the most abundant taxon, and together with spir-
orbid polychaetes showed different trends than most other invertebrates. I. nutrix was abun-
dant at almost all sites (Fig 6), although the type of habitat it dominated varied with sea-ice
duration. In areas of extended sea-ice duration it dominated horizontal surfaces, but domi-
nance shifted to vertical surfaces in areas with shorter sea-ice duration (Table 4, Fig 6). Table 3. Permutational multivariate analysis of variance (PERMANOVA) testing for differences in community structure with respect to ice cover,
depth and orientation. Significant differences (P < 0.05) are denoted in bold. Data are fourth-root transformed, and distances were based on the Bray-Curtis
similarity index. Source
df
MS
Pseudo-F
P
Between Sites(Ice)
Ice
2
53630
5.8866
0.001
Depth
1
10699
3.4144
0.009
Orientation
1
25307
12.795
0.001
Depth x Orientation
1
679.18
0.58571
0.762
Ice x Depth
2
4410.6
1.4075
0.197
Ice x Orientation
2
5159.3
2.6085
0.015
Ice x Depth x Orientation
2
888.7
0.7664
0.673
Within Sites(Ice)
Site(Ice)
6
9110.5
10.71
0.001
Site(Ice) x Depth
6
3133.6
3.6838
0.001
Site(Ice) x Orientation
6
1977.8
3.4492
0.001
Site(Ice) x Depth x Orientation
6
1159.6
2.0222
0.003
Boulder (blocking)
Boulder(Site(Ice) x Depth)
126
850.65
1.4835
0.001
Error
126
573.42
doi:10.1371/journal.pone.0168391.t003 s of variance (PERMANOVA) testing for differences in community structure with respect to ice cover,
s (P < 0.05) are denoted in bold. Data are fourth-root transformed, and distances were based on the Bray-Curtis Table 3. Permutational multivariate analysis of variance (PERMANOVA) testing for differences in community structure with respect to ice cover,
depth and orientation. Significant differences (P < 0.05) are denoted in bold. Data are fourth-root transformed, and distances were based on the Bray-Curtis
similarity index. ble 3. Permutational multivariate analysis of variance (PERMANOVA) testing for differences in community st
pth and orientation. Significant differences (P < 0.05) are denoted in bold. Data are fourth-root transformed, and dis
ilarit inde PLOS ONE | DOI:10.1371/journal.pone.0168391
January 11, 2017 9 / 20 The Roles of Sea-Ice, Light and Sedimentation in Structuring Shallow Antarctic Benthic Communities Fig 4. Multivariate patterns in boulder communities. (A) Multidimensional scaling plot (nMDS) based on Bray-Curtis similarity, showing boulder
communities as points. Shapes represent depths (circles = 6 m, triangles = 12 m), colours represent orientations (red = Vertical, green = Horizontal). Axes are scaled such that 1 unit equates to a doubling in dissimilarity between samples. (B) Correlations between species and nMDS axes. Where
species positions overlap, the more abundant species is displayed by name and other species appear as grey crosses. (C) Vector correlation between
continuous environmental covariate (sea-ice rank) and nMDS axes, and position of centroids of factor levels. (D) Contour plot of sea-ice rank, fitted by
GAM to nMDS axes. doi:10.1371/journal.pone.0168391.g004
The Roles of Sea Ice, Light and Sedimentation in Structuring Shallow Antarctic Benthic Communities Fig 4. Multivariate patterns in boulder communities. (A) Multidimensional scaling plot (nMDS) based on Bray-Curtis similarity, showing boulder The Roles of Sea-Ice, Light and Sedimentation in Structuring Shallow Antarctic Benthic Communities Table 4. Estimates, standard errors (SE), and P-values for fixed effects in GLMMs, testing the effects of sea-ice cover, depth, and substrate orientation on biota. Ice is a
semi-quantitative continuous variable, derived by ranking sites in decreasing order of seasonal sea-ice cover. Random intercepts were allowed per Boulder nested in Site, and base lev-
els for categorical fixed effects were Depth (6 m) and Surface (Horizontal). Algal cover
Invertebrate* cover
Bare space
Species richness δ
Algal richness δ
Invertebrate richness δ
Estimate
SE
P
Estimate
SE
P
Estimate
SE
P
Estimate
SE
P
Estimate
SE
P
Estimate
SE
P
(Intercept)
-7.368
0.738
<0.001
-1.494
0.393
<0.001
-0.716
0.320
0.025
2.025
0.140
<0.001
-2.066
0.484
<0.001
2.112
0.148
<0.001
Ice
0.587
0.104
<0.001
-0.248
0.059
<0.001
-0.069
0.047
0.145
-0.038
0.021
0.069
0.293
0.063
<0.001
-0.091
0.023
<0.001
Depth
2.403
0.488
<0.001
0.531
0.374
<0.001
0.375
0.143
0.009
0.112
0.118
0.343
1.653
0.397
<0.001
-0.015
0.126
0.905
Orientation
-0.552
0.235
0.019
-1.512
0.136
<0.001
0.752
0.063
<0.001
-0.607
0.139
<0.001
0.539
0.444
0.224
-0.616
0.150
<0.001
Ice: Depth
-0.224
0.066
0.001
-0.091
0.058
0.118
-0.040
0.022
0.067
-0.002
0.018
0.921
-0.148
0.048
0.002
-0.001
0.021
0.977
Ice: Orientation
0.163
0.026
<0.001
-0.131
0.035
<0.001
-0.082
0.010
<0.001
0.033
0.021
0.116
-0.047
0.053
0.373
0.003
0.025
0.910
Depth:
Orientation
-0.092
0.268
0.732
-0.803
0.200
<0.001
-0.048
0.085
0.572
-0.250
0.197
0.204
-0.956
0.541
0.077
-0.305
0.218
0.161
Ice: Depth:
Orientation
-0.062
0.030
0.042
0.252
0.044
<0.001
0.005
0.014
0.729
0.030
0.029
0.299
0.084
0.066
0.201
0.050
0.036
0.162
Inversiula nutrix
Spirorbidae δ
Sand tubes
Sponges
Encrusting algae
Arborescent algae
Estimate
SE
P
Estimate
SE
P
Estimate
SE
P
Estimate
SE
P
Estimate
SE
P
Estimate
SE
P
(Intercept)
-1.494
0.571
0.009
0.480
0.748
0.521
-2.900
0.001
<0.001
-4.052
0.589
<0.001
-11.993
1.320
<0.001
-7.171
0.997
<0.001
Ice
<0.001
0.084
0.999
0.023
0.110
0.831
-0.421
0.001
<0.001
-0.113
0.089
0.205
0.928
0.175
<0.001
0.381
0.142
0.007
Depth
-1.576
0.270
<0.001
-0.784
0.282
0.005
0.297
0.001
<0.001
1.017
0.613
0.097
6.282
0.971
<0.001
-1.608
0.796
0.043
Orientation
0.188
0.076
0.014
-1.167
0.225
<0.001
1.184
0.001
<0.001
-1.366
0.332
<0.001
-0.487
0.455
0.285
0.514
0.323
0.112
Ice: Depth
0.130
0.039
0.001
0.056
0.038
0.142
0.068
0.001
<0.001
-0.182
0.098
0.062
-0.624
0.121
<0.001
0.257
0.103
0.013
Ice: Orientation
-0.174
0.012
<0.001
0.054
0.027
0.045
0.005
0.001
<0.001
-0.079
0.057
0.166
0.176
0.047
<0.001
-0.020
0.040
0.623
Depth:
Orientation
-1.815
0.157
<0.001
-1.964
0.545
<0.001
0.103
0.001
<0.001
-0.315
0.440
0.475
0.104
0.475
0.827
-4.045
0.568
<0.001
Ice: Depth:
Orientation
0.263
0.020
<0.001
0.239
0.056
<0.001
-0.080
0.001
<0.001
0.159
0.076
0.036
-0.081
0.050
0.104
0.408
0.067
<0.001
Invertebrate* excludes Inversiula nutrix and Spirorbidae. Fig 4. Multivariate patterns in boulder communities. (A) Multidimensional scaling plot (nMDS) based on Bray-Curtis similarity, showing boulder
communities as points. Shapes represent depths (circles = 6 m, triangles = 12 m), colours represent orientations (red = Vertical, green = Horizontal). Axes are scaled such that 1 unit equates to a doubling in dissimilarity between samples. (B) Correlations between species and nMDS axes. Where
species positions overlap, the more abundant species is displayed by name and other species appear as grey crosses. (C) Vector correlation between
continuous environmental covariate (sea-ice rank) and nMDS axes, and position of centroids of factor levels. (D) Contour plot of sea-ice rank, fitted by
GAM to nMDS axes. doi:10.1371/journal.pone.0168391.g004 Spirorbid polychaetes (mostly Spirorbis nordenskjoldi) were the second most abundant inverte-
brate taxa, and were considerably more abundant at open sites than sites with more sea-ice
cover. Most sand tubes were usually inhabited by tanaids of the genus Nototanais, but some
housed other mobile invertebrates and terrebellid polychaetes. Sponge cover was highly vari-
able between sites, but showed an overall trend of greater cover with longer sea-ice duration. Encrusting algae were most abundant in the open zone, whereas arborescent algae peaked in
cover at sites with intermediate sea-ice duration (Table 4, Fig 6). Spirorbid polychaetes (mostly Spirorbis nordenskjoldi) were the second most abundant inverte-
brate taxa, and were considerably more abundant at open sites than sites with more sea-ice
cover. Most sand tubes were usually inhabited by tanaids of the genus Nototanais, but some
housed other mobile invertebrates and terrebellid polychaetes. Sponge cover was highly vari-
able between sites, but showed an overall trend of greater cover with longer sea-ice duration. Encrusting algae were most abundant in the open zone, whereas arborescent algae peaked in
cover at sites with intermediate sea-ice duration (Table 4, Fig 6). PLOS ONE | DOI:10.1371/journal.pone.0168391
January 11, 2017 PLOS ONE | DOI:10.1371/journal.pone.0168391
January 11, 2017 10 / 20 PLOS ONE | DOI:10.1371/journal.pone.0168391
January 11, 2017 Environmental gradients with sea-ice Sites that are ice-free for ~ 10% of the year have annual light budgets more than 20 times lower
than sites ice-free for ~ 50% of the year; a nonlinearity caused by the marked seasonality in
solar irradiance at high latitudes [8]. Ice-free days receive far more light in summer than in
winter, so the annual light budget is highly sensitive to the number of ice-free days around
midsummer, and relatively insensitive to the number of ice-free days closer to winter [8]. Most
variation in the timing of sea-ice breakout in the Windmill Islands occurs during summer
months, so sites represent a pronounced gradient in annual light despite only differing in their
date of sea-ice breakout by several weeks or months. Other factors that mediate effects of sea-
ice on subtidal light are ice thickness [43], snow cover, dust, and coastal topography, though
these are usually minor in comparison to sea-ice breakout. Sedimentation was also related to sea-ice cover, but in a different way to light. Sediment
flux was higher in the inner than outer areas of sites, and therefore spatially covaries with the
duration of sea-ice cover. However, the timing of peak deposition was decoupled from the tim-
ing of sea-ice breakout. Most deposition occurred in late December, around midsummer, yet
sites where sedimentation was measured typically retain their sea-ice until late January or Feb-
ruary. This implies that most sedimentation originates from the summer phytoplankton Sedimentation was also related to sea-ice cover, but in a different way to light. Sediment
flux was higher in the inner than outer areas of sites, and therefore spatially covaries with the
duration of sea-ice cover. However, the timing of peak deposition was decoupled from the tim-
ing of sea-ice breakout. Most deposition occurred in late December, around midsummer, yet
sites where sedimentation was measured typically retain their sea-ice until late January or Feb-
ruary. This implies that most sedimentation originates from the summer phytoplankton
bloom—an annual event in which suspended microalgae rapidly proliferate [44] then settle to
the seabed. The advection of phytoplankton to under-ice areas [22] then creates a region-wide
deposition event, whose timing is independent of site-specific sea-ice breakout. However, the
rate of phytoplankton settling from the water column is likely related to sea-ice breakout, since
turbulence and flow are much reduced under sea-ice [45]. Discussion This unique synthesis of light, sedimentation, and biological data provides new insight into
processes shaping nearshore Antarctic benthos. Data demonstrate that spatial variation in sea-
ice duration creates opposing gradients of light and sedimentation, which in turn shape the
structure and composition of shallow Antarctic benthic communities. Dominance shifted
from algae at sites with shorter sea-ice duration, more light and less sedimentation, to sessile
invertebrates at sites with longer sea-ice duration, less light and higher sedimentation. Impor-
tantly, however, these effects of sea-ice were mediated by habitat characteristics, such as sub-
strate orientation and depth. The relationship between sea-ice duration and benthos was
stronger on horizontal than vertical surfaces, which is explicable in that light and sedimenta-
tion are vertically oriented and affect horizontal more than vertical surfaces. The dependency
of sea-ice effects on substrate orientation supports the model that light and sedimentation are
two major pathways by which sea-ice duration shapes shallow coastal benthic communities. Shallow, horizontal habitats are most tightly linked with sea-ice dynamics, and their biota may
be useful sentinels of climate change in nearshore polar ecosystems. δ denotes Poisson GLMM and Estimates in log units; all other variables used binomial GLMM and Estimates are in logit units. Significant differences (P < 0.05) are denoted in bold. 11 / 20 The Roles of Sea-Ice, Light and Sedimentation in Structuring Shallow Antarctic Benthic Communities Fig 5. Percentage cover of algal, invertebrates, and bare space, and measures of diversity. L
plots show data from all sites, depths and orientations. Bars show the mean value, and error bars sh
standard error. Right-hand plots show predicted values of significant effects detected by GLMM. doi:10.1371/journal.pone.0168391.g005 Fig 5. Percentage cover of algal, invertebrates, and bare space, and measures of diversity. Left-hand
plots show data from all sites, depths and orientations. Bars show the mean value, and error bars show
standard error. Right-hand plots show predicted values of significant effects detected by GLMM. doi:10.1371/journal.pone.0168391.g005 Fig 5. Percentage cover of algal, invertebrates, and bare space, and measures of diversity. Left-hand
plots show data from all sites, depths and orientations. Bars show the mean value, and error bars show
standard error. Right-hand plots show predicted values of significant effects detected by GLMM. doi:10.1371/journal.pone.0168391.g005 12 / 20 PLOS ONE | DOI:10.1371/journal.pone.0168391
January 11, 2017 The Roles of Sea-Ice, Light and Sedimentation in Structuring Shallow Antarctic Benthic Communities Fig 6. Percentage cover or density of dominant taxa and groups. Left-hand plots show data fro
depths and orientations. Bars show the mean value, and error bars show standard error. Right-hand
show predicted values of significant effects detected by GLMM. doi:10.1371/journal.pone.0168391.g006 Fig 6. Percentage cover or density of dominant taxa and groups. Left-hand plots show data from all sites,
depths and orientations. Bars show the mean value, and error bars show standard error. Right-hand plots
show predicted values of significant effects detected by GLMM. d i 10 1371/j
l
0168391 006 Fig 6. Percentage cover or density of dominant taxa and groups. Left-hand plots show data from all sites,
depths and orientations. Bars show the mean value, and error bars show standard error. Right-hand plots
show predicted values of significant effects detected by GLMM. doi:10.1371/journal.pone.0168391.g006 doi:10.1371/journal.pone.0168391.g006 13 / 20 PLOS ONE | DOI:10.1371/journal.pone.0168391
January 11, 2017 The Roles of Sea-Ice, Light and Sedimentation in Structuring Shallow Antarctic Benthic Communities PLOS ONE | DOI:10.1371/journal.pone.0168391
January 11, 2017 Environmental gradients with sea-ice Therefore, we suggest that sus-
pended sediment load in the water column does not vary with sea-ice duration, but that depo-
sition and retention do, and are positively related to sea-ice duration. The positive correlation
between the sediment trap deployment period and average daily flux also implies that deposi-
tion occurs in pulse events not captured by some of the shorter deployments, which is consis-
tent with a bloom event. Some sediment may also arrive via terrestrial runoff during the
summer melt, although this is less likely when sites are covered by sea-ice, as they were when
deposition peaked in our sampling. Comparison of Windmill Island sediment fluxes with other published Antarctic near-
coastal studies reveal that sediment fluxes in the region are very low. The mean summer flux
for the Windmill Islands varies between 0.55 and 7.61 g m-2 day-1 in comparison with 43, 23.6 PLOS ONE | DOI:10.1371/journal.pone.0168391
January 11, 2017 14 / 20 The Roles of Sea-Ice, Light and Sedimentation in Structuring Shallow Antarctic Benthic Communities and 10 g m-2 day-1 at Johnson’s Dock, Livingston Island [46]; Marian Cove [47], King George
Island and Potter Cove, King George Island [48], respectively with higher sediment fluxes
reported at Potter Cove after 1992. One explanation is that these published studies are located
on islands and in the region of the South Shetlands near the Antarctic Peninsula. The Antarctic
Peninsula has a much milder and more maritime climate than continental Antarctica, whereas
our study locations were adjacent to continental Antarctica and may therefore be influenced
by different processes. Research programs established in different regions of Antarctica have focused on different
processes by which sea-ice affects benthos. Studies in Potter Cove (West Antarctic Peninsula)
found that glacial melting allowed macroalgae to colonize newly ice-free areas, but also report
reduced light penetration due to increased terrigenous sediment input from land and more
frequent ice scour [13]. Other research programs on the West Antarctic Peninsula focus on
the change in the frequency of iceberg scour with sea-ice cover, and consider this the driving
force affecting benthic communities [5, 9, 49–51]. Environmental gradients with sea-ice The structure of benthos in our study area
suggests that ice-scour in the Windmill Islands is not as frequent as at Antarctic Peninsula
study locations, possibly due to more glaciation and icebergs and a more maritime climate on
the Peninsula (more exposure to waves, currents, turbulence) against an environment which
has more stable ice, with cooler temperatures and the prevalence of offshore winds as in many
areas of continental Antarctica. Given the slow recruitment and growth of Antarctic benthic
fauna [36] our study sites did not appear to have been recently disturbed, and bare space (an
indicator of disturbance history) was lowest at sites in the open zone, where exposure to ice-
scour should be greatest. It appears that light and sedimentation are the dominant physical
processes by which sea-ice affects benthos in the Windmill islands, but in the region of the
Antarctic Peninsula these may be secondary to iceberg disturbance. Biological gradients with sea-ice There was a strong biological gradient transitioning from invertebrate to algal dominance in
the direction of decreasing sea-ice duration, but the nature of change depended on substrate
orientation and depth. Horizontal surfaces were dominated by algae in areas of shorter sea-ice
duration, and sand tubes and the bryozoan Inversiula nutrix in areas of longer sea-ice duration. Vertical surfaces were dominated by invertebrates regardless of sea-ice, but the identity of
invertebrates changed along the sea-ice gradient. Communities on horizontal substrate
showed stronger correlations to sea-ice duration than those on vertical substrate, consistent
with expected response to greater exposure to light and sedimentation. In temperate and tropi-
cal systems, non-coral invertebrates tend to occupy shaded, vertical, or down-facing habitats
[20, 31, 52], since they can be negatively affected by ultraviolet light [18] or outcompeted by
algae in well-lit areas [53, 54]. Effects of sedimentation also vary with orientation [55], as sedi-
ment is more likely to settle and accumulate on horizontal than vertical substrates. y
The relative effects of light and sedimentation can be inferred from changes in biota in rela-
tion to orientation, in areas of longer vs. shorter sea-ice duration. Assuming these physical fac-
tors affect horizontal more than vertical substrates, differences in biota between orientations at
open sites (high light, low sedimentation) most likely reflect changes in light, while differences
at closed sites (low light, high sedimentation) are more likely related to sedimentation. With
this rationale, light in our study area drives a gradient between algae and invertebrates,
whereas sedimentation facilitates fauna with sand tubes, and differentiates taxa based on mor-
phology or other mechanisms of sediment tolerance. Light and sedimentation may also have
interactive effects since negative consequences of reduced light on algae can be exacerbated The relative effects of light and sedimentation can be inferred from changes in biota in rela-
tion to orientation, in areas of longer vs. shorter sea-ice duration. Assuming these physical fac-
tors affect horizontal more than vertical substrates, differences in biota between orientations at
open sites (high light, low sedimentation) most likely reflect changes in light, while differences
at closed sites (low light, high sedimentation) are more likely related to sedimentation. With
this rationale, light in our study area drives a gradient between algae and invertebrates, whereas sedimentation facilitates fauna with sand tubes, and differentiates taxa based on mor-
phology or other mechanisms of sediment tolerance. PLOS ONE | DOI:10.1371/journal.pone.0168391
January 11, 2017 The Roles of Sea-Ice, Light and Sedimentation in Structuring Shallow Antarctic Benthic Communities under heavy sedimentation [56], and such an interaction may have contributed towards the
sharp decline in algae with increased sea-ice duration. Habitat use by the most abundant species, Inversiula nutrix, appears related to both light
and sedimentation. It is the only sessile invertebrate taxa able to reach substantial cover on
horizontal substrate at closed sites, implying a degree of tolerance to sedimentation. In con-
trast, at open sites it dominates vertical substrate, and is rare on horizontal substrate. This
switch in habitat use from horizontal to vertical substrate as sea-ice duration shortens is
likely driven by tolerance to sedimentation giving way to out-competition by algae. It
may also be more tolerant of UV light than other invertebrates, since few of the invertebrate
taxa found in the closed ice-cover zone were present in the open ice-cover zone. Spirorbid
polychaetes (mostly Spirorbis nordenskjoldi) were the other invertebrate taxa to show con-
trasting distributions to the majority of other invertebrates, being most abundant in the
open zone. Interestingly, both of these taxa were by far the most abundant in the study
region, and their ecological success may be related to an ability to exploit niches unavailable
to other invertebrates. Overall trends in algal cover are clearly driven by light, but sedimentation may contribute
to more subtle differences in the distribution of algal functional groups. Both encrusting and
arborescent algae were negatively correlated with sea-ice duration, though cover of arborescent
algae peaked in areas with some sea-ice cover. This could reflect greater tolerance to sedimen-
tation in arborescent vs. encrusting morphologies, since arborescent foliage are raised off the
substratum and remain clear of sediment accumulation [56]. Arborescent algae might experi-
ence reduced competition with encrusting algae by occupying areas of higher sedimentation. Similar patterns have been found in temperate systems, where articulated coralline algae are
advantaged relative to encrusting coralline under higher rates of sedimentation [57]. Alterna-
tively, encrusting algae may be more tolerant of the canopy-forming macroalgae that occur in
areas of shorter sea-ice duration. Macroalgae impose a range of effects on the understorey
environment, including changes in light [57], flow [58], sedimentation [56], and physical abra-
sion by canopy foliage [59]. Physical abrasion favours encrusting over arborescent forms in
understorey communities in temperate Australia [59], and similar processes may be happen-
ing in Antarctica. PLOS ONE | DOI:10.1371/journal.pone.0168391
January 11, 2017 Biological gradients with sea-ice Light and sedimentation may also have
interactive effects since negative consequences of reduced light on algae can be exacerbated PLOS ONE | DOI:10.1371/journal.pone.0168391
January 11, 2017 15 / 20 Acknowledgments We are grateful to the dive teams of Casey Station 2002–2007 for their assistance with field-
work. The authors declare no conflict of interest, financial or otherwise. Research was funded
by Australian Antarctic Science Grants 2201 and 2691. Many AAD personnel assisted with
logistics, field and laboratory work. There was an intriguing peak in bare space in the intermediate ice-cover zone, which may
reflect disturbance conditions or poor habitat quality. One hypothesis to explain the pattern is
that iceberg scour in the intermediate ice-cover zone causes regular invertebrates mortality,
but in contrast the open ice-cover zone, light is insufficient to support algal growth. Such con-
ditions would create habitat unfavourable to both invertebrates and algae. The spatial mosaic
of algae in the open zone suggests that iceberg scour is occurring and has ecological conse-
quences [60], but rates of ice-scour have not been quantified in this region like has been done
in other parts of Antarctica [29]. Quantifying ice-sour, or conducting a reciprocal transplants
of boulder communities between sea-ice cover zones, would aid in understanding the mecha-
nism driving this pattern. In summary, we found strong links between sea-ice duration and benthic community struc-
ture, and the nature of change was consistent with variation in light and sedimentation. Both
light and sedimentation vary with sea-ice duration, and their effects on biota are mediated by
the depth and orientation of habitat. Interactions between overhead sea-ice dynamics and ben-
thic habitats undoubtedly contribute to the diversity of shallow Antarctic benthic communi-
ties, and understanding these interactions is critical to forecasting climate-related change. If
climate change lengthens the period for which some areas are ice-free per year, species distri-
butions are likely to shift accordingly. Taxa currently restricted to areas of prolonged ice cover
are at risk of local extinction, which is ecologically significant because these areas contained a PLOS ONE | DOI:10.1371/journal.pone.0168391
January 11, 2017 16 / 20 The Roles of Sea-Ice, Light and Sedimentation in Structuring Shallow Antarctic Benthic Communities suite of invertebrates not found in areas of shorter sea-ice duration. Such change in benthic
communities in response to sea-ice duration has recently been observed in both the Arctic
[10] and Antarctic [13, 61], and are predicted to become more frequent with continuing
change in sea-ice dynamics [8]. Moreover, since much of the Antarctic coast lies in a relatively
narrow latitudinal band, large areas of shallow habitat may be vulnerable within an ecolog-
ically-brief period of time [62]. Communities on horizontal habitats are likely to be useful indi-
cators of climate change in shallow polar habitats, since these bear the strongest correlations
with sea-ice duration. Supporting Information S1 Table. Sampling details of sediment traps, and minimum, mean and maximum sedi-
ment flux for each site during the study period. The number of deployments represents the
number of sediment traps deployed at one site, with 2–5 sediment traps deployed at any one
time. Traps were deployed for various time intervals between 08/12/2002 and 30/12/2006. (DOCX) S2 Table. Details of sampling sites and times for the surveys of biota. At each site we col-
lected 16 boulders, 8 from 6 m depth and 8 from 12 m. (DOCX) S2 Table. Details of sampling sites and times for the surveys of biota. At each site we col-
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https://openalex.org/W2066052052
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Síndromes hipertensivas da gestação e repercussões perinatais
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Revista Brasileira de Saúde Materno Infantil
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cc-by
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Resumo Objectives: to assess perinatal effects of preg-
nancy hypertensive syndromes. Objetivos: avaliar repercussões perinatais nas
síndromes hipertensivas em gestações. Methods: hospital-based observational and
retrospective study of a tertiary Brazilian center, from
January, 1996 to October, 2003. Twelve thousand and
two hundred and seventy two (12.272) pregnancies
met the inclusion criteria. Two types of hypertension
were considered: gestational hypertension (GH) and
chronic hypertension (CH). Results of the study
demonstrated that newborns of mothers with gesta-
tional hypertension and chronic hypertension were
small for gestational age (SGA), had low Apgar score
in the 1º and 5º minutes, neonatal infection, meco-
nium aspiration syndrome (MAS), prematurity and
respiratory distress syndrome (RDS). Métodos: estudo observacional e retrospectivo, real-
izado em hospital terciário, entre janeiro de 1996 e
outubro de 2003. Um total de 12.272 gestações
preencheu critérios de inclusão. Dois tipos de
hipertensão foram considerados: hipertensão gesta-
cional (HG) e hipertensão arterial crônica (HAC). Variáveis estudadas: fetos pequenos para idade gesta-
cional (PIG), Apgar baixo no 1º e 5º minutos, infecção
neonatal, síndrome de aspiração meconial (SAM),
prematuridade, síndrome de angústia respiratória (SAR). Resultados: 1259 (10,26%) gestantes tinham
hipertensão; 344 (2,80%) foram classificadas como HG,
915 (7,45%) como HAC, havendo 11.013 (89,74%)
gestantes normotensas. HG constituiu risco elevado
para: PIG, Apgar baixo no 1º e 5º minutos, infecção
neonatal e prematuridade, mas não para SAM e SAR. HAC constituiu risco elevado para: PIG, Apgar baixo no
1º minuto, SAM, prematuridade e SAR, mas não para
Apgar baixo no 5º minuto e infecção neonatal. Quando
comparamos os riscos relativos dos grupos de HAC e
HG, houve maior risco de prematuridade no grupo de
HAC Métodos: estudo observacional e retrospectivo, real-
izado em hospital terciário, entre janeiro de 1996 e
outubro de 2003. Um total de 12.272 gestações
preencheu critérios de inclusão. Dois tipos de
hipertensão foram considerados: hipertensão gesta-
cional (HG) e hipertensão arterial crônica (HAC). Variáveis estudadas: fetos pequenos para idade gesta-
cional (PIG), Apgar baixo no 1º e 5º minutos, infecção
neonatal, síndrome de aspiração meconial (SAM),
prematuridade, síndrome de angústia respiratória (SAR). Resultados: 1259 (10,26%) gestantes tinham
hipertensão; 344 (2,80%) foram classificadas como HG,
915 (7,45%) como HAC, havendo 11.013 (89,74%)
gestantes normotensas. HG constituiu risco elevado
para: PIG, Apgar baixo no 1º e 5º minutos, infecção
neonatal e prematuridade, mas não para SAM e SAR. Síndromes hipertensivas da gestação e
repercussões perinatais Cristiane Alves de Oliveira 1
Carla Pereira Lins 2
Renato Augusto Moreira de Sá 3
Hermógenes Chaves Netto 4
Rita Guerios Bornia 5
Nancy Ribeiro da Silva 6
Joffre Amim Junior 7 Hypertensive syndromes during pregnancy
and perinatal outcomes 1,2,6 Setor de Pesquisa e Extensão. Maternidade Escola. Universidade Federal do Rio de Janeiro. Rua das Laranjeiras, 180. Laranjeiras. Rio de Janeiro, RJ. Brasil. CEP: 22.240-000 E-mail: crisalves.oliveira@openlink.com.br
3 Setor de Obstetrícia. Universidade Federal Fluminense. Niterói,
RJ, Brasil
4,5,7 Setor de Obstetrícia. Universidade Federal do Rio de Janeiro,
RJ, Brasil 1,2,6 Setor de Pesquisa e Extensão. Maternidade Escola.
Universidade Federal do Rio de Janeiro.
Rua das Laranjeiras, 180. Laranjeiras. Rio de Janeiro, RJ. Brasil.
CEP: 22.240-000 E-mail: crisalves.oliveira@openlink.com.br
3 Setor de Obstetrícia. Universidade Federal Fluminense. Niterói,
RJ, Brasil
4,5,7 Setor de Obstetrícia. Universidade Federal do Rio de Janeiro,
RJ, Brasil ARTIGOS ORIGINAIS / ORIGINAL ARTICLES ARTIGOS ORIGINAIS / ORIGINAL ARTICLES Introdução perinatais similares às de pacientes normotensas.1,4,5 A hipertensão gestacional/pré-eclâmpsia grave é
potencialmente a que apresenta pior prognóstico
materno-fetal. Conceptos de mães com PE ou PE
sobreposta têm maiores riscos de prematuridade,
ocorrência de partos de fetos pequenos para a idade
gestacional (PIG), necessidade de Unidade de Trata-
mento Intensivo (UTI) neonatal, necessidade de
suporte ventilatório e maior incidência de mortali-
dade perinatal, quando comparados aos conceptos de
mães normotensas.4 Ray et al.,5 estudaram os resul-
tados perinatais nas diferentes síndromes hiperten-
sivas, observando maiores riscos de prematuridade e
baixo peso ao nascimento nas pacientes com PE e PE
sobreposta quando comparadas à hipertensão gesta-
cional. A ocorrência de doenças associadas à prema-
turidade (SAR, hemorragia intraventricular e entero-
colite necrotizante), necessidade de ventilação assis-
tida, admissão em UTI neonatal e taxa de mortali-
dade perinatal foram maiores nas pacientes com PE
e com PE sobreposta quando comparadas às pacie-
ntes com hipertensão gestacional e HAC isolada. A hipertensão é a complicação clínica mais comum
da gestação, ocorrendo em 10 a 22% das gesta-
ções.1-3 O termo hipertensão induzida pela gestação
refere-se ao aumento da pressão arterial que se mani-
festa apenas na gravidez.4 As síndromes hipertensivas que acometem a
mulher grávida são habitualmente classificadas em
"hipertensão gestacional" (pressão arterial >140 x 90
mmHg diagnosticada pela primeira vez na gestação,
ausência de proteinúria, retorno aos níveis tencionais
até 12 semanas após o parto), "pré-eclâmpsia"
(pressão arterial >140 x 90 mmHg diagnosticada
após 20 semanas de gestação associada à proteinúria
>300 mg/24 horas), "hipertensão arterial crônica"
(pressão arterial >140 x 90 mmHg diagnosticada
antes da gestação ou antes de 20 semanas de
gestação não-atribuída à doença trofoblástica gesta-
cional ou pressão arterial >140 x 90 mmHg diagnos-
ticada após 20 semanas de gestação que persiste após
12 semanas de pós-parto), eclâmpsia (presença de
convulsão, que não pode ser atribuída a outras
causas, em mulheres com pré-eclâmpsia) e "pré-
eclâmpsia sobreposta" (surgimento de proteinúria
>300 mg/24 horas em paciente hipertensa que não
apresentava proteinúria antes de 20 semanas de
gestação ou aumento importante da proteinúria, da
pressão arterial ou plaquetas <100.000/mm3 em
gestante hipertensa com proteinúria presente antes
de 20 semanas de gestação).4 A piora do prognóstico materno-fetal está direta-
mente relacionada à gravidade da hipertensão gesta-
cional/pré-eclâmpsia. Resumo HAC constituiu risco elevado para: PIG, Apgar baixo no
1º minuto, SAM, prematuridade e SAR, mas não para
Apgar baixo no 5º minuto e infecção neonatal. Quando
comparamos os riscos relativos dos grupos de HAC e
HG, houve maior risco de prematuridade no grupo de
HAC. Results: 1259 (10.26%) had hypertension, 344
(2.80%) were classified as GH, 915 (7.45%) as CH
and 11.013 (89.74%) had no hypertension. GH was
risk factor for SGA, low Apgar score in the first and
fifth minutes, neonatal infection and prematurity, but
not for MAS and RDS. CH was a risk factor for SGA,
low Apgar score in the first minute, MAS, prematurity
and RDS, but not for low Apgar score in the fifth
minute and neonatal infection. When comparing the
relative risk between CH and GH an increased risk
for prematurity in CH was determined. Conclusões: dados sugerem que tanto HAC quanto
HG aumentaram risco para PIG, Apgar baixo no 1º e 5º
minutos, infecção neonatal, SAM, prematuridade e SAR. HAC apresentou maior risco relativo para prematuri-
dade. Conclusions: our data suggest that CH and GH
increased the risks for the outcomes studied. An
increased risk for prematurity in CH was noted. Key words Perinatal care, Hypertension, Pre-
eclâmpsia, Pregnancy, high-risk Conclusions: our data suggest that CH and GH
increased the risks for the outcomes studied. An
increased risk for prematurity in CH was noted. K
d
P
i
l
H
i
P Palavras-chave Assistência perinatal; Hiperten-
são, Pré-eclâmpsia, Gravidez de alto-risco Key words Perinatal care, Hypertension, Pre-
eclâmpsia, Pregnancy, high-risk Key words Perinatal care, Hypertension, Pre-
eclâmpsia, Pregnancy, high-risk 93 Rev. Bras. Saúde Matern. Infant., Recife, 6 (1): 93-98, jan. / mar., 2006 Oliveira CA et al. Rev. Bras. Saúde Matern. Infant., Recife, 6 (1): 93-98, jan. / mar., 2006 HAC isolada.12 Foram realizadas as análises entre o Grupo 1
(HG) e o Grupo 3 (grupo controle), entre os Grupo 2
(HAC) e Grupo 3 (grupo controle), e finalmente
foram comparados os riscos relativos obtidos para os
Grupo 1 (HG) e o Grupo 2 (HAC). Com o objetivo de estudar o impacto das
síndromes hipertensivas nos resultados perinatais,
foram avaliadas retrospectivamente gestantes que
tiveram
parto
na
Maternidade
Escola
da
Universidade Federal do Rio de Janeiro. As variáveis de estudo foram: a) parto de fetos
classificados como pequenos para a idade gesta-
cional (PIG); b) baixo índice de Apgar no 1º e no 5º
minutos após o nascimento; c) infecção neonatal; d)
síndrome de aspiração de mecônio (SAM); e) prema-
turidade; e f) síndrome de angústia respiratória
(SAR). Métodos Estudo do tipo observacional e retrospectivo, onde
foram avaliadas retrospectivamente todas as
pacientes que tiveram parto no período entre 1º de
janeiro de 1996 e 31 de outubro de 2003 na
Maternidade Escola da Universidade Federal do Rio
de Janeiro (UFRJ), Rio de Janeiro, Brasil. Considerou-se como "PIG": peso ao nascimento
abaixo do 10º percentil para a idade gestacional;
"índices de Apgar baixos": quando menores que 7;
"infecção neonatal": presença de clínica e exames
laboratoriais sugestivos de quadro infeccioso no
neonato; "SAM": visualização de mecônio na
traquéia do concepto associada à clínica de descon-
forto respiratório; "prematuridade": nascimento
antes de 37 semanas de gestação; e "SAR": necessi-
dade de ventilação assistida e/ou suplementação de
oxigênio nas primeiras 24 horas após o parto. As informações das gestações foram obtidas
através de pesquisa na Base de Dados da Mater-
nidade o Sistema Informático Perinatal do Centro
Latino Americano de Perinatologia (SIP/CLAP). Os
dados analisados foram registrados em formulário
próprio do Sistema por médicos da instituição na
ocasião da alta hospitalar de mãe e neonato após o
parto. As informações registradas foram prove-
nientes da revisão da história clínica e exames rea-
lizados pelas pacientes durante o pré-natal e inter-
nação hospitalar (anotados no prontuário). Calculou-se o risco relativo (RR) e seus limites
no intervalo de confiança a 95%. Para avaliar a asso-
ciação entre as variáveis estudadas utilizou-se o teste
do Qui-quadrado, considerado como significativo
quando p <0,05. O critério de inclusão foi parto de concepto com
idade gestacional superior a 20 semanas e gestação
única. Introdução / mar., 2006 Hipertensão na gestação e repercussões perinatais Hipertensão na gestação e repercussões perinatais Hipertensão na gestação e repercussões perinatais Resultados Definiu-se como hipertensão arterial a presença
de pressão arterial maior ou igual a 140 x 90 mmHg
em pelo menos duas ocasiões, com intervalo de
quatro horas ou mais. A pressão arterial da gestante
foi aferida após repouso mínimo de 10 minutos, com
a gestante sentada e antebraço direito posicionado
horizontalmente ao nível do coração, utilizando-se
esfignomanômetro de coluna de mercúrio. Foram
utilizados o primeiro e o quinto ruídos de Korotkoff
como limites superior e inferior da pressão arterial. O American College of Obstetricians and Gynecolo-
gists (ACOG) considera a fase V como tendo maior
precisão na avaliação da pressão diastólica. As afe-
rições da pressão arterial foram feitas durante as
consultas de pré-natal e na internação hospitalar. Foram selecionadas para o estudo 12.272 gestantes,
das quais 1259 (10,26%) apresentaram pressão arte-
rial igual ou superior a 140/90 mmHg. No Grupo 1
(HG) e no Grupo 2 (HAC) foram incluídas 344 Introdução Hauth et al.,10 compararam os
resultados perinatais em gestantes normotensas com
gestantes que desenvolveram hipertensão na gesta-
ção e observaram piores resultados no grupo de
hipertensas, principalmente nos casos graves. As
variáveis estudadas foram: parto com idade gesta-
cional inferior a 34 semanas de gestação, cresci-
mento intra-uterino restrito (CIUR), admissão em
UTI neonatal, peso ao nascimento inferior a 2500g,
síndrome de angústia respiratória (SAR) e necessi-
dade de ventilação assistida. As síndromes hipertensivas na gestação diferem
quanto à prevalência, gravidade e efeitos sobre o
feto.5 Em pacientes com HAC, a presença de PE sobre-
posta é a maior responsável pela piora da morbi-
mortalidade perinatal, havendo também maiores
taxas de resultados perinatais adversos em pacientes
com HAC grave e não-controlada.4,10,11 Rey et al.,11
compararam os resultados perinatais de pacientes
com HAC com os resultados de pacientes normoten-
sas, observando maiores taxas de mortalidade peri-
natal (45/1000 x 12/1000, p <0,001), parto prema-
turo (34,4% x 15,0%, p <0,01) e feto pequeno para a
idade gestacional (15,5% x 6,3%, p <0,01) nas pa
cientes hipertensas crônicas. Também foram mais
freqüentes no grupo com HAC o baixo peso ao
nascimento e baixos índices de Apgar no 7° e no 10°
minutos.11 A prevalência de hipertensão gestacional em
pacientes nulíparas é 6 a 17%, sendo 2 a 4% em
pacientes multíparas.1 Em 20-50% das pacientes
com hipertensão gestacional há progressão para PE,
ou seja, há desenvolvimento de proteinúria, podendo
a mesma se desenvolver antes ou após o parto.6 A prevalência de pré-eclâmpsia (PE) é normal-
mente descrita como 5 a 8%, apresentando amplas
variações na literatura. Em gestação gemelar a
prevalência de PE é de 14%, podendo chegar a 40%
em pacientes com PE prévia.3,4,7 A hipertensão arterial crônica (HAC) acomete
em torno de 5% das gestações, sendo considerada
como fator de risco para a PE. A taxa de PE sobre-
posta em pacientes hipertensas crônicas é 15 a
25%.8,9 Sibai et al.,12 observaram maior risco de prema-
turidade (56% x 25%, p <0,001) e maior mortalidade
perinatal (8% x 4%, p=0,02) nas pacientes com PE
sobreposta quando comparadas às pacientes com A maioria dos casos de hipertensão gestacio-
nal/pré-eclâmpsia leve se desenvolve próximo ao
termo e apresenta taxas de mortalidade e morbidade 94 Rev. Bras. Saúde Matern. Infant., Recife, 6 (1): 93-98, jan. Tabela 1 Tabela 1
Comparação entre Grupo 1 (hipertensão gestacional) e Grupo 3 (grupo con-
trole) quanto ao risco de desenvolvimento de complicações perinatais. RR
IC95%
c2
p*
PIG
2,10
1,77
2,48
73,948
<0,001
Apgar 1° minuto
1,26
1,05
1,52
6,198
0,012
Apgar 5° minuto
1,45
1,00
2,09
3,986
0,045
Infecção Neonatal
3,12
1,74
5,59
16,111
<0,001
SAM
1,49
0,90
2,46
2,498
0,113
Prematuridade
1,79
1,55
2,07
59,850
<0,001
SAR
1,39
0,89
2,18
2,154
0,140
RR=Risco relativo; IC95%=Intervalo de Confiança 95%; χ2=teste do Qui-
quadrado; p = p valor; PIG=Pequeno para a idade gestacional; SAM =
Síndrome de Aspiração de Mecônio; SAR=Síndrome de Angústia Respiratória;
p * considerado como significativo quando <0,05. Comparação entre Grupo 1 (hipertensão gestacional) e Grupo 3 (grupo con-
trole) quanto ao risco de desenvolvimento de complicações perinatais. As gestantes foram divididas em três grupos:
Grupo 1: gestantes portadoras de hipertensão desen-
volvida na gestação (HG) (gestantes que desen-
volveram hipertensão a partir de 20 semanas de
gestação, com e sem proteinúria); Grupo 2: gestantes
portadoras de hipertensão arterial crônica (hiper-
tensão arterial diagnosticada previamente à gestação
ou diagnosticada antes de 20 semanas de gestação) e
Grupo 3: gestantes normotensas (grupo controle). Rev. Bras. Saúde Matern. Infant., Recife, 6 (1): 93-98, jan. / mar., 2006 95 Oliveira CA et al. gestantes (2,80%) e 915 gestantes (7,45%) respecti-
vamente. No Grupo 3 (grupo controle) foram
incluídas 11.013 gestantes (89,74%). O Grupo 2 (HAC) apresentou risco relativo
aumentado para: PIG (RR=1,87, p<0,001), Apgar
baixo no 1º minuto (RR=1,65, p<0,001), para SAM
(RR=2,33, p=0,007), prematuridade (RR=2,45,
p<0,001) e SAR (RR=1,94, p=0,027), mas não para
Apgar baixo no 5º minuto (RR=1,49, p=0,165) e
infecção neonatal (RR=0,59, p=0,599) (Tabela 2). O Grupo 2 (HAC) apresentou risco relativo
aumentado para: PIG (RR=1,87, p<0,001), Apgar
baixo no 1º minuto (RR=1,65, p<0,001), para SAM
(RR=2,33, p=0,007), prematuridade (RR=2,45,
p<0,001) e SAR (RR=1,94, p=0,027), mas não para
Apgar baixo no 5º minuto (RR=1,49, p=0,165) e
infecção neonatal (RR=0,59, p=0,599) (Tabela 2). Quando comparados os Grupos 1 e 2 isolada-
mente com o Grupo 3 (grupo controle), o Grupo 1
(HG) apresentou risco aumentado para: PIG
(RR=2,10, p<<0,001), Apgar baixo no 1º minuto
(RR=1,26, p=0,012), Apgar baixo no 5º minuto
(RR=1,45, p=0,045), infecção neonatal (RR=3,12,
p<<0,001) e prematuridade (RR=1,79, p<<0,001). Entretanto, a presença de HG não se relacionou ao
maior risco para SAM (RR=1,49, p=0,113) e SAR
(RR=1,39, p=0,142) (Tabela 1). Tabela 1 Quando comparados os riscos relativos encon-
trados para as pacientes com HAC com aqueles das
pacientes com HG, verificou-se que não houve dife-
rença estatisticamente significativa entre os dois
grupos quanto ao risco relativo para ocorrência das
seguintes variáveis: PIG (RR=0,88, p=0,460), Apgar
baixo no 1º minuto (RR=1,30, p=0,084), Apgar
baixo no 5º minuto (RR=1,02, p=0,930), infecção
neonatal (RR=0,19, p=0,070), SAM (RR=1,56,
p=0,252) e SAR (RR=1,39, p=0,364). Entretanto,
houve maior risco relativo de prematuridade no
grupo de hipertensão arterial crônica (RR=1,36,
p=0,006) quando comparado com o risco relativo do
grupo com HG (Tabela 3). Tabela 2 Comparação entre Grupo 2 (hipertensão arterial crônica) e Grupo 3 (grupo
controle) quanto ao risco de desenvolvimento de complicações perinatais. Comparação entre Grupo 2 (hipertensão arterial crônica) e Grupo 3 (grupo
controle) quanto ao risco de desenvolvimento de complicações perinatais. RR
IC95%
c2
p*
PIG
1,87
1,45
2,48
18,225
< 0,001
Apgar 1° minuto
1,65
1,28
2,12
14,712
<0,001
Apgar 5° minuto
1,49
0,84
2,64
1,923
0,165
Infecção neonatal
0,59
0,08
4,27
0,275
0,599
SAM
2,33
1,24
4,40
7,220
0,007
Prematuridade
2,45
2,03
2,96
80,337
< 0,001
SAR
1,94
1,06
3,54
4,848
0,027
RR=Risco relativo; IC95%=Intervalo de Confiança 95%; χ2 = teste do Qui-
quadrado; p = p valor; PIG=Pequeno para a idade gestacional;
SAM=Síndrome de Aspiração de Mecônio; SAR=Síndrome de Angústia
Respiratória; p * considerado como significativo quando <0,05. Discussão A hipertensão está associada a um aumento signi-
ficativo de complicações perinatais. O impacto das
síndromes hipertensivas na gestação tem sido estu-
dado em vários trabalhos, havendo resultados vari-
ados na literatura. O grande tamanho amostral obtido da base de
dados pode prover uma boa visão da população estu-
dada. p
ç
g
Respiratória; p * considerado como significativo quando <0,05. A prevalência de 10,26% de pacientes hiper-
tensas na população estudada está de acordo com a
literatura sobre o assunto, que descreve esta preva-
lência entre 10 e 22%. Devido à inconsistência dos
dados obtidos através de pesquisa na Base de Dados
da Maternidade Escola da UFRJ, não foi possível
classificar os diferentes tipos de hipertensão
induzidas pela gestação (de acordo com a presença
ou não de proteinúria), sendo a análise deste estudo
limitada ao grupo de pacientes com hipertensão
desenvolvida na gestação (HG), independente da
presença de proteinúria e ao grupo de pacientes com
diagnóstico de hipertensão arterial crônica (HAC). Tabela 3 Tabela 3 Comparação dos riscos relativos para desenvolvimento de complicações peri-
natais referentes ao Grupo 2 (hipertensão arterial crônica) e ao Grupo 1
(hipertensão gestacional). (hipertensão gestacional). RR
IC95%
c2
p*
PIG
0,88
0,65
1,21
0,545
0,460
Apgar 1° minuto
1,30
0,96
1,76
2,981
0,084
Apgar 5° minuto
1,02
0,53
1,98
0,007
0,930
Infecção Neonatal
0,19
0,02
1,43
3,262
0,700
SAM
1,56
0,72
3,38
1,311
0,252
Prematuridade
1,36
1,09
1,71
7,359
0,006
SAR
1,39
0,67
2,85
0,822
0,364
RR=Risco relativo; IC95%=Intervalo de confiança 95%; χ2 = teste do Qui-
quadrado; p = p valor; PIG=Pequeno para a idade gestacional; SAM =
Síndrome de Aspiração de Mecônio; SAR = Síndrome de Angústia
Respiratória; p * considerado como significativo quando <0,05. RR
IC95%
c2
p*
PIG
0,88
0,65
1,21
0,545
0,460
Apgar 1° minuto
1,30
0,96
1,76
2,981
0,084
Apgar 5° minuto
1,02
0,53
1,98
0,007
0,930
Infecção Neonatal
0,19
0,02
1,43
3,262
0,700
SAM
1,56
0,72
3,38
1,311
0,252
Prematuridade
1,36
1,09
1,71
7,359
0,006
SAR
1,39
0,67
2,85
0,822
0,364
RR=Risco relativo; IC95%=Intervalo de confiança 95%; χ2 = teste do Qui A HAC foi mais prevalente que a HG na popu-
lação estudada (respectivamente 7,45% e 2,80% da
população total). Dentre as pacientes hipertensas
27,32% foram diagnosticadas como HG e 72,67%
como HAC. Ao contrário do presente estudo, Jain,13
tendo estudado retrospectivamente 109.428 gestan-
tes, encontrou maior prevalência de HG em relação à
HAC. Embora na maior parte da literatura as Rev. Bras. Saúde Matern. Infant., Recife, 6 (1): 93-98, jan. / mar., 2006 96 Hipertensão na gestação e repercussões perinatais prevalências de pré-eclâmpsia e de hipertensão
gestacional sejam normalmente maiores que a
prevalência de HAC, um resultado semelhante ao do
presente estudo foi encontrado por Gaio et al.,14 ao
estudar 4892 gestantes na Universidade do Rio
Grande do Sul, encontrando 4,0% de gestantes com
HAC, 0,7% com diagnóstico de hipertensão gesta-
cional e 2,3% com pré-eclâmpsia/eclâmpsia. não apresentaram maior risco de prematuridade
(parto antes de 37 semanas de gestação) quando
comparados aos de mães com hipertensão gesta-
cional; houve aumento do risco de prematuridade
nas pacientes com pré-eclâmpsia e com pré-
eclâmpsia sobreposta quando comparadas com o
grupo com hipertensão gestacional. De acordo com os resultados encontrados no
presente estudo, a HG não mostrou associação com
piores resultados perinatais quando comparada a
HAC. Tal resultado não está de acordo com a
maioria da literatura disponível. Tabela 3 Uma causa provável
dessa diferença é o fato de não terem sido diferencia-
dos os casos de HG leve daqueles com critérios de
gravidade, já que são os casos graves de hipertensão
gestacional que cursam com pior repercussão fetal,
enquanto que os casos leves têm desfechos semel-
hantes aos de gestantes normotensas.1 A presença de diagnóstico de hipertensão arterial
aumentou o risco para ocorrência de todas as
variáveis estudadas, resultado também encontrado
pelos autores acima citados. Embora a HAC não tenha aumentado significati-
vamente o risco de ocorrência de baixo índice de
Apgar no 5º minuto e de infecção perinatal, e a HG
não tenha gerado aumento significativo do risco de
SAM e SAR nos recém-natos quando comparados
aos do grupo controle (pacientes normotensas), não
houve diferença estatisticamente significativa entre
esses resultados perinatais quando os riscos relativos
dos grupos com HAC e HG foram comparados entre
si, com exceção da prematuridade, tendo sido encon-
trado maior risco relativo de prematuridade nas
pacientes hipertensas crônicas quando comparadas
às gestantes com hipertensão desenvolvida na
gestação. Ray et a.l,5 estudaram 1948 gestantes
hipertensas e compararam pacientes com hipertensão
gestacional com gestantes com as demais síndromes
hipertensivas: os recém-natos de gestantes com HAC O presente estudo permitiu concluir que as
síndromes hipertensivas na gestação, tanto a
hipertensão arterial crônica quanto a hipertensão
gestacional, aumentaram o risco para desfecho peri-
natal desfavorável (PIG, Apgar baixo no 1º e 5º
minutos, infecção neonatal, SAM, prematuridade e
SAR). Quando comparados os risco relativo para
desfecho perinatal desfavorável entre HAC e HG, a
hipertensão arterial crônica apresentou risco relativo
de prematuridade significativamente maior. 14. Gaio DS, Schmidt MI, Duncan BB, Nucci LB, Matos MC,
Branchtein L. Hypertensive disorders in pregnancy:
frequency and associated factors in a cohort of Brazilian
women. Hypertens Pregnancy 2001; 20: 269-81. Recebido em 23 de setembro de 2004
Versão final apresentada em 31 de janeiro de 2006
Aprovado em 3 de fevereiro de 2006 Rev. Bras. Saúde Matern. Infant., Recife, 6 (1): 93-98, jan. / mar., 2006 12. Sibai BM, Lindheimer MD, Hauth J, Caritis S, VanDorsten
P, Klebanoff M, MacPherson C, Landon M, Miodovnik M,
Paul R, Meis P, Dombrowski M. Risk factors for
preeclampsia, abruptio placentae, and adverse neonatal
outcomes among women with chronic hypertension. N Engl
J Med 1998; 339: 667-71. Referências 1. Sibai BM. Diagnosis and management of gestational hyper-
tension and preeclampsia. Obstet Gynecol 2003; 102: 181-
92. 7. Hnat MD, Sibai BM, Caritis S, Hauth J, Lindheimer MD,
MacPherson C, VanDorsten P, Landon M, Miodovnik M,
Paul R, Meis P, Thurnau G, Dombrowski M. Perinatal
outcome in women with recurrent preeclampsia compared
with women who develop preeclampsia as nulliparas. Am J
Obstet Gynecol 2002; 186: 422-6. 2. Livingston JC, Sibai BM. Chronic hypertension in preg-
nancy. Obstet Gynecol Clin North Am 2001; 28: 447-63. 3. ACOG
(American
College
of
Obstetricians
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Gynecologists). Committee on Practice Bulletins-
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Paulo: Atheneu; 2004. p. 247-57, 365-70. 9. ACOG
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Curet LB, Catalano PM, Morris CD. Pregnancy outcomes
in health nulliparas who developed hypertension. Obstet
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HIP: Mc Master outcome study of hypertension in preg-
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J, Lindheimer MD, Klebanoff M, VanDorsten P, Landon M,
Paul R, Miodovnik M, Meis P, Thurnau G. Adverse peri-
natal outcome are significantly higher in severe gestational
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410-16. 97 Rev. Bras. Saúde Matern. Infant., Recife, 6 (1): 93-98, jan. / mar., 2006 Oliveira CA et al. Oliveira CA et al. 13. Jain L. Effect of pregnancy-induced and chronic hyperten-
sion on pregnancy outcome. J Perinatol 1997; 17: 425-7. 13. Jain L. Effect of pregnancy-induced and chronic hyperten-
sion on pregnancy outcome. J Perinatol 1997; 17: 425-7. 98
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IVAN NIKIFOROVICH PIKSIN. The 80th anniversary of the birth
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В. И. Давыдкин Статья посвящена научной деятельности известного ученого, заслуженного деятеля
науки Российской Федерации, заслуженного деятеля науки Республики Мордовия,
заслуженного врача Республики Мордовия, лауреата Государственной премии, ру-
ководителя научно-педагогической хирургической школы «Клиническая хирургия,
изучение фундаментальных и прикладных проблем эфферентной и квантовой ме-
дицины», доктора медицинских наук, профессора Пиксина Ивана Никифоровича. И. Н. Пиксиным проведены фундаментальные исследования медико-биологи-
ческого действия квантового излучения на молекулярном, клеточном и орга-
низменном уровнях. Под его руководством исследованы показатели гомеоста-
за, иммунологического статуса, изменения ПОЛ и антиоксидантных систем,
центральной, органной и периферической гемодинамики у больных с острыми
деструктивными заболеваниями легких, щитовидной железы, гнойно-септиче-
скими заболеваниями, лактационным маститом и другой патологией. Им была
доказана эффективность применения УФО крови в комбинации с другими эф-
ферентно-квантовыми методами. Это позволило разработать новые способы экс-
тракорпоральной детоксикации (различные способы лечебного плазмафереза,
эритродесорбции, селективной и комбинированной квантовой и эфферентной
терапии, криоафереза и гепаринпреципитатафереза, озонотерапии). Коллекти-
вом научно-педагогической школы во главе с И. Н. Пиксиным проводятся иссле-
дования, связанные с диагностикой и лечением гастродуоденальных кровотече-
ний, механической желтухи, панкреатитов, нагноительных заболеваний легких
и плевры, хирургических заболеваний сердца, сосудов, профилактикой осложнений
при травмах конечностей, сахарного диабета и диабетической стопы, детской ортопе-
дии. Под руководством И. Н. Пиксина внедрены новые миниинвазивные технологии:
трансторакальное дренирование и санационная терапия гнойных полостей легких
и плевры, чрезкожная и чрезпеченочная холецистохолангиография, лечебно-ди-
агностические вмешательства при объемных образованиях брюшной полости
и щитовидной железы, методики эхоскопической диапевтики при остром деструк-
тивном панкреатите и др. Он по праву считается одним из организаторов высшего
медицинского образования в регионе, активно работает над совершенствованием
системы подготовки медицинских и научных кадров для различных регионов Рос-
сийской Федерации. Ключевые слова: ультрафиолетовое облучение крови, аутогемотрансфузия, плаз-
маферез, детоксикация, трансфузиология, эфферентная терапия. УДК 616(092) УДК 616(092) IVAN NIKIFOROVICH PIKSIN.
The 80th anniversary of the birth V. I. Davydkin ÂÅÑТÍÈÊ ÌÎÐÄÎÂÑÊÎÃÎ ÓÍÈÂÅÐÑÈТÅТÀ ÂÅÑТÍÈÊ ÌÎÐÄÎÂÑÊÎÃÎ ÓÍÈÂÅÐÑÈТÅТÀ Том 25, ¹ 2. 2015 ИВАН НИКИФОРОВИЧ ПИКСИН.
К 80-летию со дня рождения В. И. Давыдкин MORDOVIA UNIVERSITY BULLETIN MORDOVIA UNIVERSITY BULLETIN lungs, thyroid gland, purulent-septic diseases, lactational mastitis and other diseases. He have proven the particular effectiveness in use of ultraviolet blood irradiation in
combination with other appearance-quantum methods. This has allowed scientists to
develop new ways of extracorporeal detoxification (different ways of therapeutic plas-
mapheresis, erythrodesorbtion, selective and combined quantum and efferent therapy,
cryofrerezis and heparinprecipitatapheresis, ozone). A team of the scientific-pedagogical
school led by I. N. Piksin explores the problems of diagnosis and treatment of gastrodu-
odenal bleeding, mechanical jaundice, pancreatitis, suppurative diseases of the lung and
pleura, surgical diseases of the heart, blood vessels, prevention of complications of limb
injuries, diabetes and diabetic foot care, pediatric orthopedics. Under the leadership of
I. N. Piksin have introduced new minimally invasive technologies: transthoracic drain-
age and rehabilitation therapy of purulent cavities of the lung and pleura, percutaneous
and transhepatic cholecystocholangiography, diagnostic and treatment interventions in
space-occupying lesions of the abdomen and thyroid gland, ultrasound techniques of
diabet in acute destructive pancreatitis and others. He is considered as one of organizers
of higher medical education system in the region. The professor is actively working on
improvement in training of medical and scientific personnel for various regions of the
Russian Federation. Keywords: ultraviolet irradiation of blood, autologous blood transfusion, plasmapheresis,
detoxification, transfusiology, efferent therapy. В 1960–70-х гг. ученый один из пер-
вых в стране обосновал новый метод
лечения – аутогемотрансфузию (АГТ) –
переливание собственной крови боль-
ных, заготовленной до или непосредст-
венно перед операцией [24; 26; 28; 31]. В те годы операции при тиреотоксико-
зе сопровождались высоким процентом
осложнений и летальных исходов, не
было эффективных лекарств для лече-
ния больных, борьбы с тиреотоксиче-
ским кризом [29; 32–34]. Совместно
с московской и санкт-петербургской шко-
лами И. Н. Пиксиным были проведены
исследования по оценке эффективности
АГТ [35–36]. По итогам работы в 1983 г. им защищена докторская диссертация
«Аутогемотрансфузия в плановой хи-
рургии». В последующие годы АГТ ста-
ла применяться и при других плановых
и экстренных оперативных вмешатель-
ствах: ваготомии, гастрэктомии, резек-
ции желудка и легких [34], операциях
на костях, суставах, артериях, грыже-
сечениях, холецистэктомии и др. [36;
39]. Исследование морфологического
состава крови, функции системы гоме-
остаза, плазменных белков пропердина,
состояние костно-мозгового кроветво-
рения, белково-углеводного комплекса,
центральной и периферической гемоди-
намики, течение операционного и по- 1 ноября 2014 г. V. I. Davydkin The article is devoted to the scientific work of a famous scientist, honored worker of
science of the Russian Federation, honored worker of science of the Republic of Mor-
dovia, honored doctor of the Republic of Mordovia, laureate of State prize, head of
the scientific-pedagogical surgical school “Clinical surgery, the study of fundamental
and applied problems of efferent and quantum medicine”, doctor of medical Sciences
professor Piksin Ivan Nikiforovich. Professor I. N. Piksin conducted basic research of
medical-biological effects of quantum radiation on the molecular, cellular and organ-
ismal levels. Under his leadership, studying the parameters of homeostasis, immune
status, changes in peroxide oxidation of lipids and antioxidants systems, the Central
organ and peripheral hemodynamics in patients with acute destructive diseases of © Давыдкин В. И., 2015 © Давыдкин В. И., 2015 48 Vol. 25, no. 2. 2015 ÂÅÑТÍÈÊ ÌÎÐÄÎÂÑÊÎÃÎ ÓÍÈÂÅÐÑÈТÅТÀ Том 25, ¹ 2. 2015 бов фотомодификации крови; прове-
дение исследований в рамках общесо-
юзной научной программы «Человек
и свет» [21; 40]. Научное сотрудниче-
ство по этой программе позволило со-
здать новые источники света, кварцевые
кюветы для фотомодификации крови,
аппарат для фотомодификации «Свет-
1» и др. [1–2]. На базах НИИ «Человек
и свет», научно-клинических центров
эфферентной медицины и гравитацион-
ной хирургии крови творческим коллек-
тивом ученых-клиницистов, патофизио-
логов и биологов проведены фундамен-
тальные исследования медико-биологи-
ческого действия квантового излучения
на молекулярном, клеточном и организ-
менном уровнях [37–38; 42–43; 75]. слеоперационного периодов показали
значительное улучшение клинических
показателей, костномозгового кроветво-
рения, стабилизацию системы гемоста-
за, уровней глобулинов и альбуминов
[32; 36]. Впервые И. Н. Пиксиным было из-
учено
влияние
аутогемотрансфузии
на пропердин крови, который являет-
ся маркером устойчивости организма
к инфекции, показателем гуморального
звена естественного иммунитета [32]. Установлено о стимуляции иммуно-
логической реактивности организма. Положительные сдвиги, наступавшие
в организме больных при эксфузии
и реинфузии, обеспечивали благоприят-
ное течение оперативного вмешательст-
ва, обезболивание и течение послеопе-
рационного периода [41]. АГТ преду-
преждала гемодинамические, гемокоа-
гуляционные нарушения во время опе-
ративного вмешательства. Кроме того,
переливалась абсолютно совместимая
трансфузионная среда, которая с собст-
венными белками и ферментами немед-
ленно включалась в активную циркуля-
цию; аутологичная кровь не вызывала
аллергизации больного. Переливание
аутологичной
крови
способствовало
сокращению объема трансфузионных
сред. Отмечалось уменьшение числа
послеоперационных осложнений, осо-
бенно гнойно-воспалительного характе-
ра, и послеоперационной летальности. Личный опыт использования аутокрови
больных при различных хирургических
вмешательствах И. Н. Пиксин обобщил
в неимеющей аналогов монографии
«Аутотрансфузия крови при хирургиче-
ских вмешательствах» [Там же]. Лечение ультрафиолетовыми луча-
ми облученной крови проводилось с по-
мощью аппарата «Изольда» МД-73 М. Исследовались показатели гомеостаза
[75], иммунологического статуса, изме-
нения ПОЛ и антиоксидантных систем
[40; 43], центральной, органной и пери-
ферической гемодинамики у больных
с острыми деструктивными заболева-
ниями легких [3], щитовидной железы
[45], гнойно-септическими заболева-
ниями, лактационным маститом [42]
и другой патологией [21]. УФО ауто-
крови у больных с ДТЗ особенно пока-
зано при выраженных аутоиммунных
сдвигах, тяжелом тиреотоксикозе и не-
переносимости лекарственных препа-
ратов. Включение АУФОК в комплекс
лечебных
мероприятий
позволило
снизить интенсивность лекарственной
терапии, спектр и дозу применяемых
препаратов [45]. Положительный эффект АУФОК-
терапии
достигается
многофактор-
ным влиянием ультрафиолетовых лу-
чей на обменные процессы, клеточ-
ные мембраны форменных элементов
крови,
иммунологическую
реактив-
ность, улучшением органной перфузии
и реологических свойств крови [40; 45]. В зависимости от тяжести и характера
заболевания используются различные
дозы и режимы фотомодификации кро- [
]
С 1985 по 2013 г. MORDOVIA UNIVERSITY BULLETIN исполнилось 80
лет Пиксину Ивану Никифоровичу –
видному ученому, заслуженному деятелю
науки Российской Федерации, заслужен-
ному деятелю науки Республики Мор-
довия, заслуженному врачу Республики
Мордовия,
лауреату
Государственной
премии, члену Российского общества хи-
рургов, создателю научно-педагогической
хирургической школы «Клиническая хи-
рургия, изучение фундаментальных и при-
кладных проблем эфферентной и кванто-
вой медицины», доктору медицинских
наук, профессору кафедры госпитальной
хирургии с курсами травматологии, орто-
педии, глазных болезней и стоматологии. Вся научная деятельность профессо-
ра И. Н. Пиксина связана с Мордовским
университетом [67; 76–77]. После окон-
чания в 1959 г. Горьковского медицин-
ского института имени С. М. Кирова он
работал врачом-хирургом, заведовал хи-
рургическим отделением Мордовской ре-
спубликанской больницы. В эти же годы
им проводились исследования в области
хирургического лечения зоба [24–26]
и язвенной болезни [16; 22; 27]. После
защиты в 1969 г. кандидатской диссерта-
ции по результатам хирургического лече-
ния диффузного токсического зоба И. Н. Пиксин перешел работать в Мордовский
государственный университет. 49 MORDOVIA UNIVERSITY BULLETIN Vol. 25, no. 2. 2015 MORDOVIA UNIVERSITY BULLETIN ви. Особенно эффективно применение
УФО крови в комбинации с другими
экстракорпоральными методами деток-
сикации. Это позволило предложить
новые способы экстракорпоральной де-
токсикации при гнойно-деструктивных
заболеваниях легких и плевры [3; 10],
сахарном диабете [44], перитонитах
[12; 45], а также различные способы
лечебного плазмафереза, эритродесорб-
ции [6], селективной и комбинирован-
ной квантовой и эфферентной терапии,
криоафереза [12] и гепаринпреципита-
тафереза [51–52], озонотерапии [17; 23;
53; 58; 60]. Результаты исследований
кафедры госпитальной хирургии опу-
бликованы в центральной печати, под-
держаны учеными из Москвы, Санкт-
Петербурга, Нижнего Новгорода и др. научных медицинских форумов с ме-
ждународным участием по хирургии,
интенсивной терапии, эндокринной хи-
рургии [72–73] и эфферентным и кван-
товым методам лечения в клинической
медицине [79]. Он по праву считается
одним из организаторов высшего ме-
дицинского образования в регионе, ак-
тивно работает над совершенствовани-
ем системы подготовки медицинских
и научных кадров для различных реги-
онов Российской Федерации [54]. Им
внедрена сквозная рабочая программа
преподавания хирургических болезней,
на базе кафедры и клиники организован
единый научно-образовательный и кли-
нический центр. Сотрудниками возглавляемой И. Н. Пик-
синым научно-педагогической школы защи-
щены 51 кандидатская и 5 докторских дис-
сертаций, опубликованы более 600 научных
работ, 11 монографий, свыше 20 учебно-
методических пособий и рекомендаций,
21 сборник материалов научно-практи-
ческих конференций [76–77]. Более 60
научных работ студентов, выполненных
под руководством И. Н. Пиксина, яви-
лись основой для выполнения канди-
датских диссертаций. Кроме эфферентной и квантовой
гемокоррекции, коллективом научно-
педагогической школы проводятся ис-
следования по проблемам диагностики
и лечения пептических язв [16; 22; 27;
46], гастродуоденальных кровотечений
[5], механической желтухи [48], пан-
креатитов [18], нагноительных заболе-
ваний легких и плевры [10; 49; 58; 60;
68; 81], хирургических заболеваний
сердца [71], сосудов [7; 59; 62; 64; 66],
в области профилактики осложнений при
травмах конечностей [9; 11; 18], сахарно-
го диабета и диабетической стопы [8; 13;
65–66], детской ортопедии [57; 64]. р
За заслуги в развитии здравоохра-
нения региона и подготовку высокок-
валифицированных врачебных кадров
И. Н. Пиксину в 1977 г. было присво-
ено почетное звание «Заслуженный
врач Республики Мордовия». За цикл
работ «Научная разработка и внедре-
ние методов эфферентной терапии
в хирургию» и разработку нового на-
учного
направления,
существенно
улучшившего
результаты
лечения,
подготовку научных кадров и созда-
ние научно-педагогической школы ему
присвоено почетное звание «Заслужен-
ный деятель науки Республики Мордо-
вия» (1997). За разработку и внедре-
ние методов эфферентной медицины
И. Н. Пиксин в 1998 г. ÂÅÑТÍÈÊ ÌÎÐÄÎÂÑÊÎÃÎ ÓÍÈÂÅÐÑÈТÅТÀ профессор И. Н. Пик-
син заведовал кафедрой госпитальной
хирургии с курсами травматологии
и ортопедии, глазных болезней и стома-
тологии, в настоящее время – профес-
сор этой же кафедры. На протяжении
этих лет творческие усилия коллектива
под руководством И. Н. Пиксина были
направлены на разработку и внедрение
в клиническую практику новых спосо- 50 ÂÅÑТÍÈÊ ÌÎÐÄÎÂÑÊÎÃÎ ÓÍÈÂÅÐÑÈТÅТÀ Том 25, ¹ 2. 2015 Многочисленные ученики, сотруд-
ники Медицинского института и ка-
федры госпитальной хирургии сердеч-
но поздравляют Ивана Никифоровича
с юбилеем и желают ему долгих лет
плодотворной научной, педагогической
и хирургической деятельности. звание «Заслуженный деятель науки
Российской Федерации» [19]. Он также
является председателем диссертаци-
онного совета Д 212.117.08 (Саранск),
экспертом Республиканского исследо-
вательского
научно-консультативного
центра экспертизы. MORDOVIA UNIVERSITY BULLETIN стал лауреатом
Государственной премии Республики
Мордовия в области науки и техники
[20], а в 2002 г. за заслуги в научной
деятельности ему присвоено почетное Под руководством И. Н. Пикси-
на внедрены новые миниинвазивные
технологии: трансторакальное дрени-
рование [70] и санационная терапия
гнойных полостей легких и плевры
по оригинальной методике [72], чрез-
кожная и чрезпеченочная холецисто-
холангиография [78], лечебно-диагно-
стические вмешательства при объем-
ных образованиях брюшной полости
и щитовидной железы [14–15; 74],
методики эхоскопической диапевтики
при остром деструктивном панкреати-
те и др. [56; 78]. И. Н. Пиксин является инициато-
ром и организатором проведения в Ре-
спублике Мордовия 7 всероссийских И. Н. Пиксин является инициато-
ром и организатором проведения в Ре-
спублике Мордовия 7 всероссийских 51 СПИСОК ИСПОЛЬЗОВАННЫХ ИСТОЧНИКОВ 1. Авторское свидетельство, 1733012 Российская Федерация. Кювета для облучения жидкости /
И. Н. Пиксин, М. Д. Романов, А. Ф. Калязин. – 1992. 1. Авторское свидетельство, 1733012 Российская Федерация. Кювета для облучения жидкости /
И. Н. Пиксин, М. Д. Романов, А. Ф. Калязин. – 1992. 2. Авторское свидетельство, 2029507 Российская Федерация. Ретрактор-облучатель / И. Н
М. Бакайкин, А. Ф. Калязин. – 1995. 3. Влияние фотомодифицированной крови на гемодинамические параметры у больных с острыми
деструктивными заболеваниями легких / И. Н. Пиксин [и др.] // Медицинский альманах. – 2008. – № S. –
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meditsinskiy zhurnal = Kazan medical journal. 1981, no. 1, pp. 9–11. MORDOVIA UNIVERSITY BULLETIN O prisuzhdenii gosudarstvennykh premiy Respubliki Mordoviya v oblasti nauki i tekhniki za 1998 god:
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rotoxic goiter in older-older and elderly]. Kazanskiy meditsinskiy zhurnal = Kazan medical journal. 1970, no. 1, pp. 71–72. 26 Pik i I N A
f
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s yazvennoy boleznyu zheludka i 12-perstnoy kishki [Autologous blood transfusion in the surgical treatment of
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pleksnom lechenii bolnykh abstsessom legkikh [Evaluation of the effectiveness of systemic ozone therapy in
complex treatment of patients with lung abscess]. Medial = The Medial. 2012, no. 1, pp. 50–52. i 61. Piksin I. N., Akashev R. V. Method of Diabetic Foot Classification. Journal of Experimental & Clini-
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mental & Clinical Medicine. 2012, pp. 22–23. 63. Piksin I. N., Baksheev Yu. G. Patologiya pochek pri kifoskolioze u detey [Pathology of the kidney in
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blood in patients with posttraumatic complications]. Revista. Ozonoterapia. 2009, vol. 3, no. 1, pp. 116–117. ÂÅÑТÍÈÊ ÌÎÐÄÎÂÑÊÎÃÎ ÓÍÈÂÅÐÑÈТÅТÀ 78. Piksin I. N. [et al.]. Endoskopicheskie i ultrazvukovye miniinvazivnye vmeshatelstva v abdominal
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noy khirurgii [Endoscopic ultrasound and minimally invasive interventions in abdominal surgery]. Moscow,
Nauka Publ., 2011, 147 p. MORDOVIA UNIVERSITY BULLETIN Sravnitelnaya otsenka effektivnosti transtorakalnykh metodov sanatsii ostrykh ab-
stsessov legkikh: avtoref. dis. na soisk. uchen. step. kand. med. nauk [Comparative evaluation of the efficacy
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zovaniem iskusstvennogo krovoobrashcheniya v blizhayshem posleoperatsionnom periode u bolnykh pozhilogo 71. S. V. Kadakin [et al.]. Sistema transporta kisloroda posle aorto-koronarnogo shuntirovaniya s ispol
zovaniem iskusstvennogo krovoobrashcheniya v blizhayshem posleoperatsionnom periode u bolnykh pozhilogo 59 About the author: Davydkin Vasiliy Ivanovich, head of chair of Hospital Surgery with training courses of Traumatology
and Orthopedics, Ocular Diseases and Dentistry of Ogarev Mordovia State University (68, Bolshevistskaya
str., Saransk, Russia), Ph.D. (Medicine), mgu-hospital.surgery@yandex.ru 60
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The Impact of the COVID-19 Pandemic on the Tourism Sector in Jawa Tengah Province
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Entrepreneurship and Small Business Research
Volume 1, Nomor 3, p.44 - 52 Entrepreneurship and Small Business Research
Volume 1, Nomor 3, p.44 - 52 Entrepreneurship and Small Business Research
Volume 1, Nomor 3, p.44 - 52 The Impact of the COVID-19 Pandemic on the Tourism Sector
in Jawa Tengah Province
Karnowahadi1*, Endang Sulistiyani2, Dody Setyadi3 1,2,3Politeknik Negeri Semarang, Semarang, Indonesia
*Corresponding author: Karnowahadi
Corresponding Email : karnowahadi@polines.ac.id 1,2,3Politeknik Negeri Semarang, Semarang, Indonesia
*Corresponding author: Karnowahadi
Corresponding Email : karnowahadi@polines.ac.id 1,2,3Politeknik Negeri Semarang, Semarang, Indonesia
*Corresponding author: Karnowahadi
Corresponding Email : karnowahadi@polines.ac.id ARTICLE INFO
Article history:
Received 04 March 2022
Accepted 21 September 2022
Available Online 05 December 2022 ARTICLE INFO
Article history:
Received 04 March 2022
Accepted 21 September 2022
Available Online 05 December 2022 ABSTRACT
The COVID-19 pandemic has resulted in economic paralysis in
various countries around the world. One of the provinces in
Indonesia, namely Jawa Tengah Province which is a mainstay
of world tourist destinations, cannot be separated from the
impact of COVID-19. The tourism sector in Jawa Tengah
Province contributes less than 10% of the Gross Regional
Domestic Product. The COVID-19 pandemic has resulted in a
decrease in the contribution of the tourism sector to Jawa
Tengah's Gross Regional Domestic Product by more than 50%. The number of tourists who fell during the pandemic resulted
in decreased tourism sector income. Although the average
length of stay did not decrease, hotel occupancy rates
continued to decline during the pandemic. The social impacts
caused by COVID-19 include people preferring to stay at home
instead of talking to their neighbors, people don't like crowds,
community groups are more protective of their environment
(by making environmental entrance gates). The Jawa Tengah
Provincial Government with the permission of the Central
Government took a policy of regeneration of the tourism sector
without neglecting the protection of public health. Keywords:
Tourism, lodging facilities, room
occupancy, length of stay, COVID-
19 © 2022 The Author(s). Published by International Ecsis
Association. This is an open access article under the Creative
Commons Attribution-ShareAlike 4.0 International License. https://doi.org/10.55980/esber.v1i3.50 https://doi.org/10.55980/esber.v1i3.50 1. Introduction The Covid-19 pandemic has hit the world since 2020. Apart from the health sector, the
sector affected is the tourism sector (Boto-García & Mayor, 2022; Mariolis et al., 2021). The
impact of the Covid-19 pandemic on tourism around the world is very wide (Huang et al., 2021). Before covid-19, the tourism sector contributed to alleviating poverty in many developing
countries (Zhao & Xia, 2020). Tourism is the most vulnerable sector to the impact of pandemic
deterrence measures, such as restrictions on social mobility (Gössling et al., 2020; Li et al., 2020). Sectors such as hotels, restaurants and aviation received the most impact (Fan et al., 2022; Uğur
& Akbıyık, 2020). Although the weights are different, the world's tourism sector is paralyzed. Similarly, tourist destinations in Indonesia are experiencing total paralysis. Bali, which is a world
tourist destination, has experienced the same thing. The impact of covid-19 on Bali tourism
contributes to reducing Bali's achievements in poverty alleviation (Bhaskara & Filimonau, 2021;
Okafor et al., 2022; Pham & Nugroho, 2022). Tourism is an economic sector that is being actively built by the Indonesian government
since 2014, including tourism sector in Jawa Tengah Province (Rhama, 2022). During the Covid-
19 pandemic, tourist destinations in Jawa Tengah Province were closed to the public, because
public area is the most high-risk place (Das et al., 2022; Goffman, 2020; Lei et al., 2021). In fact, 44 | P a g e 44 | P a g e Entrepreneurship and Small Business Research
Volume 1, Nomor 3, p.44 - 52 all residential entrances are also closed to the public (Chen et al., 2022). This is done to break the
chain of transmission of Covid-19. all residential entrances are also closed to the public (Chen et al., 2022). This is done to break the
chain of transmission of Covid-19. Tourism is one of the sectors of the economy that has been most affected by the Covid-19
pandemic but has been slow to recover (Rafidola Mareta Riesa & Haries, 2022). According to
UNWTO (2021), international tourist arrivals fell by 80%. When the Covid-19 pandemic emerged,
no one was able to predict the end of the pandemic. The increasing death rate due to the Covid-19
pandemic has made all state policies directed at the public health sector. The Covid-19 pandemic
has changed the pattern of people's lives in various fields. 1. Introduction Social distancing is a term that is
familiar to the general public. The residential area becomes an exclusive area and is closed to
other communities. Masks covering the mouth and nose are mandatory items for everyone. Hand
sanitizer is a liquid that is always carried by everyone wherever they go. There is no handshake
between individuals in society. In the business sector, all transaction services are carried out on-
line. The closure of all tourist destinations in Indonesia during the Covid-19 pandemic has not
only had an impact on the economic downturn. The increase in environmental damage in the
natural tourist areas of national parks occurred due to decreased supervision and conservation
measures during the lockdown (Andréfouët et al., 2021; Cahyadi & Newsome, 2021). This
research explores the various impacts of the Covid-19 pandemic on the tourism sector in the
Jawa Tengah Province, Indonesia. Some of the discussions presented included the impact on the
number of tourists, the contribution of tourism to Regional Income, the impact on the tourism
sector workforce. 2. Methods The method used in this study is a quantitative method. The data was obtained from
secondary data sources, namely the Central Statistics Agency (BPS, 2022), Bank Indonesia (Bank
Indonesia, 2022), and several other data sources. The sample data analyzed were data before the
pandemic (in 2018 and 2019) and data during the pandemic (in 2020 and 2021). The analytical
tools used in this study are descriptive analysis and analysis of the mean difference test (Chang &
Wu, 2021). Descriptive analysis is used to explain the various data that have been collected. Analysis of the mean difference test was used to see the difference between conditions before and
during the Covid-19 pandemic. The hypothesis in this study states that the Covid-19 pandemic
has a negative impact on the tourism sector in Jawa Tengah Province. Figure 1: Number of Foreign Tourists in Jawa Tengah in 2018-2021 The number of domestic tourists still dominates visitors in Jawa Tengah Province. In 2018
the number of domestic tourists was 48.9 million tourists. In 2019 it increased by 18.3% to 57.9
million tourists. Meanwhile, in 2020 the number of tourists decreased by 60.92% to 22.63 million
tourists. In 2021 the number of tourists decreased by 5.7% to 21.3 million tourists (Figure 2). Figure 2: Number of Domestic Tourists in Jawa Tengah in 2018-2021
One of the supporters of tourism development is lodging facilities. In 2019 there was an
increase in the hotel room occupancy rate in the Jawa Tengah Province by 0.63% compared to
2018. In 2020 the hotel room occupancy rate decreased by 37.45%. In 2021 the hotel room
occupancy rate will increase again by 10.51% (Figure 3). 48.943.607
57.900.863
22.629.085
21.332.409
-
10.000.000
20.000.000
30.000.000
40.000.000
50.000.000
60.000.000
70.000.000
2018
2019
2020
2021 Figure 2: Number of Domestic Tourists in Jawa Tengah in 2018-2021
48.943.607
57.900.863
22.629.085
21.332.409
-
10.000.000
20.000.000
30.000.000
40.000.000
50.000.000
60.000.000
70.000.000
2018
2019
2020
2021 Figure 2: Number of Domestic Tourists in Jawa Tengah in 2018-2021 One of the supporters of tourism development is lodging facilities. In 2019 there was an
increase in the hotel room occupancy rate in the Jawa Tengah Province by 0.63% compared to
2018. In 2020 the hotel room occupancy rate decreased by 37.45%. In 2021 the hotel room
occupancy rate will increase again by 10.51% (Figure 3). One of the supporters of tourism development is lodging facilities. In 2019 there was an
increase in the hotel room occupancy rate in the Jawa Tengah Province by 0.63% compared to
2018. In 2020 the hotel room occupancy rate decreased by 37.45%. In 2021 the hotel room
occupancy rate will increase again by 10.51% (Figure 3). Figure 3: hotel room occupancy rate in Jawa Tengah Province 2018-2021
45,82
46,11
28,84
31,87
0
10
20
30
40
50
2018
2019
2020
2021 Figure 3: hotel room occupancy rate in Jawa Tengah Province 2018-2021
Judging from the length of stay of foreign tourists (Figure 4), in 2018 the average stay of
foreign tourists in Jawa Tengah was 2.22 days. Length of stay decreased in 2019, in star hotels
decreased by 19.4% and in non-star hotels decreased by 14.4%. However, in 2021 it will increase
by 25.14% for star hotels and 91% for non-star hotels. 3.1. Development of the Tourism Sector in Jawa Tengah 3.1. Development of the Tourism Sector in Jawa Tengah
Indonesia is a country that has various forms of tourism destinations. Visitors can choose
the desired tourist destinations, including nature tourism, religious tourism , business tourism,
beach tourism, mountain tourism, underwater tourism, and so on. Likewise, tourism destinations
in Jawa Tengah Province, religious destinations (Borobudur Temple, Cetho Temple, Demak
Mosque, Sunan Muria Tomb, Sam Po Kong, Maria Keteb Cave, etc.), cultural destinations
(Surakarta Palace, Semarang Old Town, Batik Pekalongan, Wayang, etc.) (Matteucci et al., 2022),
mountain destinations (Merapi, Merbabu, Sindoro, Sumbing, etc.), beach destinations (Tegal
Beach, Pekalongan Beach, Semarang Beach, Wonogiri Beach, etc.), and other forms of tourism
destinations. In 2018, the number of foreign tourists was 677,168 foreign tourists. Meanwhile, in 2019
the number of foreign tourists increased by 2.15% to 691,699 foreign tourists. In 2020 there was
a decline in foreign tourist arrivals of 88.9% from 2019. In 2021 there was a decrease of 97.7%
compared to 2020. (Figure 1). 45 | P a g e
677.168
691.699
78.290
1.793
-
200.000
400.000
600.000
800.000
2018
2019
2020
2021 45 | P a g e Entrepreneurship and Small Business Research
Volume 1, Nomor 3, p.44 - 52 Entrepreneurship and Small Business Research
Volume 1, Nomor 3, p.44 - 52 Entrepreneurship and Small Business Research
Volume 1, Nomor 3, p.44 - 52 Figure 1: Number of Foreign Tourists in Jawa Tengah in 2018-2021 45,82
46,11
28,84
31,87
0
10
20
30
40
50
2018
2019
2020
2021 Figure 3: hotel room occupancy rate in Jawa Tengah Province 2018-2021 Judging from the length of stay of foreign tourists (Figure 4), in 2018 the average stay of
foreign tourists in Jawa Tengah was 2.22 days. Length of stay decreased in 2019, in star hotels
decreased by 19.4% and in non-star hotels decreased by 14.4%. However, in 2021 it will increase
by 25.14% for star hotels and 91% for non-star hotels. Judging from the length of stay of foreign tourists (Figure 4), in 2018 the average stay of
foreign tourists in Jawa Tengah was 2.22 days. Length of stay decreased in 2019, in star hotels
decreased by 19.4% and in non-star hotels decreased by 14.4%. However, in 2021 it will increase
by 25.14% for star hotels and 91% for non-star hotels. Judging from the length of stay of foreign tourists (Figure 4), in 2018 the average stay of
foreign tourists in Jawa Tengah was 2.22 days. Length of stay decreased in 2019, in star hotels
decreased by 19.4% and in non-star hotels decreased by 14.4%. However, in 2021 it will increase
by 25.14% for star hotels and 91% for non-star hotels. 46 | P a g e Entrepreneurship and Small Business Research
Volume 1, Nomor 3, p.44 - 52
Figure 4: Length of Stay of Foreign Tourists in Jawa Tengah 2018-2021 (Days)
2,22
1,79
1,80
2,42
2,22
1,90
2,23
3,63
-
1,00
2,00
3,00
4,00
2018
2019
2020
2021
Foreign: Star
Foreign: Non-Star Entrepreneurship and Small Business Research
Volume 1, Nomor 3, p.44 - 52
Figure 4: Length of Stay of Foreign Tourists in Jawa Tengah 2018-2021 (Days)
2,22
1,79
1,80
2,42
2,22
1,90
2,23
3,63
-
1,00
2,00
3,00
4,00
2018
2019
2020
2021
Foreign: Star
Foreign: Non-Star Entrepreneurship and Small Business Research
Volume 1, Nomor 3, p.44 - 52 Entrepreneurship and Small Business Research
Volume 1, Nomor 3, p.44 - 52 Entrepreneurship and Small Business Research
Volume 1, Nomor 3, p.44 - 52 Figure 4: Length of Stay of Foreign Tourists in Jawa Tengah 2018-2021 (Days) The average stay for domestic tourists (Figure 5), in 2018 was 1.35 days. In 2019 it
decreased by 4.4% for star hotels and 20% for non-star hotels. However, until 2021 the average
stay of domestic tourists has increased by 4.7% for star hotels. Figure 1: Number of Foreign Tourists in Jawa Tengah in 2018-2021 In 2018 and
2019, the distribution of tourism sector income to the GRDP of Jawa Tengah Province was 0.02%. In 2020, it decreased by 50% to 0.01%. The distribution of tourism sector income to GRDP can be seen in Figure 8. In 2018 and
2019, the distribution of tourism sector income to the GRDP of Jawa Tengah Province was 0.02%. In 2020, it decreased by 50% to 0.01%. The distribution of tourism sector income to GRDP can be seen in Figure 8. In 2018 and
2019, the distribution of tourism sector income to the GRDP of Jawa Tengah Province was 0.02%. In 2020, it decreased by 50% to 0.01%. Figure 8: Distribution of the Tourism Sector to the GRDP of Jawa Tengah 2018-2021
0,020%
0,020%
0,010%
0,010%
0,000%
0,005%
0,010%
0,015%
0,020%
0,025%
2018
2019
2020
2021 Figure 8: Distribution of the Tourism Sector to the GRDP of Jawa Tengah 2018-2021 Figure 1: Number of Foreign Tourists in Jawa Tengah in 2018-2021 Meanwhile, the average stay for
non-star hotels until 2021 has decreased by 1.9%). The average stay for domestic tourists (Figure 5), in 2018 was 1.35 days. In 2019 it
decreased by 4.4% for star hotels and 20% for non-star hotels. However, until 2021 the average
stay of domestic tourists has increased by 4.7% for star hotels. Meanwhile, the average stay for
non-star hotels until 2021 has decreased by 1.9%). Figure 5: Length of Stay of Domestic Travelers in Jawa Tengah 2018-2021 (Days)
1,35
1,29
1,31
1,35
1,35
1,08
1,07
1,06
-
0,20
0,40
0,60
0,80
1,00
1,20
1,40
1,60
2018
2019
2020
2021
Domestic: Star
Domestic: Non-Star Figure 5: Length of Stay of Domestic Travelers in Jawa Tengah 2018-2021 (Days) 3.2. Contribution of the Tourism Sector to GRDP 3.2. Contribution of the Tourism Sector to GRDP
The gross domestic regional product (GRDP) of Jawa Tengah Province has increased from
year to year, except in 2020 (Figure 6). In 2019, the GRDP of Jawa Tengah Province increased by
7.3% compared to 2018. However, in 2020 it decreased by 0.96%. In 2021 GRDP will increase by
5.4% compared to 2020. 47 | P a g e
Figure 6 : Jawa Tengah GRDP 2018-2021 (Billion IDR)
1.268.261,17
1.360.960,13
1.347.922,69
1.420.799,91
1.150.000,00
1.200.000,00
1.250.000,00
1.300.000,00
1.350.000,00
1.400.000,00
1.450.000,00
2018
2019
2020
2021 47 | P a g e Entrepreneurship and Small Business Research
Volume 1, Nomor 3, p.44 - 52 Entrepreneurship and Small Business Research
Volume 1, Nomor 3, p.44 - 52 Jawa Tengah Province's income from the tourism sector increased by 4.3% in 2019 compared to
2018. In 2020 there was a decline in revenue from the tourism sector by 50.5% compared to
2019. In 2021 it decreased by 6.9% compared to year 2020 (Figure 7). Jawa Tengah Province's income from the tourism sector increased by 4.3% in 2019 compared to
2018. In 2020 there was a decline in revenue from the tourism sector by 50.5% compared to
2019. In 2021 it decreased by 6.9% compared to year 2020 (Figure 7). Figure 7: Jawa Tengah Tourism Sector Revenue 2018-2021
301,62
314,73
155,81
145,07
-
50,00
100,00
150,00
200,00
250,00
300,00
350,00
2018
2019
2020
2021 Figure 7: Jawa Tengah Tourism Sector Revenue 2018- The distribution of tourism sector income to GRDP can be seen in Figure 8. 3.3. Conditions Before and During the Covid-19 Pandemic The Covid-19 pandemic has changed the way people live around the world. Under normal
conditions, every time you meet other people you always give greetings by shaking hands or
hugging, but during a pandemic this is a prohibition. During the pandemic everyone is required to
wear a mask that covers the mouth and nose. Movement outside the home of every human being
is very limited. Changes in the pattern of life greatly affect the condition of the world economy. Similarly, economic conditions in Jawa Tengah, several economic indicators have changed
significantly. Table 1 shows some of the tourism economic indicators that have been affected by the
Covid-19 pandemic. The number of foreign tourist visits to Jawa Tengah Province during the
pandemic decreased significantly. The mean foreign tourist visit before the pandemic was
684,433.50 people, while during the pandemic it was only 40,041.50 people per year. The number
of domestic tourists also experienced a significant decline during the pandemic. Before the
pandemic, the mean number of domestic tourists was 53,422,235 people, while during the
pandemic it was 21,980,747 people per year. 48 | P a g e Entrepreneurship and Small Business Research
Volume 1, Nomor 3, p.44 - 52 Entrepreneurship and Small Business Research
Volume 1, Nomor 3, p.44 - 52 Table 1. Results for Compare Means Before and During Covid-19 Pandemic
Variables
Condition
Mean
Sig. Foreign Visitors
Before the pandemic
684.433,50
,004
During the pandemic
40.041,50
Domestic Visitors
Before the pandemic
53.422.235,00
,020
During the pandemic
21.980.747,00
Hotel
room
occupancy
rate
Before the pandemic
45,97
,009
During the pandemic
30,36
Foreigners at Star Hotel
Before the pandemic
2,0068
,811
During the pandemic
2,1100
Foreigners at Non-Star Hotel
Before the pandemic
2,0618
,350
During the pandemic
2,9300
Indonesian at Star Hotel
Before the pandemic
1,3181
,762
During the pandemic
1,3300
Indonesian at Non-Star Hotel
Before the pandemic
1,2131
,382
During the pandemic
1,0650
Tourism Income (Billion)
Before the pandemic
308,1741
,003
During the pandemic
150,4397
PDRB (Billion IDR)
Before the pandemic
1.314.610,65
,358
During the pandemic
1.384.361,30
Contribution (%)
Before the pandemic
,023454
,004
During the pandemic
,010885 Table 1. Results for Compare Means Before and During Covid-19 Pandemic The hotel room occupancy rate in conditions before the Covid-19 pandemic was 45.97%. During the Covid-19 pandemic, the hotel room occupancy rate decreased by 34% when compared
to conditions before the pandemic. The hotel room occupancy rate during the pandemic was
30.36%. 3.3. Conditions Before and During the Covid-19 Pandemic The means length of stay of foreign tourists in star and non-star hotels has increased but
the increase is not significant, which is between 2 to 2.9 days per visit. Likewise with domestic
tourists, the stay at star hotels has not increased significantly, which is between 1.31 to 1.33
days per visit. However, the means stay of domestic tourists in non-star hotels has decreased,
although not significantly, between 1.21 to 1.06 days per visit. g
g
y
y
The average income of Jawa Tengah Province from the tourism sector before the pandemic
was IDR 308.17 billion, while during the pandemic it was IDR 150.44 billion. Revenue from the
tourism sector has decreased significantly during the Covid-19 pandemic. The average regional
income of Jawa Tengah Province (GRDP) has increased during the pandemic, although not
significantly. The average GRDP before the pandemic was IDR 1,314,610.65 billion, while during
the pandemic it increased to IDR 1,384,361.30 billion. In terms of the contribution of tourism
sector income to GRDP, during the pandemic there was a significant decline. 4. Discussion The Covid-19 Pandemic period began in 2020 until 2021, even now (in 2021) it is still in the
process of recovery. The Covid-19 pandemic has had a huge impact on human civilization
worldwide. The impact is felt by the Province of Jawa Tengah. Foreign tourist visits to tourism
destinations in Jawa Tengah during the Covid-19 pandemic (2020-2021) experienced a very
drastic decline compared to before the pandemic, which was more than 90%. This is due to
various Indonesian government rules and policies which were handed down to Jawa Tengah
Provincial regulations related to securing public health due to the Covid-19 Pandemic. Social
movement of the community is very limited. All trips abroad and from abroad are closed gradually
according to the health conditions of the world community. The number of domestic tourists has
decreased by around 56.4% from 2018 to 2021. y
The decline in the number of tourists was followed by a decrease in hotel room occupancy
rates throughout Jawa Tengah. From 2018 to 2021 the hotel room occupancy rate decreased by 49 | P a g e | P a g e 49 | P a 49 Entrepreneurship and Small Business Research
Volume 1, Nomor 3, p.44 - 52 Entrepreneurship and Small Business Research
Volume 1, Nomor 3, p.44 - 52 37%. The decline in the hotel room occupancy rate is smaller than the decrease in the number of
tourists. This is because during the Covid-19 pandemic, some hotels offer services for self-
quarantine (Dogru et al., 2023; Leroi-Werelds et al., 2021). 37%. The decline in the hotel room occupancy rate is smaller than the decrease in the number of
tourists. This is because during the Covid-19 pandemic, some hotels offer services for self-
quarantine (Dogru et al., 2023; Leroi-Werelds et al., 2021). q
(
g
,
;
,
)
One of the critical things that supports tourism development is lodging facilities (Liu et al.,
2022). Management strategy plays an important role in responding to fluctuating hotel demand
during the pandemic transition to recovery (Salem et al., 2021; Wang et al., 2020). In 2019 there
was an increase in the hotel room occupancy rate in the Jawa Tengah Province by 0.63%
compared to 2018. In 2020 the hotel room occupancy rate decreased by 37.45%. 4. Discussion In 2021 the
hotel room occupancy rate will increase again by 10.51% However, there is a difference in
demand patterns between hotels with economic rates and hotels with luxury facilities (Ozdemir et
al., 2021). Hotel demand is likely to fluctuate while surging throughout recovery, operators must
continually adjust their capacity management strategies to address evolving demand. Including
strategies in providing transportation services in tourist destinations (Domènech et al., 2022). The
decline in the number of tourists was not directly proportional to the means length of stay of
tourists in hotels, both star and non-star hotels. Although not significant, the length of stay of
tourists in hotels has increased. This shows that the average length of stay of tourists to Jawa
Tengah Province is not affected by pandemic conditions. The GRDP of Jawa Tengah Province increased by more than 12% from 2018 to 2021. When
compared between conditions before and during the Covid-19 pandemic, the increase that
occurred was still not a significant increase. This is supported by the value of the contribution of
income from the tourism sector which has not been significant to the value of GRDP. Revenue
from the tourism sector has decreased significantly but this has no effect on the GRDP of Jawa
Tengah Province. 5. Conclusion Before the Covid-19 pandemic hit the world, tourism conditions in Jawa Tengah Province
experienced an increase, both in terms of the number of visitors, contribution to GDP, hotel
occupancy rates, and so on. However, during the Covid-19 pandemic, the situation changed
significantly. The number of foreign and domestic tourists decreased. Hotel room occupancy rates
have decreased. However, the average length of stay of tourists remains relatively unchanged. Revenue from the tourism sector has decreased significantly. On the other hand, the GRDP of
Jawa Tengah Province has increased during the Covid-19 pandemic. Likewise, the contribution of
tourism sector income to GRDP has decreased by more than 50%. This means that the Covid-19
pandemic has significantly affected the tourism sector in the Jawa Tengah Province. (Switzerland), 13(14). https://doi.org/10.3390/su13147610 (Switzerland), 13(14). https://doi.org/10.3390/su13147610 Chen, C.-M., Jih, C.-Y., Lin, Y.-C., & Hung, W.-H. (2022). The impact of border control policy on
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Volume 1, Nomor 3, p.44 - 52 (Switzerland), 13(14). https://doi.org/10.3390/su13147610 https://doi.org/10.1016/j.futures.2022.103013 Okafor, L., Khalid, U., & Gopalan, S. (2022). COVID-19 economic policy response, resilience and
tourism recovery. Annals of Tourism Research Empirical Insights, 3(2), 100073. https://doi.org/10.1016/j.annale.2022.100073 Ozdemir, O., Dogru, T., Kizildag, M., Mody, M., & Suess, C. (2021). Quantifying the economic
impact of COVID-19 on the U.S. hotel industry: Examination of hotel segments and
operational structures. Tourism Management Perspectives, 39(November 2020), 100864. https://doi.org/10.1016/j.tmp.2021.100864 Pham, T., & Nugroho, A. (2022). Tourism-induced poverty impacts of COVID-19 in Ind 51 | P a g e
Pham, T., & Nugroho, A. (2022). Tourism-induced poverty impacts of COVID-19 in Indonesia. Entrepreneurship and Small Business Research
Volume 1, Nomor 3, p.44 - 52 Annals of Tourism Research Empirical Insights, 3(2), 100069. https://doi.org/10.1016/j.annale.2022.100069 Annals of Tourism Research Empirical Insights, 3(2), 100069. https://doi.org/10.1016/j.annale.2022.100069 Annals of Tourism Research Empirical Insights, 3(2), 100069.
https://doi.org/10.1016/j.annale.2022.100069 Rafidola Mareta Riesa, & Haries, A. (2022). Investigation of people familiarity towards virtual
tourism as an alternative to travel. Economics, Business, Accounting & Society Review, 1(3),
179–185. https://doi.org/10.55980/ebasr.v1i3.30 Rhama, B. (2022). Local communities’ and tourists’ adaptation to pandemic-induced social
disruption: Comparing national parks and urban destinations. International Journal of
Disaster Risk Reduction, 82(February), 103380. https://doi.org/10.1016/j.ijdrr.2022.103380 Salem, I. E., Elbaz, A. M., Elkhwesky, Z., & Ghazi, K. M. (2021). The COVID-19 pandemic: The
mitigating role of government and hotel support of hotel employees in Egypt. Tourism
Management, 85(June 2020), 104305. https://doi.org/10.1016/j.tourman.2021.104305 Uğur, N. G., & Akbıyık, A. (2020). Impacts of COVID-19 on global tourism industry: A cross-
regional comparison. Tourism Management Perspectives, 36(January), 100744. https://doi.org/10.1016/j.tmp.2020.100744 Wang, X., Guchait, P., & Pasamehmetoglu, A. (2020). Anxiety and gratitude toward the
organization: Relationships with error management culture and service recovery
performance. International Journal of Hospitality Management, 89(June), 102592. https://doi.org/10.1016/j.ijhm.2020.102592 Zhao, L., & Xia, X. (2020). Tourism and poverty reduction: Empirical evidence from China. Tourism Economics, 26(2), 233–256. https://doi.org/10.1177/1354816619863266 52 | P a g e
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Shelley's Defences of Poetry
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1 1 1 Shelley’s Defences of Poetry1 Shelley’s Defences of Poetry1 Michael O’Neill
Durham University Michael O’Neill The Poet of Alastor (1816), Shelley’s enigmatic poem of driven and disappointed
quest, might illustrate the dangers of what in the poem’s Preface is described as “self-centred
seclusion” (92). But the poem’s narrative form prevents any simply moralistic reading from
enjoying un-interrogated sway. Told by a Narrator, who expresses deep admiration for the
Poet as a “surpassing Spirit” (714), and whose unsatisfied longing for communion with
nature tallies with the Poet’s failed attempt to find an embodied form of the “veilèd maid”
(151) of a particularly vivid dream, Alastor ricochets between unconvinced acceptance of
“Nature’s vast frame” (719) and despairing longing for something beyond “Art and
eloquence, / And all the shows ’o the world” (710-11). It comes to a close without closing
off an openness to all that resists final closure. appeal. The Poet of Alastor (1816), Shelley’s enigmatic poem of driven and disappointed
quest, might illustrate the dangers of what in the poem’s Preface is described as “self-centred
seclusion” (92). But the poem’s narrative form prevents any simply moralistic reading from
enjoying un-interrogated sway. Told by a Narrator, who expresses deep admiration for the
Poet as a “surpassing Spirit” (714), and whose unsatisfied longing for communion with
nature tallies with the Poet’s failed attempt to find an embodied form of the “veilèd maid”
(151) of a particularly vivid dream, Alastor ricochets between unconvinced acceptance of
“Nature’s vast frame” (719) and despairing longing for something beyond “Art and
eloquence, / And all the shows ’o the world” (710-11). It comes to a close without closing
off an openness to all that resists final closure. As in many of Shelley’s poems, the ambiguities of Alastor owe much to Shelley’s complex
response to a precursor poet, in this case Wordsworth, whose solemnly melodious blank
verse and themes of solitude and relationship with nature in The Excursion and Tintern Abbey
provide the frame within and against which the younger poet works. Shelley’s dealings with
Wordsworth are not merely antagonistic. He may question aspects of the older poet’s creed
and perceived ideology. Yet Shelley’s poetic remodelling implies the importance of
Wordsworth’s mode of vision. Wordsworth is probing central questions, even if his answers
do not compel Shelley’s assent. Michael O’Neill Michael O’Neill Percy Bysshe Shelley (1792-1822) was a poet who possessed, in his own words, “the power
of communicating and receiving intense and impassioned conceptions respecting man and
nature.” 2 Yet the greatness of his poetry, this essay will argue, does not essentially reside in
his capacity to articulate his strong libertarian beliefs. These beliefs may be the ground of
his conscious intellectual being. They show the influence of many thinkers, including that
enshrined in the Enquiry Concerning Political Justice (1793), written by his father-in-law,
William Godwin. But the supposition that Shelley uses poetry as the vehicle for the
endorsement of a system of ideas is fundamentally erroneous, as he himself argues in two
important places for understanding his poetics: the Preface to Prometheus Unbound, where
he asserts that “Didactic poetry is my abhorrence” (232) and A Defence of Poetry, where he
develops a sophisticated theory of poetry’s primary appeal to the imagination and argues that
“A Poet ... would do ill to embody his own conceptions of right and wrong, which are usually
those of his place and time, in his poetical creations, which participate in neither” (682). William Godwin. But the supposition that Shelley uses poetry as the vehicle for the
endorsement of a system of ideas is fundamentally erroneous, as he himself argues in two
important places for understanding his poetics: the Preface to Prometheus Unbound, where
he asserts that “Didactic poetry is my abhorrence” (232) and A Defence of Poetry, where he
develops a sophisticated theory of poetry’s primary appeal to the imagination and argues that
“A Poet ... would do ill to embody his own conceptions of right and wrong, which are usually
those of his place and time, in his poetical creations, which participate in neither” (682). Shelley’s importance and achievement as a poet derive from the way in which he tests,
dramatises, anatomizes and enacts the processes involved in belief or, indeed, doubt. He
turns out, surprisingly given the terms of his reputation as a poet hurrying always to exalt
principles of liberty, love, and equality, to be one of the major exemplars of Keats’s ideal of
“Negative Capability” (Keats Letters 1. 193). Shelley is often prepared to open his poetry to
differing interpretations, to allow the reader’s mind to be the final courtroom of the poetry’s 2 appeal. Michael O’Neill The “Hymn” creates a lyric form that in its longer and shorter lines, and subtle rhyming,
mimes the coming and going of the “Power” and the waxing and waning of the poet’s
confidence. It plays its own variations on Wordsworth’s theme of visionary loss and
subsequent recovery in his “Ode: Intimations of Immortality.” Whereas Wordsworth writes a
poetry of plangent lament, Shelley, as he switches in his third stanza, say, from deriding
orthodox “Frail spells” to launching his own “Frail spell” in the direction of “Thy light
alone” (29, 32) displays a beautifully unprotected abruptness. The unadvertised swiftness of
movement from scepticism to self-generated faith is typical of the drama going on in and
animating his poetry. In “Mont Blanc” (version A), he again brings Wordsworth to mind, and
“The everlasting universe of things” (1) that “Rolls through the mind” seems to participate in
the “motion and a spirit” (102) that “rolls through all things” (103) in Tintern Abbey (quoted
from Gill (ed.)). But if Wordsworth celebrates “a sense sublime / Of something far more
deeply interfused” (96-7), Shelley offers a warier, more sceptical view.. And yet at the
poem’s close, responding to his own more deeply sustained sense of the mind’s power
(shown in the echoes of Coleridge’s “Kubla Khan” at the close of section 4, echoes that offset
the grimmer naturalism of preceding lines), Shelley suggests the dependence of any sense of
sense on “the human mind’s imaginings” (143). The “Hymn” creates a lyric form that in its longer and shorter lines, and subtle rhyming,
mimes the coming and going of the “Power” and the waxing and waning of the poet’s
confidence. It plays its own variations on Wordsworth’s theme of visionary loss and
subsequent recovery in his “Ode: Intimations of Immortality.” Whereas Wordsworth writes a
poetry of plangent lament, Shelley, as he switches in his third stanza, say, from deriding
orthodox “Frail spells” to launching his own “Frail spell” in the direction of “Thy light
alone” (29, 32) displays a beautifully unprotected abruptness. The unadvertised swiftness of
movement from scepticism to self-generated faith is typical of the drama going on in and
animating his poetry. Michael O’Neill Wordsworth is the precursor brought to mind yet redefined
in two major shorter poems written the year Alastor was published: “Hymn to Intellectual
Beauty” and “Mont Blanc.” In the former, Shelley is at once the radical atheist of The
Necessity of Atheism (1811) and the Notes to the early Queen Mab (1813) who contends that
“Every reflecting mind must acknowledge that there is no proof of the existence of a Deity”
(81) and the visionary individualist who redefines “God” in his unfinished On Christianity. There, Shelley reprises a central conviction of the “Hymn to Intellectual Beauty” when he As in many of Shelley’s poems, the ambiguities of Alastor owe much to Shelley’s complex
response to a precursor poet, in this case Wordsworth, whose solemnly melodious blank
verse and themes of solitude and relationship with nature in The Excursion and Tintern Abbey
provide the frame within and against which the younger poet works. Shelley’s dealings with
Wordsworth are not merely antagonistic. He may question aspects of the older poet’s creed and perceived ideology. Yet Shelley’s poetic remodelling implies the importance of
Wordsworth’s mode of vision. Wordsworth is probing central questions, even if his answers
do not compel Shelley’s assent. Wordsworth is the precursor brought to mind yet redefined
in two major shorter poems written the year Alastor was published: “Hymn to Intellectual
Beauty” and “Mont Blanc.” In the former, Shelley is at once the radical atheist of The
Necessity of Atheism (1811) and the Notes to the early Queen Mab (1813) who contends that
“Every reflecting mind must acknowledge that there is no proof of the existence of a Deity”
(81) and the visionary individualist who redefines “God” in his unfinished On Christianity. There, Shelley reprises a central conviction of the “Hymn to Intellectual Beauty” when he 3 writes: “There is a power by which we are surrounded, like the atmosphere in which some
motionless lyre is suspended, which visits with its breath our silent chords, at will” (Shelley
Prose 251). If “intellectual beauty” is experienced within the mind, its ultimate source may
be intra- or extra-human (“visits” and “power” lend support to the latter possibility), and is
certainly beyond what in A Defence of Poetry Shelley calls “the determination of the will”
(696). Michael O’Neill He is a poet of desire, of the longing for change, for “some world far from
ours” (“To Jane [‘The keen stars were twinkling’]” 21-22) that is our world redeemed. But he
is also a poet who writes compellingly about all that thwarts desire. He is to his fingertips a
poet of criss-crossing perspectives; if his poetry “enlarges the circumference of the
imagination by replenishing it with thoughts of ever new delight” (Defence of Poetry 682), it
continually attunes itself to what in the Preface to The Cenci he calls “sad reality” (314). Shelley is a poet of emotional and conceptual extremes conveyed in verse of great distinction,
force and subtlety. He is a poet of desire, of the longing for change, for “some world far from
ours” (“To Jane [‘The keen stars were twinkling’]” 21-22) that is our world redeemed. But he
is also a poet who writes compellingly about all that thwarts desire. He is to his fingertips a
poet of criss-crossing perspectives; if his poetry “enlarges the circumference of the
imagination by replenishing it with thoughts of ever new delight” (Defence of Poetry 682), it
continually attunes itself to what in the Preface to The Cenci he calls “sad reality” (314). “Two Spirits: An Allegory,” for all (or possibly because of) its editorial quandaries, is a
quintessential Shelleyan lyric of divided impulses. The poem exists only as a rough draft with
multiple cancellations and unclear indications in places of final or near-final choices, but
what is manifest is that it has found a precise lyric configuration of its own, in its stanzaic
structure, rhymes, and images. Although the poem dramatizes the clash between caution and
idealistic desire, it immediately complicates both positions; the First Spirit warns but sees the
lure of desire; the second persists in desire, but sees the dangers he is facing. The poem sets
exalted terror against exhilarated commitment to “the flood of the tempest dark” (26), and
arrives, in its coda, at a fine balance between suggestions of loss and recovery. It ends with a
“traveller” (43) who “awakes on the fragrant grass” (47) and “finds night day” (48), just
tilting the poem towards a residual trust in the heart’s best hopes, even as it characteristically
puts the reader on the spot, implicitly asking him or her why ”day” and “night” should be
invested with stable and hierarchical symbolic significances. Michael O’Neill In “Mont Blanc” (version A), he again brings Wordsworth to mind, and
“The everlasting universe of things” (1) that “Rolls through the mind” seems to participate in
the “motion and a spirit” (102) that “rolls through all things” (103) in Tintern Abbey (quoted
from Gill (ed.)). But if Wordsworth celebrates “a sense sublime / Of something far more
deeply interfused” (96-7), Shelley offers a warier, more sceptical view.. And yet at the
poem’s close, responding to his own more deeply sustained sense of the mind’s power
(shown in the echoes of Coleridge’s “Kubla Khan” at the close of section 4, echoes that offset
the grimmer naturalism of preceding lines), Shelley suggests the dependence of any sense of
sense on “the human mind’s imaginings” (143). 4 Shelley is a poet of emotional and conceptual extremes conveyed in verse of great distinction,
force and subtlety. He is a poet of desire, of the longing for change, for “some world far from
ours” (“To Jane [‘The keen stars were twinkling’]” 21-22) that is our world redeemed. But he
is also a poet who writes compellingly about all that thwarts desire. He is to his fingertips a
poet of criss-crossing perspectives; if his poetry “enlarges the circumference of the
imagination by replenishing it with thoughts of ever new delight” (Defence of Poetry 682), it
continually attunes itself to what in the Preface to The Cenci he calls “sad reality” (314). “Two Spirits: An Allegory,” for all (or possibly because of) its editorial quandaries, is a
quintessential Shelleyan lyric of divided impulses. The poem exists only as a rough draft with
multiple cancellations and unclear indications in places of final or near-final choices, but
what is manifest is that it has found a precise lyric configuration of its own, in its stanzaic
structure, rhymes, and images. Although the poem dramatizes the clash between caution and
idealistic desire, it immediately complicates both positions; the First Spirit warns but sees the
lure of desire; the second persists in desire, but sees the dangers he is facing. The poem sets
exalted terror against exhilarated commitment to “the flood of the tempest dark” (26), and
arrives, in its coda, at a fine balance between suggestions of loss and recovery. Michael O’Neill It ends with a
“traveller” (43) who “awakes on the fragrant grass” (47) and “finds night day” (48), just
tilting the poem towards a residual trust in the heart’s best hopes, even as it characteristically
puts the reader on the spot, implicitly asking him or her why ”day” and “night” should be
invested with stable and hierarchical symbolic significances. Shelley is a poet of emotional and conceptual extremes conveyed in verse of great distinction,
force and subtlety. He is a poet of desire, of the longing for change, for “some world far from
ours” (“To Jane [‘The keen stars were twinkling’]” 21-22) that is our world redeemed. But he
is also a poet who writes compellingly about all that thwarts desire. He is to his fingertips a
poet of criss-crossing perspectives; if his poetry “enlarges the circumference of the
imagination by replenishing it with thoughts of ever new delight” (Defence of Poetry 682), it
continually attunes itself to what in the Preface to The Cenci he calls “sad reality” (314). “Two Spirits: An Allegory,” for all (or possibly because of) its editorial quandaries, is a
quintessential Shelleyan lyric of divided impulses. The poem exists only as a rough draft with
multiple cancellations and unclear indications in places of final or near-final choices, but
what is manifest is that it has found a precise lyric configuration of its own, in its stanzaic
structure, rhymes, and images. Although the poem dramatizes the clash between caution and
idealistic desire, it immediately complicates both positions; the First Spirit warns but sees the
lure of desire; the second persists in desire, but sees the dangers he is facing. The poem sets
exalted terror against exhilarated commitment to “the flood of the tempest dark” (26), and
arrives, in its coda, at a fine balance between suggestions of loss and recovery. It ends with a
“traveller” (43) who “awakes on the fragrant grass” (47) and “finds night day” (48), just
tilting the poem towards a residual trust in the heart’s best hopes, even as it characteristically
puts the reader on the spot, implicitly asking him or her why ”day” and “night” should be
invested with stable and hierarchical symbolic significances. Shelley is a poet of emotional and conceptual extremes conveyed in verse of great distinction,
force and subtlety. Michael O’Neill Something comparable occurs at the close of “Ode to the West Wind,” Shelley’s major short
poem. There, Shelley concludes his wrestling with the angel of the wind, at once verbally
embodied “breath of Autumn’s being” (2), and image of revolutionary inspiration, by
shaping an apparently rhetorical question out of the final couplet of the five that turn 5 5 headlong terza rima invocations and pleadings into vertiginous sonnets. Apparently
rhetorical, since Shelley prompts the reader to ask why there is any reason other than human
desire that the spiritual, poetic, and political should model their processes of change on those
evident in the cycles of the seasons. The compulsion to honour the drive towards betterment
impels the poem through its progressive uncovering of the poet’s “sore need” (52), a need for
the wind’s revivifying power to re-animate his words. In the last section, the poet rises,
phoenix-like, out of the ashes of his sprawled abasement at the hand of chastising experience,
experience that forces him to exclaim, “I fall upon the thorns of life! I bleed!” (54). True to
the reversals and shifts that make the ode a record of shaped but inwardly conflicted struggle
rather than a polemical exhortation, Shelley, in this last section, pleads with the wind in such
a way that plea becomes impassioned command. He cries: “Drive my dead thoughts over
the universe / Like withered leaves to quicken a new birth” (63-4). The lines concoct their
own, very Shelleyan blend of pathos and power, out of their ability to combine assertion,
evident in the strong stress on “Drive,” with awareness of failure, seen in the reference to
“dead thoughts,” and hope for the future, shown in the reference to the emphasized “new
birth.” headlong terza rima invocations and pleadings into vertiginous sonnets. Apparently
rhetorical, since Shelley prompts the reader to ask why there is any reason other than human
desire that the spiritual, poetic, and political should model their processes of change on those
evident in the cycles of the seasons. The compulsion to honour the drive towards betterment
impels the poem through its progressive uncovering of the poet’s “sore need” (52), a need for
the wind’s revivifying power to re-animate his words. Michael O’Neill In the last section, the poet rises, phoenix-like, out of the ashes of his sprawled abasement at the hand of chastising experience,
experience that forces him to exclaim, “I fall upon the thorns of life! I bleed!” (54). True to
the reversals and shifts that make the ode a record of shaped but inwardly conflicted struggle
rather than a polemical exhortation, Shelley, in this last section, pleads with the wind in such
a way that plea becomes impassioned command. He cries: “Drive my dead thoughts over
the universe / Like withered leaves to quicken a new birth” (63-4). The lines concoct their
own, very Shelleyan blend of pathos and power, out of their ability to combine assertion,
evident in the strong stress on “Drive,” with awareness of failure, seen in the reference to
“dead thoughts,” and hope for the future, shown in the reference to the emphasized “new
birth.” “Ode to the West Wind” wishes to “Scatter” (66) the poet’s “words among mankind” (67),
literally to disseminate them, “Ashes and sparks” (67) that will, the poem hopes, have the
effect that, in A Defence of Poetry¸ Shelley ascribes to Dante’s work and all “high poetry”: a
capacity to serve as prompts to “inextinguishable thought” (693). This sense of poetry as
working on the reader’s imagination is the spring of Shelley’s poetic practice in Prometheus
Unbound. It is not enough, for example, for us to dismiss Jupiter as “the tyrant of the world”
(3. 4. 183); through Prometheus’ double-signifying “recalling” of his curse, we have to
recognise how tyranny takes two to dance its savage tango. The very hatred and contempt 6 which Prometheus expresses towards Jupiter establish the two figures as caught up in a
strangely twinned alliance, from which Prometheus can only break once he realizes the
psychodynamics of his dependence on hatred. At the same time, enslaving as hatred can be,
its expression is also a necessary first step in the eventual liberation imagined in the lyrical
drama. As it moves beyond the rocky dungeon of the Caucasus, Prometheus Unbound
continually appeals to the reader’s desire to imagine the new, the transfigured, the yet to be. It does so through lyrical measures that appeal to yet brilliantly frustrate and redirect the
senses into “thought’s wildernesses” (1. 742), as in the “Life of Life!” (2. 5. Michael O’Neill 48) lyric chanted
by a “Voice in the Air” to the transformed Asia; through dialogic scenes, as when Asia
catechizes Demogorgon about the origins of evil in 2. 5; through verse whose negations
memorably allow the altered and the old to occupy the same poetic space, as when the Spirit
of the Hour proclaims that after the fall of Jupiter and the coming of a renovated world “None
wrought his lips in truth-entangling lines” (3. 4. 142); and through visions whose re-
organizing energy seems to mime the work of the imagination itself, as when Panthea in Act
4 depicts the earth as a “multitudinous Orb” (253) that “Grind the bright brook into an azure
mist / Of elemental subtlety, like light” (254-5). That “azure mist / Of elemental subtlety”
has a self-reflexive dimension and might describe the vision to which the poem tends. Yet,
even at the end, Demogorgon is at hand to advise of the need, should tyranny return, “to
hope, till Hope creates / From its own wreck the thing it contemplates” (4. 573-4); pushed
into the rhyme position, “creates” assumes a special, self-generating heroism. At the same time, in the roughly contemporaneous Julian and Maddalo, the Shelleyan which Prometheus expresses towards Jupiter establish the two figures as caught up in a
strangely twinned alliance, from which Prometheus can only break once he realizes the
psychodynamics of his dependence on hatred. At the same time, enslaving as hatred can be,
its expression is also a necessary first step in the eventual liberation imagined in the lyrical
drama. As it moves beyond the rocky dungeon of the Caucasus, Prometheus Unbound
continually appeals to the reader’s desire to imagine the new, the transfigured, the yet to be. It does so through lyrical measures that appeal to yet brilliantly frustrate and redirect the
senses into “thought’s wildernesses” (1. 742), as in the “Life of Life!” (2. 5. 48) lyric chanted
by a “Voice in the Air” to the transformed Asia; through dialogic scenes, as when Asia
catechizes Demogorgon about the origins of evil in 2. 5; through verse whose negations
memorably allow the altered and the old to occupy the same poetic space, as when the Spirit
of the Hour proclaims that after the fall of Jupiter and the coming of a renovated world “None
wrought his lips in truth-entangling lines” (3. 4. Michael O’Neill 142); and through visions whose re-
organizing energy seems to mime the work of the imagination itself, as when Panthea in Act
4 depicts the earth as a “multitudinous Orb” (253) that “Grind the bright brook into an azure
mist / Of elemental subtlety, like light” (254-5). That “azure mist / Of elemental subtlety”
has a self-reflexive dimension and might describe the vision to which the poem tends. Yet,
even at the end, Demogorgon is at hand to advise of the need, should tyranny return, “to
hope, till Hope creates / From its own wreck the thing it contemplates” (4. 573-4); pushed
into the rhyme position, “creates” assumes a special, self-generating heroism. At the same time, in the roughly contemporaneous Julian and Maddalo, the Shelleyan
surrogate Julian asserts, “Where is the love, beauty and truth we seek / But in our mind?”
(374-5), only to be told by his friend and intellectual adversary, the Byronic Maddalo, “You
talk Utopia” (179). The poem, subtitled “A Conversation,” uses its deftly modulating, 7 frequently enjambed couplets to create a poem of gripping interest. If we sway between
Julian’s optimism and Maddalo’s electrifyingly authoritative pessimism, we are also taken in
the depths of a third speaker’s mind, the Maniac. Torn between a desire to repress and speak
of emotional trauma, the Maniac speaks “Of the mind’s hell” (441). He does so in tones that
veer between near-paranoia and the confessionally anguished, even as he asserts that “to
myself I do not wholly owe / What now I suffer” (321-2). The very syntax of that phrasing
suggests the difficulty of understanding the self (and others). It is a difficulty enacted with
great humaneness by a poem that at its close places us uncomfortably close to “the cold
world” which “shall not know” (617), Julian decides, what happened between the Maniac and
his Lady. The libertarian hopefulness that is apparent in the Prometheus Unbound volume of 1820
concedes that hope’s major guarantor is hope itself, a state inseparable from self-aware
commitment to the imagination. In later poems Shelley gives greater prominence to the
potentially perilous nature of the imagination. Michael O’Neill The Triumph of Life, the poem on which he
was working at the time of his death by drowning, uses its Dantescan form, a dream-vision in
fluid terza rima, to reassess the gap between aspiration and reality, between what “glimmers”
as possibilities (see lines 33 and 431, and what appears to erase such possibilities. Different
lights blaze, shine, and gleam through the poem, now evoking the stars that give some form
of approach to hope, now suggesting the ordinary light of the sun that threatens to obliterate
star-light, and now describing the blinding glare cast by the Car (or chariot) in which the
ominous figure of Life sits. Over and over, history presents the poet-narrator with the
spectacle of good undoing itself; life overcomes virtually all who live with the exceptions, it
would seem, of Jesus and Socrates, exemplary figures who fled back to their “native noon”
(131). In the midst of this desolate vision, the poet-narrator meets Rousseau, kindler of 8 revolutionary flames, and author of idealising erotic fiction and anguished confession, and
through Rousseau’s narrative we are offered, in condensed symbolic form, a version of the
archetypal Romantic figure. Like Shelley and Byron, in their different ways, Rousseau has
“suffered what [he] wrote, or viler pain!” (279). Rousseau’s account of his encounter with
the “shape all light” (352) reruns yet looks freshly at Shelley’s career-long pursuit of ideals;
the shape’s muse-like quality suggests that whatever drives the poetic imagination may be
disturbingly ambivalent. Yet what the shape is or stands for we find hard finally to tell; what
we do know is what Rousseau experiences; and what he experiences, an experience in which
the shape seems to blot out his thoughts, yet vanishes until she becomes a yearned-for,
glimmering absence, he does not fully understand; and the poetry is able to evoke this
incomplete understanding with ruthlessly vigilant empathy. Shelley, then, is a poet who knows that many readers (and perhaps an aspect of himself)
would agree with Maddalo that he “talk[s] Utopia”: he makes poetic capital out of this
knowledge by allowing for alternative perspectives (as in Julian and Maddalo), or by coming
at his obsessions from unusual, re-invigorating angles. Michael O’Neill The Witch of Atlas, for instance, a
poem of prodigal, bewitchingly comic fantasy, almost sends up the typical Shelleyan
concern with a veiled ideal, when it describes the witch weaving a veil that will serve as “A
shadow for the splendour of her love” (152). Shelley is not a poet who writes the same poem
over and over; his poems are particular and differentiated, and subtly alert to their own mode
of being. So Prometheus Unbound explores its own particular world in Act I, when Earth
speaks of a shadow-world that includes “Dreams and the light imaginings of men, / And all
that faith creates or love desires” (200-01). That first line momentarily stands at an angle to
the lyrical drama, itself an embodiment of “Dreams and the light imaginings of men,” even as 9 9 the next line switches perspective, and mounts an implicitly strong defence of “All that faith
creates.” Nor would it be right to ignore the various plausible poets we can extract from the overall
oeuvre. There is Walter Bagehot’s Shelley, a poet of “peculiar removed essences of lyrical
rapture” (Bagehot, 4.121). There is the tough-minded, politically radical Shelley, idealistic,
yet aware of “the unbending realities of actual life” (A Philosophical View of Reform 664). And there is the deconstructive Shelley decentering centres, calling into question all essences. These Shelleys overlap. “The Cloud” combines playful lyric mythmaking with contemporary
scientific knowledge to produce a poem which comes at its big themes lightly. In the last
lines of “The Cloud”, “convex” (79) is right for an observer looking down at the earth’s
atmosphere; the point brings out the “peculiar removed” position of the cloud, a speaker who
stands apart from the human and yet who is at the same time turned by the jauntily anapaestic
verse into something like the elusive principle of life itself. The cloud is turned too, into a
symbol of libertarian hope, with its refusal to be squashed, and of the imagination, with its
capacity for transformation. In its light-hearted yet triumphant way “The Cloud” answers the
riddle of mutability which haunts the Prometheus Unbound volume. In that volume Shelley
comes up in “The Sensitive Plant” with the seemingly Platonic assertion that “For love, and
beauty, and delight, / There is no death nor change” (Conclusion, 21-22). Michael O’Neill “The Sensitive
Plant” has, by this stage, turned from enchanting if tremulous fable into vividly relished
nightmare; beauty has been destroyed in the third part, and Shelley’s response is to create
from its wreckage a knowingly fictive and “modest creed” (Conclusion 13) based in the fact
that “nothing is, but all things seem” (Conclusion 11). Riskily and affectingly he removes
himself, or appears to remove himself, from the world of process which in “The Cloud” he Nor would it be right to ignore the various plausible poets we can extract from the overall
oeuvre. There is Walter Bagehot’s Shelley, a poet of “peculiar removed essences of lyrical
rapture” (Bagehot, 4.121). There is the tough-minded, politically radical Shelley, idealistic,
yet aware of “the unbending realities of actual life” (A Philosophical View of Reform 664). And there is the deconstructive Shelley decentering centres, calling into question all essences. These Shelleys overlap. “The Cloud” combines playful lyric mythmaking with contemporary
scientific knowledge to produce a poem which comes at its big themes lightly. In the last
lines of “The Cloud”, “convex” (79) is right for an observer looking down at the earth’s
atmosphere; the point brings out the “peculiar removed” position of the cloud, a speaker who
stands apart from the human and yet who is at the same time turned by the jauntily anapaestic
verse into something like the elusive principle of life itself. The cloud is turned too, into a
symbol of libertarian hope, with its refusal to be squashed, and of the imagination, with its
capacity for transformation. In its light-hearted yet triumphant way “The Cloud” answers the
riddle of mutability which haunts the Prometheus Unbound volume. In that volume Shelley
comes up in “The Sensitive Plant” with the seemingly Platonic assertion that “For love, and
beauty, and delight, / There is no death nor change” (Conclusion, 21-22). “The Sensitive
Plant” has, by this stage, turned from enchanting if tremulous fable into vividly relished
nightmare; beauty has been destroyed in the third part, and Shelley’s response is to create
from its wreckage a knowingly fictive and “modest creed” (Conclusion 13) based in the fact
that “nothing is, but all things seem” (Conclusion 11). Riskily and affectingly he removes
himself, or appears to remove himself, from the world of process which in “The Cloud” he 10 10 celebrates. Michael O’Neill “I change but cannot die” (“The Cloud” 76) strikes a note which, for all its
playfulness, is at the centre of Shelley’s view of life and poetry. Yet the longing for a state of certainty beyond process is typical too, and out of that quarrel
will emerge much of the drama that animates the concluding section of Adonais, Shelley’s
elegy for Keats. In that poem, the desire to affirm that an imagined absolute (“The One”) is
superior to experienced process is seemingly strong, as when Shelley asserts that “The One
remains, the many change and pass; / Heaven’s light forever shines, Earth’s shadows fly; /
Life, like a dome of many-coloured glass, /Stains the white radiance of Eternity, / until Death
tramples it to fragments.” (460-4). In fact, the quotation reveals how Shelley moves beyond
and away from the tone of calm philosophical statement of the opening line. The famous
image brings with it fraught complexities, brought to a focus in the emphasis placed on the
pivotal verb “Stains.” If life disfigures “the white radiance of Eternity,” another meaning of
the verb acknowledges that life also enriches the radiance. Death may destroy the screen
between us and the white radiance, yet its trampling brutalism cannot but elicit a degree of
flinching; suddenly the “many-coloured dome” possesses a fragile beauty. In keeping with
this compelling dramatization of to-and-fro impulses, Shelley asks apparently rhetorical
questions that turn out not be rhetorical at all (see lines 459 and 469), and finally depicts
himself as questing after an absolute which he seems forever fated not to reach, as he is
“borne darkly, fearfully, afar” (492). In its final resurrection the cloud says, “I arise and unbuild it [i.e. the blue dome of air which
is its cenotaph] again” (“The Cloud” 84). “[U]nbuilding” is a crucial activity in Shelley’s
work, which decreates in order to recreate, and bears on the vexed and rich question of
Shelleyan belief. Should one, for instance, we see the Power of “Mont Blanc”, the One in 11 Adonais and, say, Liberty in “Ode to Liberty” as secularized mirror images of the God of
Christian theology? The answer must surely be “no” to the degree that Shelley’s affirmations
are acutely aware of their fictional status. Michael O’Neill Shelleyan answers are imaginatively created and
dynamically provisional, as when we are told how, “burning through the inmost veil of
Heaven, / The soul of Adonais, like a star, / Beacons from the abode where the Eternal are”
(Adonais 493-95). The poem may end with the word “are”, a word suggestive of being, but it
ends too with a typical hint of veils within veils; if the poems glimpses what it would be like
to abide with Adonais, it knows too that there are miles to go before it sleeps. When “Liberty” is invoked as “Thou heaven of earth!” (“Ode to Liberty” 166), the phrasing
uses religious language to enforce its humanist and historical view that Liberty exists only as
realized by human beings within particular societies. But Shelley’s humanism sees no reason
why it should deny itself the intensity of feeling often associated with religion. In
Epipsychidion Shelley borrows religious language to describe the idealized “Emily” in the
passage beginning “The glory of her being, issuing thence, / Stains the dead, blank, cold air
with a warm shade / Of unentangled intermixture, made / By Love, of light and motion” (91-
3), and proceeding to an intuition of “one intense / Diffusion, one serene Omnipresence”
(93-4). Throughout the poem, Shelley is aware, like T. S. Eliot’s Sweeney, that he has
“gotta use words” (Eliot 135) and he continually confronts the limits of expression. Yet here,
as elsewhere, his sense of language’s limitations frustrates much less than it enables his
poetry. He may have jotted, at the end of his essay “On Love”, that “These words inefficient
and metaphorical—Most words so—No help—” (Major Works 821, n. 632). Yet the fact that
words are “metaphorical” is one way he finds of freeing himself from their traditional
associations; so in the Epipsychidion passage, the process of redefinition works so
bewilderingly and yet purposefully that we have, in reading, to give up the notion that we can 12 identify Emily by seeing her as a solely a person or merely an idea (such as an incarnation of
Intellectual Beauty;]the way she is described, rather, illustrates Shelley’s point that the poetry
seeks to mark the “before unapprehended relations of things” (A Defence of Poetry 676). Michael O’Neill The
poetry involves the reader in the process of seeking to comprehend how Emily can be
“Scarce visible from extreme loveliness” (104), even as we are instructed to “See where she
stands!” (112), an instruction that demands we see through, not simply with, the eye. Or, as
Harold Bloom puts the matter memorably, “The problem is to describe a secularized
epiphany that cannot be described, but Shelley was a specialist in the indescribable” (Bloom
51). Indeed, as he presents “a mortal shape indued / With love and life and light and deity”
(112-13), Shelley even prompts to us to redefine our understanding of what a “secularized
epiphany” might be. All categories are under something close to assault through a use of
words whose micro-behaviours conduce towards erosion of separating barriers; so, here, the
assonance involved in the last three nouns persuades them to co-habit a newly existing space. identify Emily by seeing her as a solely a person or merely an idea (such as an incarnation of
Intellectual Beauty;]the way she is described, rather, illustrates Shelley’s point that the poetry
seeks to mark the “before unapprehended relations of things” (A Defence of Poetry 676). The
poetry involves the reader in the process of seeking to comprehend how Emily can be identify Emily by seeing her as a solely a person or merely an idea (such as an incarnation of
Intellectual Beauty;]the way she is described, rather, illustrates Shelley’s point that the poetry
seeks to mark the “before unapprehended relations of things” (A Defence of Poetry 676). The
poetry involves the reader in the process of seeking to comprehend how Emily can be
“Scarce visible from extreme loveliness” (104), even as we are instructed to “See where she
stands!” (112), an instruction that demands we see through, not simply with, the eye. Or, as
Harold Bloom puts the matter memorably, “The problem is to describe a secularized
epiphany that cannot be described, but Shelley was a specialist in the indescribable” (Bloom
51). Indeed, as he presents “a mortal shape indued / With love and life and light and deity”
(112-13), Shelley even prompts to us to redefine our understanding of what a “secularized
epiphany” might be. Michael O’Neill All categories are under something close to assault through a use of
words whose micro-behaviours conduce towards erosion of separating barriers; so, here, the
assonance involved in the last three nouns persuades them to co-habit a newly existing space. Harold Bloom puts the matter memorably, The problem is to describe a secularized
epiphany that cannot be described, but Shelley was a specialist in the indescribable” (Bloom
51). Indeed, as he presents “a mortal shape indued / With love and life and light and deity”
(112-13), Shelley even prompts to us to redefine our understanding of what a “secularized
epiphany” might be. All categories are under something close to assault through a use of
words whose micro-behaviours conduce towards erosion of separating barriers; so, here, the
assonance involved in the last three nouns persuades them to co-habit a newly existing space. It is pointless for the critic to reproach Shelley, as F. R. Leavis did, for failing to possess a
firm grip on the actual since for Shelley the actual does not exist until defined in the light of
the mind’s sense of its potential (194). In Shelley’s greatest poetry his terms bear witness to
his attempt to redefine; so in the passage just referred to from Epipsychidion familiar
abstractions take on new life, coming together, briefly, into an original “intermixture,” which
both reflects “The glory of her being” and is only a staining of it. The passage sets going a
characteristic and energizing friction between the sense (just round the corner here: see 123
and following lines) that words are inadequate and that they can, at full stretch, just glimpse
what it is they wish to say, between the impulse to come to rest on some assured absolute
(here “that Beauty,” 102) and the swerving away from that, between the poetic self- 13 consciousnesss and the other-awareness which coalesce in a phrase such as “Scarce visible
from extreme loveliness” (104) which describes the poem and “The glory of her being.” Such an emphasis on self-aware fictionality makes Shelley a Romantic forerunner of Wallace
Stevens. Shelley is never so coolly dandified as the poet who writes, “The final belief is to
believe in a fiction, which you know to be a fiction, there being nothing else. The exquisite
truth is to know that it is a fiction and that you believe in it willingly” (Stevens 163). Michael O’Neill But the
poet who ends “Mont Blanc” with a question that lays the burden for making meaning on
“the human mind’s imaginings” (143), who at the end of “The Sensitive Plant” offers us an
extremely immodest assertion in the form of a modest creed we might find pleasant to
consider, who in mid-course in Adonais revises a previous stanza’s doubt to assert that the
dead Keats “is not dead, he doth not sleep” (343), who argues that the “desire to be for ever
as we are . . . is . . . the secret persuasion which has given birth to the opinion of a future
state” (133 rev) and who in a note to “Hellas” says that it is the poet’s province “to attach
himself to those ideas which exalt and ennoble humanity” (Major Works 585), has little to
learn from Stevens about fictiveness. Yet Shelley does not, for all his scepticism and self-
awareness about the workings of desire, abandon the idea that there is possibly a true state of
things whose light exceeds the might of our obscure organs. Thus, the close of Epipsychidion
leaves unresolved whether the failure of the poet’s quest to ascend “Into the height of love’s
rare Universe” (589) can be laid at the door of language’s inadequacy to do justice to the
concept of some ineffable union, or whether the notion of some such union can only ever be
asserted, never wholly and fully experienced. It is, however, for the daring and originality with which Shelley’s poems explore the
possibilities of poetry that they claim our admiration. Their generic experimentation is 14 central to this daring and originality. Shelley remodels many forms, producing in Hellas, for
instance, his second major example of a lyrical drama, which draws on Greek classical
practice (Aeschylus’s The Persians) to produce a poem that speaks to the immediate
historical present and future as it addresses the War of Greek Independence, and sets
contemporary political struggle in the context of a cyclical vision of historical and cosmic
change. Again, propaganda is held in abeyance. In the final choral lyric, Shelley sets a
cyclical view of history, according to which “The world’s great age begins anew” (1060),
against a “weariness” induced by all cycles, “The world is weary of the past,” the poem
finishes, “O might it die or rest at last!” (1100-1). Michael O’Neill Once again, a Shelleyan poem concludes
not in a state of final certitude, but on a more suspended note that awakens doubts and
uncertainties. Yes, the poem is partisan, but it sets the Greek struggle for liberty in the
context of an awareness that all struggle might or must involve “hate and death” (1096) and
that there may be an unbridgeable gulf between an immutable idea of Hellenic culture and its
value, one “based on the crystalline sea / Of thought and its eternity” (698-9), and the
actualities of contemporary conflict. In “Ozymandias,” Shelley uses the sonnet-form not to
monumentalize, but to declare the folly of all monuments, especially those built by tyrants in
their own vainglorious self-praise. The poem itself must take the place of the sculptured
wreck it interprets as an emblem of tyranny’s inevitable passing away, but how art is to avoid
complicity with power is also a theme of the poem. For one thing, the artist who “well those
passions read” (6) was able to insinuate criticism even as he ostensibly affirmed tyrannical
greatness. Thus art is always capable of freedom, even if the full realization of that freedom
may lie in the minds of later beholders. More disquietingly, though, is the implicit question
whether art’s creations are themselves subject to inevitable decay. Yet here, as he traces
democratizing, erasing vistas of empty “sands” (14), Shelley hints at the ongoing role for
poets, since their achievement is always in need of renewal, of rediscovery, and of recreation. 15 Cultural entropy is inevitable, we are told at the start of A Defence of Poetry, “and then, if no
new poets should arise to create afresh the associations which have been thus disorganized,
language will be dead to all the nobler purposes of human intercourse” (Major Works 676). The drama and dynamism which this essay has sought to trace in Shelley’s poetry finds its
eloquent apologia in these words by him. In turn, his multifaceted influence on poets as
diverse as Browning and T. S. Eliot bears witness to the fact that his work has gone on
stimulating new and diverse forms of creativity. Michael O’Neill Cultural entropy is inevitable, we are told at the start of A Defence of Poetry, “and then, if no
new poets should arise to create afresh the associations which have been thus disorganized,
language will be dead to all the nobler purposes of human intercourse” (Major Works 676). The drama and dynamism which this essay has sought to trace in Shelley’s poetry finds its
eloquent apologia in these words by him. In turn, his multifaceted influence on poets as
diverse as Browning and T. S. Eliot bears witness to the fact that his work has gone on
stimulating new and diverse forms of creativity. 1 This essay has been published as ‘Percy Bysshe Shelley, Poetry’ in The Encylopedia of Romantic Literature,
ed. Frederick Burwick (3 vols., Malden, MA: Wiley-Blackwell, 2012). The author would like to thank
Frederick Burwick, Marilyn Gaull, and Wiley-Blackwell for their agreement to reprint the essay in The
Wordsworth Circle. He would also like to acknowledge the late Jonathan Wordsworth for inviting him to speak
on a panel about Shelley at the Wordsworth Summer Conference in Grasmere in 1992; the essence of this
essay’s thinking about the “essential Shelley” was drafted for or thought about in connection with that panel. WORKS CITED Bagehot, Walter. The Collected Works of Walter Bagehot. Vol 4. Ed. Forrest Morgan and
Richard Holt Hutton. 5 vols. Routledge, 1995; Bloom, Harold, “Visionary Cinema of
Romantic Poetry,” The Ringers in the Tower: Studies in Romantic Tradition. The University
of Chicago Press, 1971; Eliot, T. S., Collected Poems. Faber and Faber, 1974; Jones, Steven
E. (ed.), The “Julian and Maddalo” Notebook. Vol. XV of The Bodleian Shelley
Manuscripts. Garland, 1990; Keats, John. The Letters of John Keats 1814-1821, ed. Hyder
Edward Rollins. 2 vols. Harvard University Press; Leavis, F. R, Revaluation: Tradition and
Development in English Poetry. 1936. Penguin, 1972; Shelley, Percy Bysshe, The Major
Works. Ed. Zachary Leader and Michael O’Neill. Oxford University Press, 2003, 2009 corr;
The Prose Works of Percy Bysshe Shelley. Vol. 1. Ed. E. B. Murray. Clarendon Press, 1993;
Stevens, Wallace, Opus Posthumous. Knopf, 1957; Wordsworth, William, The Major Works,
ed. Stephen Gill. Oxford University Press, 2000. 16 1 This essay has been published as ‘Percy Bysshe Shelley, Poetry’ in The Encylopedia of Romantic Literature,
ed. Frederick Burwick (3 vols., Malden, MA: Wiley-Blackwell, 2012). The author would like to thank
Frederick Burwick, Marilyn Gaull, and Wiley-Blackwell for their agreement to reprint the essay in The
Wordsworth Circle. He would also like to acknowledge the late Jonathan Wordsworth for inviting him to speak
on a panel about Shelley at the Wordsworth Summer Conference in Grasmere in 1992; the essence of this
essay’s thinking about the “essential Shelley” was drafted for or thought about in connection with that panel. 2 A Defence of Poetry, in Percy Bysshe Shelley: The Major Works. Ed. Zachary Leader and Michael O’Neill. Oxford: Oxford University Press, 2003, 2009 corr.. All quotations from Shelley, unless otherwise stated, are
from this volume. Page numbers for prose are supplied parenthetically.
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DISEMINASI INKLUSI KEUANGAN DAN PENGEMBANGAN KUALITAS SUMBER DAYA MANUSIA KOPERASI SMP 83
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Jurnal Pengabdian Dharma Laksana
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DISEMINASI INKLUSI KEUANGAN DAN PENGEMBANGAN
KUALITAS SUMBER DAYA MANUSIA KOPERASI SMP 83
1Arif Surahman, 2Agustina Mogi, 3Muhammad Yuda Alhabsyi,
4Eko Cahyadi, 5Hengki Hermawan
Dosen Fakultas Ekonomi Universitas Pamulang
Email : 1arifsurahman9@gmail.com 1Arif Surahman, 2Agustina Mogi, 3Muhammad Yuda Alhabsyi,
4Eko Cahyadi, 5Hengki Hermawan
Dosen Fakultas Ekonomi Universitas Pamulang
Email : 1arifsurahman9@gmail.com ABSTRAK Permasalahan utama yang dihadapi oleh Koperasi SMP 83 adalah sebagai
berikut : A. Koperasi menghadapi masalah publikasi dan eksistensi dalam
keragaman produk pembiayaan kepada anggotanya. B. Koperasi perlu lebih banyak
mengetahui mengenai inklusi keuangan. C. Koperasi memiliki mutu sumberdaya
yang rendah dalam mengelola pembiayaan yang telah diberikan. Beberapa langkah
yang ditempuh sebagai metode untuk melaksanakan solusi terhadap permasalahan
yang dihadapi yayasan akan dilakukan dengan beberapa hal berikut ini:1. Sosialisasi Program 2. Koordinasi 3. Pelaksanaan Program 4. Evaluasi Program. Hasil dari pengabdian kepada masyarakat ini adalah berubahnya pola pikir
dan wawasan dari peserta yang mengikuti penyuluhan sebagaimana dibuktikan
dengan adanya games dan challenge untuk menguji kemampuan dan wawasan
peserta PKM. P-ISSN : 2621-7155
E-ISSN : 2621-7147 P-ISSN : 2621-7155
E-ISSN : 2621-7147 P-ISSN : 2621-7155
E-ISSN : 2621-7147 Jurnal Pengabdian
Dharma Laksana Mengabdi Untuk Negeri Jurnal Pengabdian
Dharma Laksana Mengabdi Untuk Negeri PENDAHULUAN Untuk memiliki keunggulan dalam
pengelolaan kredit, maka para pengelola dana yang meminjam harus
merevolusionerkan caranya belajar dengan menggunakan metode belajar yang bisa
meningkatkan kemampuannya untuk mencerna informasi dengan baik. Oleh karena
itu, kami dari Dosen Universitas Pamulang bermaksud untuk melakukan
pengabdian masyarakat dengan judul "Diseminasi Inklusi Keuangan dan
Pengembangan Kualitas Sumber Daya Manusia Koperasi SMP 83."
Gambar 1. Presentasi p
p
p
Disisi lain, koperasi juga perlu untuk mengembangkan kualitas sumberdaya
anggotanya, supaya pinjaman-pinjaman yang macet dapat berkurang. Pengetahuan
didunia barat telah memodernisasi bagaimana cara seseorang untuk belajar. Seseorang yang bernama Tony Buzan telah
menemukan
cara
revolusioner
untuk
meningkatkan kemampuan belajar. Peningkatan
kualitas sumber daya manusia ini juga bagus
bagi reputasi kerjasama antara koperasi dengan
dunia perbankan. Sehingga apabila kualitas
kredit koperasi sudah bagus, maka bank tidak
akan berpikir dua kali untuk bekerjasama Gambar 1. Presentasi g
p
Kerjasama dengan perbankan akan
semakin
menguntungkan,
sehingga
uang
koperasi bisa ditaruh di bank yang melakukan
kerjasama dan para anggota koperasi bisa
langsung dipotong gajinya oleh bank untuk
dimasukkan kedalam rekening koperasi untuk
selanjutnya disalurkan dalam bentuk pendanaan kepada anggota koperasi. Pengetahuan tentang inklusi keuangan perlu didesiminasikan oleh dunia
akademisi, supaya semua orang paham dan bisa memperoleh pinjaman untuk
melakukan bisnis dan keluar dari kemiskinan. Untuk memiliki keunggulan dalam
pengelolaan kredit, maka para pengelola dana yang meminjam harus
merevolusionerkan caranya belajar dengan menggunakan metode belajar yang bisa
meningkatkan kemampuannya untuk mencerna informasi dengan baik. Oleh karena
itu, kami dari Dosen Universitas Pamulang bermaksud untuk melakukan
pengabdian masyarakat dengan judul "Diseminasi Inklusi Keuangan dan
Pengembangan Kualitas Sumber Daya Manusia Koperasi SMP 83."
Gambar 1. Presentasi Kerjasama dengan perbankan akan
semakin
menguntungkan,
sehingga
uang
koperasi bisa ditaruh di bank yang melakukan
kerjasama dan para anggota koperasi bisa
langsung dipotong gajinya oleh bank untuk
dimasukkan kedalam rekening koperasi untuk
selanjutnya disalurkan dalam bentuk pendanaan kepada anggota koperasi. Gambar 1. Presentasi Gambar 1. Presentasi Pengetahuan tentang inklusi keuangan perlu didesiminasikan oleh dunia
akademisi, supaya semua orang paham dan bisa memperoleh pinjaman untuk
melakukan bisnis dan keluar dari kemiskinan. Untuk memiliki keunggulan dalam
pengelolaan kredit, maka para pengelola dana yang meminjam harus
merevolusionerkan caranya belajar dengan menggunakan metode belajar yang bisa
meningkatkan kemampuannya untuk mencerna informasi dengan baik. Oleh karena
itu, kami dari Dosen Universitas Pamulang bermaksud untuk melakukan
pengabdian masyarakat dengan judul "Diseminasi Inklusi Keuangan dan
Pengembangan Kualitas Sumber Daya Manusia Koperasi SMP 83." PENDAHULUAN Didalam Undang-Undang Dasar 1945 koperasi merupakan lembaga
perekonomian yang diamanatkan untuk menjadi kekuatan ekonomi masyarakat. Cita-cita luhur dari koperasi Indonesia adalah membebaskan masyarakat dari
jeratan hutang rentenir dan cita-cita ini terus menghadapi tantangan yang beragam
dari tahun ke tahun (Sitepu & Hasyim, 2018). Dunia perbankan dapat berkolaborasi
dengan koperasi sehingga terdapat win-win solution yang sama-sama
menguntungkan bagi kedua belah pihak. " Ketua Koperasi "Oryza Sativa" Dinas
Pertanian dan Kehutanan Kabupaten Bogor, Engkus Kusminar mengatakan
meskipun koperasi tidak memiliki agunan, terdapat dua gagasan yang perlu
dipertimbangkan oleh pihak bank dalam melihat peluang terjalinnya harmonisasi
kerjasama dengan koperasi. Pertama, bank bertindak sebagai executing berarti
pihak bank memberikan pinjaman kepada koperasi berupa saldo yang kemudian
kami pinjamkan ke anggota dengan sistem pemotongan gaji langsung untuk
membayarnya.Yang kedua, kata Engkus adalah dengan cara geneling, yang berarti
setiap anggota koperasi kepegawaian bisa secara langsung meminjam sejumlah
dana kepada bank seperti biasa namun pihak bank memberikan syarat menyertakan
keanggotaan koperasi dan pihak bank mengadakan bagi hasil dengan koperasi
(Susanti&Fajar, 2016)". Akibat dari sinergi ini, maka baik dunia perbankan maupun 41 Vol. 3, No. 1, Juli 2020 Dharma Laksana P-ISSN : 2621-7155
E-ISSN : 2621-7147 Jurnal Pengabdian
Dharma Laksana Mengabdi Untuk Negeri Jurnal Pengabdian
Dharma Laksana Mengabdi Untuk Negeri dunia koperasi dapat menghasilkan produk-produk pembiayaan dan tabungan yang
lebih bervariasi dan lebih inovatif sehingga sama-sama akan mengembangkan
kualitas produk dari koperasi dan kualitas produk dari bank. kualitas produk dari koperasi dan kualitas produk dari bank. Disisi lain, koperasi juga perlu untuk mengembangkan kualitas sumberdaya
anggotanya, supaya pinjaman-pinjaman yang macet dapat berkurang. Pengetahuan
didunia barat telah memodernisasi bagaimana cara seseorang untuk belajar. Seseorang yang bernama Tony Buzan telah
menemukan
cara
revolusioner
untuk
meningkatkan kemampuan belajar. Peningkatan
kualitas sumber daya manusia ini juga bagus
bagi reputasi kerjasama antara koperasi dengan
dunia perbankan. Sehingga apabila kualitas
kredit koperasi sudah bagus, maka bank tidak
akan berpikir dua kali untuk bekerjasama
dengan koperasi. Kerjasama dengan perbankan akan
semakin
menguntungkan,
sehingga
uang
koperasi bisa ditaruh di bank yang melakukan
kerjasama dan para anggota koperasi bisa
langsung dipotong gajinya oleh bank untuk
dimasukkan kedalam rekening koperasi untuk
selanjutnya disalurkan dalam bentuk pendanaan kepada anggota koperasi. Pengetahuan tentang inklusi keuangan perlu didesiminasikan oleh dunia
akademisi, supaya semua orang paham dan bisa memperoleh pinjaman untuk
melakukan bisnis dan keluar dari kemiskinan. Jurnal Pengabdian
Dharma Laksana Mengabdi Untuk Negeri METODE PELAKSANAAN KEGIATAN Metode pelaksanaan akan ditempuh dengan melakukan penyuluhan dan
pelatihan kepada anggota koperasi SMP 83. Bentuk partisipasi dari koperasi yang
diharapkan pada waktu kegiatan ini adalah penyediaan perlengkapan dan sarana
prasarana pendukukung yang dibutuhkan untuk melaksanakan kegiatan PKM. Setelah rangkaian acara kegiatan PKM dilakukan, maka akan dilakukan
proses evaluasi. Bentuk dari evaluasi yang akan dilakukan adalah dengan
pengunjungan berkala dan pelatihan yang lanjutan dalam bentuk Pengabdian
Kepada Masyarakat. Vol. 3, No. 1, Juli 2020 Dharma Laksana P-ISSN : 2621-7155
E-ISSN : 2621-7147 Jurnal Pengabdian
Dharma Laksana Mengabdi Untuk Negeri Jurnal Pengabdian Gambar 2. Foto Bersama UNPAM dengan SMP 83 Gambar 2. Foto Bersama UNPAM dengan SMP 83 Beberapa langkah yang ditempuh untuk melaksanakan solusi terhadap
permasalahan yang akan dilakukan adalah beberapa hal berikut ini : 1. Sosialisasi Program
Cara yang akan ditempuh untuk mensosialisasikan program penyuluhan adalah
dengan mengunjungi koperasi dan memberitahu bahwa akan ada pelatihan dan
penyuluhan berupa Pengabdian Kepada Masyarakat dari Dosen UNPAM. 2. Koordinasi
Koordinasi dilakukan antara panitia PKM Dosen UNPAM dengan Koperasi
SMP 83. Perihal yang dikoordinasikan adalah rancangan kegiatan yang
diajukan serta kesepakatan untuk pelaksanaan program. 3. Pelaksanaan Program
Program kegiatan PKM akan dilakukan di koperasi SMP 83 dengan cara
melakukan penyuluhan dan pelatihan. 4. Evaluasi
Evaluasi dilakukan ditempat dengan menggunakan games-games dan kuis-kuis
berhadiah. HASIL DAN PEMBAHASAN Berikut ini adalah hasil dan pembahasan dan kegiatan pengabdian
masyarakat yang telah dilakukan pada bulan Desember 2019 : 1. Acara dibuka dengan upacara pagi, dimana pada saat ini diberikan berbagai
macam sambutan, baik dari pihak SMPN 83 maupun dari pihak UNPAM. Setelah itu, acara dilanjutkan didalam ruangan dengan memberikan pembekalan
materi kepada para peserta PKM yang terdiri dari para guru dan anggota koperasi
SMPN 83. 2. Materi yang pertama diberikan oleh narasumber adalah tentang Inklusi
keuangan. Yaitu pengetahuan tentang produk perbankan dan pengetahuan
tentang bisnis E-Commerce. Materi selanjutnya yang diutarakan oleh
narasumber yang sama yaitu Arif Surahman, adalah mengenai pengembangan Vol. 3, No. 1, Juli 2020 Dharma Laksana P-ISSN : 2621-7155
E-ISSN : 2621-7147 Jurnal Pengabdian
Dharma Laksana Mengabdi Untuk Negeri sumber daya manusia dengan mengajarkan cara mengingat dengan
menggunakan metode Mnemonics dan metode lainnya yang dikembangkan
sendiri oleh narasumber. 3. Setelah semua materi diutarakan, maka dilakukan quis dan permainan-
permainan. Tujuannya adalah untuk menguji pemahaman peserta mengenai
materi yang sudah diberikan. 3. Setelah semua materi diutarakan, maka dilakukan quis dan permainan-
permainan. Tujuannya adalah untuk menguji pemahaman peserta mengenai
materi yang sudah diberikan. Gambar 3. Simbolis Pemberian Plakat Gambar 3. Simbolis Pemberian Plakat Jurnal Pengabdian
Dharma Laksana Mengabdi Untuk Negeri KESIMPULAN DAN SARAN Perlu adanya program ekstrakulikuler dilingkungan koperasi SMPN 83 yang
dapat digunakan untuk mengembangkan lebih lanjut mengenai kerjasama dengan
dunia perbankan dan kerjasama lebih lanjut mengenai pengembangan mutu sumber
daya manusia dengan Universitas Pamulang. Selain itu diperlukan juga untuk
mengundang praktisi perbankan untuk kegiatan PKM berikutnya, supaya potensi
koperasi SMPN 83 untuk melakukan kolaborasi dengan dunia perbankan dapat
langsung terfasilitasi. Saran yang dapat kami berikan terhadap PKM selanjutnya adalah perlu
diadakan sesi tanya jawab yang lebih panjang dan waktu pemberian materi yang lebih
lama. Namun memang hal ini terhalang dengan kesibukan mengajar di SMP tersebut. Vol. 3, No. 1, Juli 2020 Dharma Laksana P-ISSN : 2621-7155
E-ISSN : 2621-7147 Jurnal Pengabdian
Dharma Laksana Mengabdi Untuk Negeri Jurnal Pengabdian
Dharma Laksana Mengabdi Untuk Negeri 45 45 DAFTAR PUSTAKA A Sobarna, S Hambali, S Sutiswo, D Sunarsi. (2020). The influence learning used
ABC run exercise on the sprint capabilities. Jurnal Konseling dan
Pendidikan 8 (2), 67-71 Astutik, Y. ( 2019, Oktober 3). https://www.cnbcindonesia.com. Dipetik 2019, dari
https://www.cnbcindonesia.com/market/20191015210700-17-107298/ojk-
yakin-target-inklusi-keuangan-75-tercapai-tahun-ini. D Sunarsi. (2014). Pengaruh Gaya Kepemimpinan, Motivasi dan Disiplin Kerja
Terhadap Kinerja Pendidik. Universitas Pamulang Fajar, S. (2016, Oktober 3). https://kalsel.antaranews.com/. Diambil kembali dari
https://kalsel.antaranews.com/berita/39614/koperasi-perlu-kerjasama-
perbankan-tanpa-agunan. Gumilar, I., Sunarsi, D. (2020). Comparison of financial performance in banking
with high car and low car (Study of banks approved in the kompas 100 index
for the period 2013-2017). International Journal of Psychosocial
Rehabilitation. Volume 24 - Issue 7 Maddinsyah, A., Sunarsi, D., Hermawati, R., Pranoto. (2020). Analysis of location
selection effect on the user decision that influcence the success of the service
business of micro, small and medium enterprise (MSME) in bandung timur
region. International Journal of Advanced Science and Technology. Vol. 29
No. 06 Purwanti, P., Sarwani, S., & Sunarsi, D. (2020). Pengaruh Inovasi Produk Dan
Brand Awareness Terhadap Keputusan Pembelian Konsumen Pada PT. Unilever Indonesia. Inovasi, 7(1), 24-31. Sunarsi, D., & Asmalah, L. (2018). Pelatihan Manajemen Pengembangan Diri Bagi
Penerima Beasiswa RZIS UGM Dan Dompet Shalahuddin Jogjakarta. Jurnal Pengabdian Dharma Laksana, 1(1), 51-60. Sunarsi, D., & Erlangga, A. (2020). The Effect of Leadership Style and Work
Environment on the Performance of Stationary Pump Operators in the Water
Resources Office of West Jakarta City Administration. International Journal
of Advances in Social and Economics, 2(3). Sunarsi, D., Kustini, E., Lutfi, A. M., Fauzi, R. D., & Noryani, N. (2019). Penyuluhan Wirausaha Home Industry Untuk Meningkatkan Ekonomi
Keluarga Dengan Daur Ulang Barang Bekas. BAKTIMAS: Jurnal
Pengabdian pada Masyarakat, 1(4), 188-193. Sunarsi, D., Kusjono, G., & Nuryana, I. (2019). Pelatihan Manajemen Penguasaan
Kelas Dan Pembuatan Bahan Ajar Bagi Tenaga Pengajar Sukarela Taman
Belajar Kreatif Mekarsari. Jurnal Pengabdian Dharma Laksana, 2(1), 41-44. Vol. 3, No. 1, Juli 2020 Dharma Laksana
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https://openalex.org/W4206976151
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https://bmcoralhealth.biomedcentral.com/track/pdf/10.1186/s12903-021-01446-x
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English
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Root canal curvature as a prognostic factor influencing the diagnostic accuracy of radiographic working length&nbsp;determination&nbsp;and postoperative canal axis modification: An in vitro comparative study.&nbsp;
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Research Square (Research Square)
| 2,021
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cc-by
| 7,180
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Faraj BMC Oral Health (2021) 21:90
https://doi.org/10.1186/s12903-021-01446-x Faraj BMC Oral Health (2021) 21:90
https://doi.org/10.1186/s12903-021-01446-x Abstract Background: Radiographic analysis of tooth morphology is mandatory for accurate calibration of the degree of canal
curvature angle and radiographic working length to its real dimensions in case difficulty assessment protocols. This
study aimed to determine the impact of the degree of root canal curvature angle on maintaining the real working
length and the original canal axis of prepared root canals using a reciprocating rotary instrumentation technique. Methods: Radiographic image analysis was performed on 60 extracted single-rooted human premolar teeth with a
moderate canal curvature (10°–25°) and severe canal curvature (26°–70°). Working length and longitudinal canal axis
were determined using cone-beam computed tomography (CBCT) and digital periapical radiography. The real canal
length was determined by subtracting 0.5 mm from the actual canal length. Root canals were prepared using the
WaveOne Gold reciprocating file (Dentsply Maillefer, Ballaigues, Switzerland). Results: There was no significant relation of the degree of canal curvature angle to the accuracy of radiographic
working length estimated on CBCT and digital periapical radiographic techniques (P > 0.05). Postinstrumentation
changes in the original canal axis between moderate and severe canal curvature angles, assessed on CBCT and peri-
apical digital radiographic images were statistically non-significant (P > 0.05). Conclusions: A standardized digital periapical radiographic method performed similarly to the CBCT technique near
to its true working length. No significant interaction exists between the diagnostic working length estimation, post-
operative root canal axis modification, and the degree of canal curvature angle, using reciprocating rotary instrumen-
tation technique. Keywords: Canal curvature, Cone‐beam computed tomography, Canal axis, Digital periapical image, Real canal
length Root canal curvature as a prognostic
factor influencing the diagnostic accuracy
of radiographic working length determination
and postoperative canal axis modification:
an in vitro comparative study Bestoon Mohammed Faraj* Background Root canal cleaning and shaping is a fundamental step
in clinical endodontics, and it is directly related to sub-
sequent disinfection and filling procedures. It must
enhance a funnel shape from the cervical to the apical
portion, preserving the preoperative canal curvature *Correspondence: bestoon.faraj@univsul.edu.iq
College of Dentistry, Conservative Department, University of Sulaimani,
Madame Mitterand Street 30, 46001 Sulaimani, Kurdistan Region, Iraq Root canal working length determination is a funda-
mental component of the endodontic examination and
treatment planning procedure. Conventional 2-dimen-
sional (2D) radiographs provide a cost-effective, high-res-
olution image, which continues to be the most commonly
used method in daily restorative dental practice. How-
ever, intraoral radiography has some limitations because
of its 2-dimensional nature, information may be difficult
to interpret, especially in challenging conditions when
the anatomy of the root canal system is complex [6, 7].h Cone-beam computed tomography (CBCT) is a use-
ful device that produces multiplanar reformatted images
and allows two-dimensional views in all three dimensions
(axial, coronal, and sagittal planes). It has been used as
a research tool for various aspects of endodontics, such
as analysis of angulation of root canal curvature and the
efficacy of various instrumentation techniques on the
outcome of shaping. Cone-beam computed tomogra-
phy is confirmed to be more accurate than conventional
x-rays in determining root canal systems, as well as have
an impact on treatment planning [24, 25]. The validity of the working length plays a fundamental
role in determining the success of root canal treatment
and could be a predictor of possible complications [8]. Measurement of the working length may be much more
difficult in curved canals, which makes measuring diffi-
cult [9]. Overestimation of the working length may give
rise to over instrumentation beyond the apical foramen,
and as a consequence, pain and discomfort can occur,
whereas underestimation of the working length may
result in incomplete root canal cleaning and shaping [10,
11]. No clear evidence in the literature exists links quan-
tity evaluation of canal curvature with working length
estimation and shaping ability of reciprocating rotary
instrumentation procedure. Although several studies
conducted and assessed the association of instrumen-
tation outcomes with many different factors, including
the degree of canal curvature, and preoperative working
length estimation. The experimental study of each par-
ticular parameter, in a curved canal, on extracted teeth,
has yet to be precisely addressed, particularly in case dif-
ficulty assessment protocols. This study aimed first to
investigate the influence of the degree of root canal cur-
vature on the radiographic working length estimation on
extracted single-rooted human premolar teeth. © 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://crea-
tivecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdo-
main/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Faraj BMC Oral Health (2021) 21:90 Faraj BMC Oral Health (2021) 21:90 Page 2 of 9 to maintain the preoperative root canal geometry or to
straighten the curves [18, 19]. angle and the location of the major apical foramen [1, 2]. These technical objectives are of great significance and
have always been much more challenging in curved root
canals. Uncentered and extensive canal axis modification
can result in iatrogenic errors such as strip perforation,
ledge formation, and insufficient disinfection of prepared
root canals [3, 4]. However, the use of rotary nickel-tita-
nium (NiTi) files has improved the overall shaping out-
come and reduced the possibility of procedural errors
with a subsequently lower incidence of canal curvature
straightening [5]. WaveOne Gold (Dentsply Maillefer, Ballaigues, Swit-
zerland) instrument is a single-file system with a recipro-
cating motion manufactured using a post-manufacturing
thermal process that produces a file with superelastic
nickel-titanium metal properties. This improvement in
mechanical characteristics exhibits a unique alternating
off-centered parallelogram-shaped cross-section and a
progressively decreasing percentage taper design. The
Primary WaveOne Gold instrument is more resistant
to cyclic fatigue, more flexible with a better cutting effi-
ciency than the conventional Primary WaveOne file [20,
21]. Several studies concluded that canal shaping using a
single instrument with different kinematics did not com-
promise canal cleanliness and took less working time
to prepare the canals compared with the conventional
multi-instrument systems [22, 23]. Secondly,
to determine a link between the degree of root canal cur-
vature angle and the postoperative change in longitudinal
root canal axis, using a reciprocating rotary instrumenta-
tion technique through a quantitative evaluation, under
the scrutiny of CBCT and digital radiographic imaging. A better outcome of root canal treatment achieved
when the original canal axis is preserved and without
creating iatrogenic events, such as instrument fracture,
canal transportation, ledges, or perforations [12]. The
evaluation of changes in canal shape after instrumenta-
tion has been suggested as a reliable process to assess the
ability of a shaping technique to preserve the original axis
of the root canal in its place [13]. In clinical endodontics, several technical parameters
were applied to investigate the canal shape before and
after radicular dentine instrumentation. The quantita-
tive evaluation of postoperative root canal changes can
be estimated by the “centering ratio” method on original
root canal curvature angle [14, 15], or by measuring pre-
and postoperative radicular dentine thickness, on super-
imposing radiographic images before and after shaping
using CBCT scanning or digital radiographic imaging
techniques[16, 17]. Furthermore, analysis of change
in canal curvature angle after shaping has been widely
used in the literature to evaluate the tendency of a tech-
nique, or the mechanical properties of an instrument, Canal curvature measurements
A
d
l
d An experienced oral and maxillofacial radiologist
obtained all the CBCT and digital radiographic images
and performed the measurements. The change in canal
axis was determined as the difference between canal cur-
vature before and after instrumentation. For the CBCT
evaluation, scan images from the clear sagittal view were
selected depending on the multiplanar imaging-refor-
matted sections. The slices were first reproduced in a ver-
tical position to visualize the tooth cusp, pulp chamber,
apical foramen, and the complete view of the root canal
pathway. All images converted for viewing with image
analysis software (NNT Software, Verona, Italy) to meas-
ure the canal curvature angle.h Real working length measurement on extracted teeth g
g
A standard straight-line access opening was prepared for
all the teeth. A size #10 or #15 K-File (Dentsply Maillefer,
Ballaigues, Switzerland) was passively advanced until its
tip was seen at the level of the coronal most boundary of
the major apical foramen, by the aid of a magnifying lens
(Keeler, Windsor, UK, ×3 magnification). The distance
between the reproduced coronal reference point and the
tip of the file was measured with an electronic digital cal-
iper (Mitutoyo Corp., Japan) to the nearest (0.01 mm.),
and documented as the actual working length. The real
working length was determined by subtracting 0.5 mm
from the actual canal length [27]. Ethical approval and sample selection.h The ethics protocol for this study was confirmed
and accepted by the Ethical Committee at Sulaimani Faraj BMC Oral Health (2021) 21:90 Faraj BMC Oral Health (2021) 21:90 Page 3 of 9 Pre‑instrumentation working length estimation on CBCT
and digital periapical radiographic images Pre‐instrumentation digital periapical imaging Each tooth was embedded in the radiolucent polysilox-
ane putty dental impression material (3 M ESPE, St. Paul,
MN, USA), and encoded with a number. The digital radi-
ographical examination was carried out for all the teeth
in two directions (buccolingual and mesiodistal), using
a standardized parallel technique. A high-frequency oral
x-ray machine (EzRay Air W; Vatech, Korea), were used
with an exposure time of 0.367 seconds (60 kV, 4 mA). The target– receptor distance was increased to compen-
sate for image magnification and to ensure that only the
most parallel rays were directed toward the tooth and
the X-ray sensor (EzSensor Classic, Vatech, Korea). As a
result, a long (16-inch) target–receptor distance was used
[27]. Pre‑instrumentation working length estimation on CBCT
and digital periapical radiographic images University (Protocol Number; 392/2020). The meth-
odology applied has followed the CRIS guidelines as
considered in the 2014 concept note [26]. A total of 60
curved single-rooted human premolar teeth with vary-
ing degrees of root curvature were selected for this study. The teeth with uncommon extreme variations were
excluded, like twisted or fused roots. Endodontically
treated teeth, internal or external root resorption were
also excluded. Whereas those with completely formed
apices, single canal with one apical foramen, and ≥ 10°
canal curvature were included. The patients’ age, gen-
der, tooth’s quadrant, or reason for extraction were not
documented. Specimens were stored in 10 % formalin for
disinfection for a maximum of 2 weeks. Tissue fragments
and calcified debris were removed manually by using a
hand scaler, then washed in running water. Finally, they
were stored in normal saline at room temperature until
the time of the investigation. Traceable calibration was performed in the center of the
pulpal cavity and followed each visible canal deviation,
thus allowing for the measurements of curved canals. The
radiographic tooth length was determined on the CBCT
and digital periapical images as the distance between the
tip of the cusp and the major apical foramen. The radio-
graphic working length for all the specimens was meas-
ured separately on digital periapical and CBCT images
after subtraction of 1mm from the radiographic tooth
length [27]. Pre‑instrumentation CBCT scans Two custom-made wood boxes were used for the mount-
ing of the teeth and to confirm the standardization for the
CBCT images. Each tooth was embedded in cold-cure
clear acrylic resin (Vertex Castavaria, Netherland) with
a technical specification of 9 minutes dough time and 6
minutes working time at 55°C, using a cylindrical plas-
tic container .Thereafter, they quoted with MS3 master
die separator (Ivoclar Vivadent, USA) to enable precise
repositioning during pre and post-instrumentation scans. Ten teeth were mounted in each template consistently
by using dental stone plaster (Rident, Rajasthan, India). Each mold was horizontally fitted to the chin support of
the CBCT machine (NewTom, Giano, Verona, Italy) in a
way that the occlusal plane was arranged parallel to the
plate, and scanned with 90 kVp, 3 mA, voxel size: 0.125
mm, exposure time: 5.4 s, by using FOV 8 cm by 11 cm
[27, 28]. The Schneider method was applied for the estimation
of the degree of canal curvature before and after instru-
mentation. Two straight lines of equal lengths were used. The first line represented the continuity of the apical
region, and the second line followed the middle and cor-
onal thirds of the root canal. The angle between the radii
was geometrically measured, and the canal curvature was
expressed in degrees (Figs. 1, 2).The formed canal angle
was named according to the degree of root canal curva-
ture into moderate (10-25°) and severe (26-70°). All scan
images captured before and after instrumentation were Faraj BMC Oral Health (2021) 21:90 Faraj BMC Oral Health Page 4 of 9 Fig. 1 Representative digital periapical images a pre-instrumentation
canal curvature angle measurement b post- instrumentation canal
axis modification analyzed with image analysis software (NNT Software,
Verona, Italy), to determine the canal curvature changes
[27, 29]. Root canal cleaning and shaping An endodontist, experienced in the use of the Primary
WaveOne Gold Reciprocating file (DentsplyMaillefer,
Ballaigues, Switzerland), performed all procedures. The
analysis of the radiographic images reproduced in three
dimensional for the canal curvatures and shaping ability
was carried out by an oral and maxillofacial radiologist
who was blind in respect of all experimental groups. p
p
g
p
The access coronal cavity was prepared using a round
carbide bur #4 (Dentsply Maillefer, Ballaigues, Switzer-
land), and the canal patency checked with a #15 K-type
hand file (Dentsply Maillefer, Ballaigues, Switzerland). The coronal third flared with Gates-Glidden drills 2 and
3 (Dentsply Maillefer, Ballaigues, Switzerland). Work-
ing length was confirmed manually with a #15 K-File
using a standard protocol. A glide path created using a
ProGlider instrument (16/02) (Dentsply Maillefer, Bal-
laigues, Switzerland) carried to the working length. A
handpiece generated by an electric motor (Silver, VDW,
Munich, Germany) was used for instrumentation. The
speed, torque, and file sequence were applied according
to the manufacturer’s instructions. Only five canals were
instrumented at each time interval to minimize opera-
tor fatigue. After each file sequence, the prepared canal
was washed out with 3 ml of 3 % NaOCl solution (Techno
Dent, Greece), followed by a 5 ml solution of %17 EDTA
(SPIDENT, Korea). Then, the canals were irrigated with
5 ml of 3 % NaOCl as a final rinse. Root canal irriga-
tions were performed by using a 5 mL disposable plastic
syringe with a 30 gauge side opening needle (Optimus,
SP, Brazil) at room temperature. The needle was inserted
inside the canal without binding and the solutions were Fig. 1 Representative digital periapical images a pre-instrumentation
canal curvature angle measurement b post- instrumentation canal
axis modification Fig. 1 Representative digital periapical images a pre-instrumentation
canal curvature angle measurement b post- instrumentation canal
axis modification Fig. 2 Representative CBCT images demonstrating a post-instrumentation canal curvature angle measurement Fig. 2 Representative CBCT images demonstrating a post-instrumentation canal curvature angle measurement CBCT images demonstrating a post-instrumentation canal curvature angle measurement Faraj BMC Oral Health (2021) 21:90 Page 5 of 9 introduced slowly and passively allowing adequate
back-flow. introduced slowly and passively allowing adequate
back-flow. within the investigated parameters between moderate
and severe curvature angles. introduced slowly and passively allowing adequate
back-flow. Post‐instrumentation CBCT and digital periapical image
analysis The values of the mean and standard deviation of work-
ing canal length concerning the degree of canal curvature
angle close to its real clinical canal length are summa-
rized in (Tables 1, 2). There was no remarkable role of the
degree of canal curvature angle on the precise estima-
tion of radiographic canal length (P > 0.05). Regarding the
CBCT image scanning, there was a tendency for under-
estimation of the working canal length from − 1.2mm
to − 0.1mm (− 0.538 ± 0.303) in 52 (86.7%) of the exam-
ined specimens. Whereas, overestimation from 0.1mm to After instrumentation, each tooth was repositioned in its
previous position inside a plaster block. The post-CBCT
and digital periapical radiographs were obtained with
the same parameter applied in the pre- instrumentation
phase. Longitudinal axial canal axis and canal curvature
angle were determined (Figs. 1, 2). The percentage of the
change in canal curvature angle after instrumentation
(canal axis modification) was calculated using the follow-
ing formula [29]; Canal curvature angle after instrumentation‑canal
curvature angle before instrumentation Table 2 Estimation
accuracy
of
working
length
measurement closest to real values in a range of 0.5mm
tolerance level recorded on CBCT and digital radiographic
images
*Chi- square test
**Not significant
Tolerance level
Degree of curvature
Total
%
P value*
Moderate
10°–25°
Severe
26°–70°
CBCT image 0.5 mm
Yes
26
60.5%
7
41.2%
33
55%
0.18**
No
17
39.5%
10
58.8%
27
45%
Digital image 0.5 mm
Yes
25
48.8%
7
41.18%
32
53.3%
0.92**
No
18
51.2%
10
58.82%
28
46.7% Table 2 Estimation
accuracy
of
working
length
measurement closest to real values in a range of 0.5mm
tolerance level recorded on CBCT and digital radiographic
images Statistical analysis Statistical analysis of data obtained in this study per-
formed using IBM SPSS Statistics for Windows, version
24.0 (Armonk, NY: IBM) software. A P-value < 0.05 was
considered a statistically significant level. The sample size
was determined with the Sealed Envelope software for a
power of 80 %. The normal distribution of the data was
tested using the Shapiro-Wilk test. Chi-square tests were
used to compare the frequencies of qualitative variables. When the distribution of variables was normal, an inde-
pendent sample t-test was used to compare the results Table 1 Comparison of mean and standard deviation values of working length estimated on CBCT and digital
adiographic images in relation to the degree of canal curvature angles and standard deviation values of working length estimated on CBCT and digital
o the degree of canal curvature angles Table 1 Comparison of mean and standard deviation values of working length estimated on CBCT and digital
radiographic images in relation to the degree of canal curvature angles
*Independent t test
**Not significant
Degree of curvature
Mean ± SD
P value *
Moderate
10°–25°
Severe
26°–70°
Total
%
Digital image
Overestimation
Closest to real length
Minimun to Maximun
N
20.86 ± 1.63
0.624 ± 0.409
0.2 to 2.3
43
20.43 ± 0.83
0.658 ± 0.293
0.3 to 1.2
17
20.72 ± 1.43
0.635 ± 0.374
0.2 to 2.3
60(100%)
0.27 **
0.75**
CBCT image
Overestimation
Closest to real length
Minimun to Maximun
N
19.43 ± 1.22
0.675 ± 0.892
0.1 to 2.0
4
20.3 ±
0.80 ±
− 0.8
1
19.6 ± 1.13
0.7 ± 0.775
0.1 to 2.0
5(8.3%)
0.47**
0.91**
Underestimation
Closest to real length
Minimun to Maximun
N
19.83 ± 1.72
− 0.494 ± 0.306
− 1.0 to − 0.1
36
19.41 ± 1.02
− 0.638 ± 0.280
− 1.2 to − 0.2
16
19.70 ± 1.54
− 0.538 ± 0.303
− 1.2 to − 0.1
52(86.7%)
0.81**
0.12** 1 Comparison of mean and standard deviation values of working length estimated on CB
graphic images in relation to the degree of canal curvature angles Faraj BMC Oral Health (2021) 21:90 Page 6 of 9 Faraj BMC Oral Health Fig. 3 Box plots comparing the relation of the degree of root canal
curvatures with the postoperative canal axis modifications assessed
on CBCT images indicated an accuracy of 55% (33) within a range of + 0.5
mm tolerance level (Fig. 3). Statistical analysis The canal preparation led to a decrease of the root
canal curvature for both moderate and severe canal cur-
vature angles collectively (Tables 3, 4). The difference
between before and after instrumentation shows sta-
tistically no significant deviation of curvature between
moderate and severe canal curvature angles reproduced
on CBCT and digital periapical radiographic images
(p < 0.75) (Figs. 3, 4). Discussion In the present study, the correlation between the diag-
nostic working length calculations, the parameter of the
shaping outcome (canal axis modification) in root canal
preparation, and its degree of canal curvature angle were
investigated under ex vivo conditions. Several techniques
are available for the comparative evaluation of the preop-
erative radiographic working length determination and
shaping outcome of reciprocating rotary Nickel-Titanium
(NiTi) instruments in curved canals. The comparison of
the pre-and post-instrumentation radiographic images
of the outlines of the longitudinal root canal axis ena-
bled the evaluation of the most relevant parameters of
root canal preparation. Furthermore, assessment of their
diagnostic validity and the possible influence of other Fig. 3 Box plots comparing the relation of the degree of root canal
curvatures with the postoperative canal axis modifications assessed
on CBCT images Fig. 3 Box plots comparing the relation of the degree of root canal
curvatures with the postoperative canal axis modifications assessed
on CBCT images 2.0mm (0.7 ± 0.775) were reported in only 5 teeth (8.3 %). The periapical radiographic images slightly overesti-
mated the real canal length measurement (0.635 ± 0.374). On the whole, this technique revealed an accuracy of
53.3 % (32), within the range of + 0.5 mm tolerance
level (Table 2). The results from CBCT image scanning Table 3 The mean and standard deviation (SD) values of the change in the canal curvature angles after shaping
in both moderate and sever curvature groups, calculated on CBCT images
* Independent t test
** Not significant
Moderate
10°–25°
Severe
26°–70°
P-value*
N
Mean
SD
N
Mean
SD
Before instrumentation
43
21.32
4.26
17
29.95
4.143
0.688**
After instrumentation
43
18.541
4.143
17
22.84
2.693
0.1**
Curvature change
43
− 12.721
10.016
17
− 23.202
10.131
0.691** Table 3 The mean and standard deviation (SD) values of the change in the canal curvature
in both moderate and sever curvature groups, calculated on CBCT images Table 4 The mean and standard deviation (SD) values of the change in the canal curvature angles after shaping
in both moderate and sever curvature groups, calculated on digital periapical radiographic images
*Independent t test
**Significant
***Not significant
Moderate
10°–25°
Severe
26°–70°
P-value*
No. Mean
SD
No. Discussion Mean
SD
Before instrumentation
43
18.24
4.06
17
28.85
2.13
0.013**
After instrumentation
43
15.62
3.82
17
24.75
2.14
0.023**
Curvature change
43
− 16.82
7.71
17
− 18.82
5.76
0.388*** d standard deviation (SD) values of the change in the canal curvature angles after shaping
sever curvature groups, calculated on digital periapical radiographic images Table 4 The mean and standard deviation (SD) values of the change in the canal curvature
in both moderate and sever curvature groups, calculated on digital periapical radiographic images Page 7 of 9 Page 7 of 9 Faraj BMC Oral Health (2021) 21:90 Faraj BMC Oral Health Fig. 4 Box plots comparing the relation of the degree of root canal
curvatures with the postoperative canal axis modifications assessed
on digital periapical radiographic images more accurate when performed for root canals having a
moderate canal curvature. While, there was no statisti-
cally significant difference in mean values of root canal
length measured with an extensive degree of curvature
angle, in comparison to its real length. These outcomes
are most probably related to the direction and nature
of root angulation and anatomic noise, which results
in the missing of some details of the root canal anat-
omy assessed with the periapical digital radiography. However, CBCT scanning overcomes this distortion
in image quality by maintaining the multiplanar recon-
struction, which leads to a clear 3D visualization of the
root canal configuration. i
Primary WaveOne Gold reciprocating file was selected
for this study since clear evidence in the literature exists
to support its efficiency in the preparation of different
root canal morphology [37–39].In the present study, we
chose not to include conventional stainless steel K-files as
a control group. The superiority of NiTi instruments in
maintaining the original canal shape better than K-files is
already established in the literature [40, 41]. The results
from the present study confirmed the ability of rotary
NiTi instruments to maintain the original canal curva-
ture, even in extremely curved canals, and showed that
none of the tested moderate and severe canal curvature
samples reached the aforementioned critical level of
canal straightening, keep its original canal axis. Similar
findings were observed in previous studies performed on
root canals with severe curvature [38, 39].The increased
flexibility of NiTi instruments is behind their good cen-
tering ability and maintaining the original canal axis [40]. Discussion According to the manufacturer instructions, the technol-
ogy applied in WaveOne Gold files expands the flexibility
and strength of the instrument [42], as well as enhancing
its elasticity and its reciprocating motion, which main-
tains the central canal path while shaping it and reduced
the incidence of procedural errors [43, 44]. Fig. 4 Box plots comparing the relation of the degree of root canal
curvatures with the postoperative canal axis modifications assessed
on digital periapical radiographic images associated factors such as degree of canal curvature can
be performed using appropriate computer software [30].h The major advantage of the use of extracted teeth in
experimental investigations is the reproduction of the
clinical situation, an inspection of root canal morphol-
ogy, and the establishment of a satisfactory real positive
finding. A human premolar tooth with a single root canal
was selected in the present investigation to eliminate var-
iables specific to root canal configuration. However, it is
difficult to standardize variables such as root canal con-
figuration, and the nature of canal curvatures. The reli-
ability, accuracy, and certainty of these study protocols
had previously been approved in the literature reviews
[31–33]. In the present methodology, the measurement of root
canal curvature was calibrated by using CBCT scanning. This allows improved precision and helps to increase the
evaluation accuracy of the cross-sectional shape of pre-
pared root canals through a three-dimensional radio-
graphic image with no destruction of the specimens [34,
35]. With this technique, it is feasible to compare the
longitudinal canal axis of the root canal before and after
shaping. Due to these considerations, the worksheet was
made for only 60 teeth. Attempts were made to ensure
the standardization of the selected teeth through several
inclusion and exclusion criteria.hi The results of the present study revealed that the use of
the WaveOne Gold reciprocating file results in a higher
mean value of the postoperative canal axis modifica-
tion in teeth with severe canal curvature than moderate
curvature. This outcome might be related to the fact
that canal curvature may have caused the non-uniform
distribution of stress on the instrument and increased
transportation values [21–23]. Another promising find-
ing was that the limited canal axis modification in this
study might be related to the good centralization capacity
of the instruments in the canal, especially in the apical-
and middle third. CBCT: Cone-beam computed tomography; FOV: Field of view; SD: Standard
deviation; KVp: Kilovoltage peak; mA: Milliampere; 2D: Two-dimensional; 3D:
Three-dimensional. Funding This research was supported by the University of Sulaimani (Kurdistan Region,
Iraq). The funder had a role in the decision to publish of the manuscript. Availability of data and materials Availability of data and materials
The datasets used and/or analysed during the current study are available from
the corresponding author on reasonable request. The datasets used and/or analysed during the current study are available from
the corresponding author on reasonable request. Competing interests The sample size in this study was small, and the
process of scanning and reconstructing radiographic
images were limited to 60 teeth. Larger sample size will
compensate for anatomical variables and may show if
there are differences in findings regarding more com-
plex canal morphology focusing on the number and
classes of canal configuration. It is also important to be
aware of possible sources of errors to avoid over inter-
pretation of radiographic images. However, based on
the protocol applied in this work for working length
confirmation, the instrument prepared root canals to
acceptable good clinical standards. The clinical rele-
vance of radiographic working length confirmation to
the degree of root canal curvature concerning the ulti-
mate shaping outcome of instrumentation is likely to be
critical in case difficulty assessment protocols. The author declares that there is no conflict of interest regarding the publica-
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b y
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images. Braz Dent J. 2008;19:114–18. •
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English
| null |
Eye images do not promote prosocial behaviour or norm compliance in an online Dictator Game
| null | 2,017
|
cc-by
| 7,388
|
Eye images do not promote prosocial behaviour or norm compliance in an online Dictator
Game
Nichola J Raihani1* & Redouan Bshary2 1. Department of Experimental Psychology, 26 Bedford Way, University College London, London
5
WC1H 0AP. 1. Department of Experimental Psychology, 26 Bedford Way, University College London, London
5
WC1H 0AP. 2. Institut de Biologie, Université de Neuchâtel, Neuchâtel , CH-2000, Switzerland. * Corresponding author: nicholaraihani@gmail.com 1. Department of Experimental Psychology, 26 Bedford Way, University College London, London
5
WC1H 0AP. 2. Institut de Biologie, Université de Neuchâtel, Neuchâtel , CH-2000, Switzerland. * Corresponding author: nicholaraihani@gmail.com * Corresponding author: nicholaraihani@gmail.com Abstract
10 Abstract
10
Evidence for the effect of eye images on prosocial behavior is mixed: some studies have found
that images of eyes enhance cooperative behavior while others have not replicated this effect. In a recent meta-analysis, Nettle et al. (2013) argued that previous null results have occurred
because data have been analysed incorrectly. Specifically, the authors predicted that eye images
either (i) reduce variance in donation amount by reducing the likelihood that the Dictator will
15
keep the entire endowment or (ii) increase compliance with cooperative norms. Since several
previous studies have only looked at the effect of eye images on mean donation amount, they
have not tested these predictions. We test both hypotheses here using a Dictator Game (n = 779)
conducted over Amazon Mechanical Turk, a setup that has previously yielded negative results. We provided players with two types of descriptive norm information, by telling them how much
20
others typically give in this setting. We compared donations under the norm treatments with
decisions made under control conditions, where no norm information was supplied. In each
setting, subjects were additionally shown images of eyes or a control image (flowers) so that any
additional effect of eye images on prosocial behavior could be examined. Eye images did not increase Dictator Game giving, regardless of whether 'giving' was defined as mean donation or
25 25 simply the tendency to make a non-zero donation. Furthermore, eye images did not increase
compliance with a descriptive norm in this setting. Due to these negative results, we conclude
that the conditions and underlying mechanisms that lead to positive effects of eyes on prosocial
behaviour remain elusive. simply the tendency to make a non-zero donation. Furthermore, eye images did not increase
compliance with a descriptive norm in this setting. Due to these negative results, we conclude
that the conditions and underlying mechanisms that lead to positive effects of eyes on prosocial
behaviour remain elusive. 30
Introduction
Empirical evidence abounds to support the idea that people are more cooperative when they know
that others will observe their behaviour (1-4), and it has been proposed that images of eyes evoke a
similar response as being observed by a real person. Several laboratory and real-world experiments
have suggested that cooperative behaviour is increased when subjects are exposed to eye images,
35
relative to neutral controls (reviewed in (5,6)). Abstract
10 Nevertheless, there have also been several prominent
failures to replicate the positive effect of eye images on cooperative behaviour, with a recent meta-
analysis suggesting that the current evidence is consistent with the hypothesis that there is no main
effect of eye images on prosocial behaviour (6). An alternative explanation for these failed
replications is that the watching eye effect is context-specific. Indeed, Nettle et al. (5) have
40
suggested that watching eyes do not increase pro-sociality per se but instead increase the tendency
to comply with an established norm of cooperation. We test this hypothesis here. 30 Introduction A common paradigm for investigating the effect of eye images on cooperative behaviour under
laboratory settings is the Dictator Game (7). This is a two-player game where one player, the Dictator,
45
is given an endowment and is allowed to give any share of the endowment to the other player, the
Receiver. In this game, the Receiver must accept any share offered by the Dictator. An income-
maximising, self-interested individual is expected to keep the entire endowment but humans rarely
behave this way. In most cases, Dictators give around 28 % of the endowment to the Receiver,
although there is also usually substantial variation around this mean, with many players choosing to
50 50 keep the entire endowment or to give half of the money to the partner (see (8) for a meta-analysis). Several studies have identified factors that might influence Dictator Game giving, including the effect
of presenting the Dictator with eye images during the game. While some studies have found that eye
images motivate more generous allocations to the Receiver in this game (e.g. (9-12)), others studies
have not replicated this effect (13). keep the entire endowment or to give half of the money to the partner (see (8) for a meta-analysis). Several studies have identified factors that might influence Dictator Game giving, including the effect
of presenting the Dictator with eye images during the game. While some studies have found that eye
images motivate more generous allocations to the Receiver in this game (e.g. (9-12)), others studies
have not replicated this effect (13). 55 Nettle et al. (5) suggested that the ambiguity over the effect of eye images on cooperative behavior
arises because researchers have not asked the right questions with their data. In Dictator Game
settings, researchers have typically asked whether eye images increase the mean donation made to
receivers. Nettle et al. (5) suggested that eye images might instead make people less prone to extreme
60
strategies in social settings. we should observe reduced variance in donation amount when people
view eye images than when they view control images (5). Indeed, where this effect is explicitly
measured, it is observed that the presence of eye images reduces variance in Dictator donations (5,9). An alternative hypothesis is that eye images only promote prosocial behavior when prosocial behavior
is normative in that context (5). Introduction In the long interval since writing it and not getting
it accepted anywhere, some studies have started to address this question. For example, a recent study
by Bateson et al. (15) where a local littering norm was manipulated (by experimentally adding or
reducing litter) and the presence of eyes on further littering in these two norm scenarios was
80
measured. In this setting, the authors found no evidence to suggest that eye images increased
cooperative behaviour (by reducing littering) or increased compliance with a social norm (by littering
only when litter was already present). There was a non-significant interaction between the presence
of eye images and the number of observers on littering behaviour: curiously, eye images apparently 80 only reduced littering behaviour when there was a minimum of six observers present. This result is
85
puzzling: further investigation (preferably with a pre-registered study) is warranted to explore
whether this was a statistical anomaly or a trend that is truly representative of real-world patterns of
behaviour. A more recent study asked whether eye images increased tendency to comply with
normative information to pay for newspapers under an honour system, finding that normative information alone ('the majority of our readers pay for their copy') was more effective than the norm
90
information combined with eye images at promoting honesty (16). Since the predictions that eye
images (i) reduce variance in donation amount or (ii) increase compliance with experimentally created
norms were originally formulated with respect to Dictator Game giving, it makes sense to test these
hypotheses in that setting. We do this here. 95 Introduction As such, in the context of the Dictator Game, it has been argued that
65
eye images should not be expected to increase the amount donated to the receiver but instead to
increase the propensity of giving any non-zero donation (5). This is based on the (albeit unverified)
idea that giving something to the Receiver is an established social norm in this game. Indeed, in some
studies, it does seem to be the case that eye images decrease the tendency of the dictator to keep the
entire endowment (e.g. (5,9,10) but see (14)). However, it is not clear from these studies whether the
70
dictators perceived that the norm in this setting was to give some of the endowment to the receiver. 60 Based on the predictions outlined in the metastudy by Nettle et al., (2013), here we tested the
hypotheses that (i) eye images make people more likely to comply with a descriptive social norm,
and (ii) eye images reduce variance in dictator donations. When we first wrote this paper, no studies
75
had experimentally manipulated the presence or absence of a local descriptive norm and then asked Based on the predictions outlined in the metastudy by Nettle et al., (2013), here we tested the
hypotheses that (i) eye images make people more likely to comply with a descriptive social norm,
and (ii) eye images reduce variance in dictator donations. When we first wrote this paper, no studies
75
had experimentally manipulated the presence or absence of a local descriptive norm and then asked how eye images affect compliance with that norm. In the long interval since writing it and not getting
it accepted anywhere, some studies have started to address this question. For example, a recent study
by Bateson et al. (15) where a local littering norm was manipulated (by experimentally adding or
reducing litter) and the presence of eyes on further littering in these two norm scenarios was
80
measured. In this setting, the authors found no evidence to suggest that eye images increased
cooperative behaviour (by reducing littering) or increased compliance with a social norm (by littering
only when litter was already present). There was a non-significant interaction between the presence
of eye images and the number of observers on littering behaviour: curiously, eye images apparently how eye images affect compliance with that norm. Methods This project was approved by the University College London ethics board under the project number
3720/001. All data were collected in March 2014 using the online labour market, Amazon Mechanical
Turk (MTurk, www.mturk.com). No deception was used in this study and subjects were not debriefed
as to the purpose of the study after the game. Participation in this study was voluntary and subjects
100
could choose to opt in or out of the game (by choosing to click the hyperlink to access the study) after
reading a brief description of the task. Since participation was voluntary and subjects participated 100 anonymously, our ethics approval does not stipulate that we obtain written or verbal consent from
subjects recruited from MTurk. Mechanical Turk is now a widely accepted tool for conducting
behavioural experiments online (see (17) for a review) and the results of many classic studies,
05
originally performed in laboratory settings, have been replicated several times using MTurk (17). anonymously, our ethics approval does not stipulate that we obtain written or verbal consent from
subjects recruited from MTurk. Mechanical Turk is now a widely accepted tool for conducting
behavioural experiments online (see (17) for a review) and the results of many classic studies,
05
originally performed in laboratory settings, have been replicated several times using MTurk (17). 105 We recruited 1,600 MTurk workers to play a Dictator Game. Of these workers, 800 were randomly
assigned to the 'dictator' role and 800 to the 'receiver' role in the game, although the more neutral
terms 'Player 1' and 'Player 2' were used in the information seen by subjects and are used hereafter. 110
Receivers generated no data in this task and thus are not included in any analyses. In the game, Player 1 was endowed with a sum of $0.50 (c.f. (18)) and was told that they could choose
to give as much of this sum as they wanted to Player 2 (see online supplementary information for
game instructions). MTurk workers are identified by a unique 14- digit code rather than their names. 115
Workers were told that their ID would not be revealed to their partner in the game, thus ensuring
anonymity. Player 1 was asked to select one of 11 donations to send to Player 2, from $0.00 to $0.50,
increasing in $0.05 increments. After taking part in the Dictator Game, subjects were asked to state
their age and gender. Methods We asked whether the presence of eyes versus flowers was associated with reduced
variance in donation amount or reduced propensity to keep the entire endowment, as predicted by
Nettle et al. (5). Since the presence of normative information in the instructions could also have
influenced these variables, for these tests we restricted the analysis to data collected under control
conditions (although the analyses were repeated including all data and were not qualitatively
5
different). donation? We asked whether the presence of eyes versus flowers was associated with reduced
variance in donation amount or reduced propensity to keep the entire endowment, as predicted by
Nettle et al. (5). Since the presence of normative information in the instructions could also have
influenced these variables, for these tests we restricted the analysis to data collected under control 155 (ii) Do eye images increase compliance with a descriptive norm? We ran two General Linear Mixed Models (GLMMs) to ask whether compliance with each of the
descriptive norms was affected by image type. In the first model, we used the data from the “Give
160
Half” norm group, with associated control, resulting in a sample size of 392 for analysis. We created
a binary response term called 'Complied' which was set as '1' if Player 1 gave $0.25 or more to Player
2 and '0' if Player 1 gave < $0.25 to Player 2. This term was set as the response term in a binomial
GLMM with the terms 'Image (Eyes / Flowers)', 'Treatment (Norm / Control)', and the two-way
interaction 'Treatment x Image' included as explanatory terms in the model. ‘Image ID’ was included
165
as a random effect. In the second model, we used the data from the “Give Something” group, with
associated control. The response term 'Complied' was set as '1' if Player 1 gave any non-zero donation
to Player 2 and '0' if Player 1 kept the entire endowment for themselves. The same explanatory terms
were fitted to this model as in the model above, resulting in a sample size of 387 for analysis. We ran two General Linear Mixed Models (GLMMs) to ask whether compliance with each of the
descriptive norms was affected by image type. In the first model, we used the data from the “Give
160
Half” norm group, with associated control, resulting in a sample size of 392 for analysis. We created
a binary response term called 'Complied' which was set as '1' if Player 1 gave $0.25 or more to Player
2 and '0' if Player 1 gave < $0.25 to Player 2. This term was set as the response term in a binomial
GLMM with the terms 'Image (Eyes / Flowers)', 'Treatment (Norm / Control)', and the two-way interaction 'Treatment x Image' included as explanatory terms in the model. ‘Image ID’ was included
165
as a random effect. In the second model, we used the data from the “Give Something” group, with
associated control. The response term 'Complied' was set as '1' if Player 1 gave any non-zero donation
to Player 2 and '0' if Player 1 kept the entire endowment for themselves. The same explanatory terms
were fitted to this model as in the model above, resulting in a sample size of 387 for analysis. 170 Methods 120 All subjects were required to answer three comprehension questions correctly. If a subject answered
any of the comprehension questions incorrectly, s(he) was excluded from taking part in the study. Ultimately, 21 workers assigned to the dictator role were excluded, so our analyses are based on
n=779 dictators (age range: 18-67, mean: 30 ±0.4; 494 males, 282 females, 2 did not specify). 125 125 different eye images and six different flower images in this study (sourced from Google Images;
130
Figure 1). Flowers were used as the control image following previous studies (e.g. (18-20)). In the
norm treatments, the participant received one of two descriptive norms (see below). In the control
treatments, we included the number '25' and the number '50', respectively, to control for the possibility
that these numbers might have acted as an anchor (21) for donation amounts in the norm treatments. 130 Anchoring occurs when the presentation of the first piece of numerical information influences
135
judgements and decisions made thereafter (22). “Give Half” norm: Data show that Player 1 typically gives $0.25 to Player 2 (eyes, n = 99 / flowers,
n = 98). “Give Something” norm: Data show that Player 1 rarely keeps the entire $0.50 (eyes, n = 99 / flowers,
140
n = 96). “Give half” control: Data show that most MTurk workers are over 25 years old (eyes, n = 99 / flowers,
n = 96). “Give something” control: Data show that most MTurk workers are under the age of 50 (eyes, n = 95
/ flowers, n = 97). 145 “Give something” control: Data show that most MTurk workers are under the age of 50 (eyes, n = 95
/ flowers, n = 97). 145 145 Data analyses
We asked the following main questions: (i) Do eye images reduce variance in donation amount or propensity to keep the entire
150 150 donation? We asked whether the presence of eyes versus flowers was associated with reduced
variance in donation amount or reduced propensity to keep the entire endowment, as predicted by
Nettle et al. (5). Since the presence of normative information in the instructions could also have
influenced these variables, for these tests we restricted the analysis to data collected under control
conditions (although the analyses were repeated including all data and were not qualitatively
5
different). donation? (iii) Do eye images increase donation amounts? 180
Rather than testing a null hypothesis, the relative degree of support for each model from the candidate
set is calculated (24). Initially, we specified a global model which included all the explanatory and
random terms. The input variables were then standardized according to (25), allowing the relative
strength of parameter estimates to be interpreted. We used the package MuMIn (26) to derive and
compare submodels from the initial global model. Models were compared to one another using
185
Akaike's Information Criterion corrected for small sample sizes (AICc). A subset of 'top models' were
defined by taking the best model (the model with the lowest AICc value) and any models within
2AICc units of the best model (following (24)). Using this subset of 'top' models, we computed the
average parameter estimates for each term included in the subset of models, as well as the relative 180 importance of the term. Importance is calculated by summing the Akaike weights of all models where
190
the term in question is included in the model. Akaike weights represent the probability of a given
model being the true model (compared to other candidate models in the set) (24). Importance can
therefore be thought of as the probability that the term in question is a component of the best model
(27). In the results section, we only present the parameter estimates from the top models (those that
were within 2 AICc units of the best model). All data and R code are available at Figshare (doi:
195
10.6084/m9.figshare.5459266). (iii) Do eye images increase donation amounts? Finally, we asked whether the presence of eye images (in the absence of normative information)
resulted in increased mean donations in this game. Donations were extremely right-skewed. Hence,
we transformed donation into a three-level, ordinal categorical variable, according to the interquartile
range of the data. We used only data from the control conditions, resulting in n= 387 for analysis. 175 175 All data were analysed using R version 3.4.1 (http://www.r-project.org). To determine the relative
importance of the explanatory terms included in each model, we used an information-theoretic
approach with model averaging as described in (23). Under an information- theoretic approach, a
series of candidate models are generated, with each model representing a biological hypothesis. 180
Rather than testing a null hypothesis, the relative degree of support for each model from the candidate
set is calculated (24). Initially, we specified a global model which included all the explanatory and
random terms. The input variables were then standardized according to (25), allowing the relative
strength of parameter estimates to be interpreted. We used the package MuMIn (26) to derive and
compare submodels from the initial global model. Models were compared to one another using
185
Akaike's Information Criterion corrected for small sample sizes (AICc). A subset of 'top models' were
defined by taking the best model (the model with the lowest AICc value) and any models within
2AICc units of the best model (following (24)). Using this subset of 'top' models, we computed the
average parameter estimates for each term included in the subset of models, as well as the relative
importance of the term. Importance is calculated by summing the Akaike weights of all models where
190
the term in question is included in the model. Akaike weights represent the probability of a given
model being the true model (compared to other candidate models in the set) (24). Importance can
therefore be thought of as the probability that the term in question is a component of the best model All data were analysed using R version 3.4.1 (http://www.r-project.org). To determine the relative
importance of the explanatory terms included in each model, we used an information-theoretic
approach with model averaging as described in (23). Under an information- theoretic approach, a
series of candidate models are generated, with each model representing a biological hypothesis. Results The mean amount Player 1 donated to Player 2 in the current study was $0.13 ± 0.00 (the same values
were obtained when calculated separately for eyes and flowers conditions), corresponding to 26 % of
the stake. The median donation was $0.10 (the same value was obtained when calculated separately 200 for eyes and flowers conditions) and the modal donation for each image type was $0.00 (Figure 1). for eyes and flowers conditions) and the modal donation for each image type was $0.00 (Figure 1). (i) Do eye images reduce variance in donation amount or propensity to keep the entire
donation? Our data did not support the hypothesis that variance in donation amount is reduced when
205
subjects view eye images as opposed to control images (Levene's Test for homogeneity of variance:
df = 1, F value = 0.61, P = 0.43) Neither did the presence of eye images significantly reduce Player
1's tendency to keep the entire endowment. Using data just from the control conditions, 84 / 194 (43.3
%) participants who saw eye images donated nothing to the receiver, compared with 79 / 193 (40.9
%) who saw flowers (Chi-squared test: X-squared = 0.14, df = 1, P = 0.71). 210 (ii) Do eye images increase compliance with a descriptive norm? First, we established how successful our control treatments were at evoking compliance relative to
the matched control treatment. In the “Give Half” norm treatment 72 / 197 (36.5 %) players
complied with this norm, which was not significantly different to the 65 / 195 (33.3 %) who gave
215
half the matched control (Chi-squared test: X-squared = 0.32, df = 1, p = 0.57). For the “Give
Something” norm treatment, however, our norm information did alter players' behaviour relative to
the matched control: 134/195 (68.7 %) players who saw the norm information complied compared
with 111/192 (57.8 %) players in the matched control (Chi-squared test: X-squared = 4.49, df = 1, p
= 0.03). 220 Compliance with the “Give Half” norm was not affected by the presence of eye images, or by an
interaction between seeing the image and the norm in conjunction (Table 1). There was a slight
positive effect of the norm information on the tendency to give at least half the endowment to the Compliance with the “Give Half” norm was not affected by the presence of eye images, or by an
interaction between seeing the image and the norm in conjunction (Table 1). There was a slight
positive effect of the norm information on the tendency to give at least half the endowment to the
receiver, but the confidence intervals associated with this effect included zero. These patterns were
225
largely the same for the model investigating compliance with the “Give Something' norm (Table 2). Compliance with the “Give Half” norm was not affected by the presence of eye images, or by an
interaction between seeing the image and the norm in conjunction (Table 1). There was a slight
positive effect of the norm information on the tendency to give at least half the endowment to the
receiver, but the confidence intervals associated with this effect included zero. These patterns were
225
largely the same for the model investigating compliance with the “Give Something' norm (Table 2). (iii) Do eye images increase mean donations? Looking at just the control data, eye images had a slightly negative effect on the donation amount
(Table 3), though the confidence intervals associated with this effect are also consistent with the
230
hypothesis that image type has no effect on donation amount. 230 Discussion This study adds to the growing literature on the effect (or lack thereof) of eye images on prosocial
behaviour (6). In contrast to the prediction outlined in (5), which suggested that eye images encourage
235
people to comply with social norms rather than to make higher donations per se, here we found no
evidence that eye images promote normative behaviour. While we were able to alter players'
behaviour (in one of the experimental conditions) by giving information about how other people
typically behave in this game, the addition of eye images to this norm information did not increase This study adds to the growing literature on the effect (or lack thereof) of eye images on prosocial
behaviour (6). In contrast to the prediction outlined in (5), which suggested that eye images encourage
235
people to comply with social norms rather than to make higher donations per se, here we found no
evidence that eye images promote normative behaviour. While we were able to alter players'
behaviour (in one of the experimental conditions) by giving information about how other people
typically behave in this game, the addition of eye images to this norm information did not increase
compliance with the norm relative to neutral images of flowers. Moreover, we found no evidence to
240
suggest that eye images increased the propensity to make a non-zero donation to the receiver, or that
eye images reduced variation in donation amount. These results, together with those from other
studies (reviewed in (6)), call into question the idea that artificial surveillance sues promote prosocial
behaviour and suggest that, if they do, they only do so under a restricted set of condition (which so
far remain elusive). 245 compliance with the norm relative to neutral images of flowers. Moreover, we found no evidence to
240
suggest that eye images increased the propensity to make a non-zero donation to the receiver, or that
eye images reduced variation in donation amount. These results, together with those from other
studies (reviewed in (6)), call into question the idea that artificial surveillance sues promote prosocial
behaviour and suggest that, if they do, they only do so under a restricted set of condition (which so
far remain elusive). 245 compliance with the norm relative to neutral images of flowers. Discussion Moreover, we found no evidence to
240
suggest that eye images increased the propensity to make a non-zero donation to the receiver, or that
eye images reduced variation in donation amount. These results, together with those from other
studies (reviewed in (6)), call into question the idea that artificial surveillance sues promote prosocial
behaviour and suggest that, if they do, they only do so under a restricted set of condition (which so
far remain elusive). 245 Eye images did not increase compliance with either the 'give half' or the 'give something' norm. The
analyses suggest that our “Give Half” information was unsuccessful at promoting compliance. On the
other hand, our other “Give Something” norm manipulation was effective: telling players that most
people give at least some of the endowment to the receiver in this game did decrease the dictator’s
250
tendency to keep the entire endowment. Adding eye images to this information, however, did not 250 have an additive effect on compliance. It is not clear why our 'give something' norm was more
effective at increasing compliance than the 'give half' norm but one plausible explanation is that
people can comply with a ‘give something’ norm for a smaller cost than is involved in complying
with a ‘give half’ norm. Previous work has shown that subject compliance with normative information
255
declines as the cost of compliance increases (28). have an additive effect on compliance. It is not clear why our 'give something' norm was more
effective at increasing compliance than the 'give half' norm but one plausible explanation is that
people can comply with a ‘give something’ norm for a smaller cost than is involved in complying
with a ‘give half’ norm. Previous work has shown that subject compliance with normative information
255
declines as the cost of compliance increases (28). 255 It is not yet clear why some studies find a positive effect of eye images on prosocial behavior, while
others fail to replicate this effect. It has been suggested that Dictator Games with low stakes typically
lead to a high proportion of players making a non-zero donation and so additional effects of eye
260
images on giving behavior would be difficult to detect (5). Discussion Notwithstanding that stake size may indeed
affect dictator donations (29), the data from this study showed that 296 / 779 of the players (38.0 %)
kept the entire endowment, which is a larger percentage than in the original Haley & Fessler study
(9) (where 30.6 % kept all the money), where a significant positive effect of eyes on probability of
giving something was detected. Thus, the small stakes used in MTurk studies are unlikely to be the
265
driving force behind the null effect of eye images in this setting. Another reason for the apparent inconsistency in watching eye effects is that the sample size is
typically far larger when recruiting subjects from the internet and so type I errors are far less likely
to occur than when smaller sample sizes are used. Studies conducted over the internet might also
270
differ in some fundamental way to those that are conducted under laboratory and field settings. For
example, Sparks & Barclay (30) suggest that frequent internet users' exposure to advertisements
online render them adept at screening out stimuli that are prominent but irrelevant to the task at hand
(e.g. our eye and flower images in this task). If this were the case then it might explain why data collected using MTurk have consistently yielded no meaningful effect of eye images on Dictator
275
Game giving. Alternatively, eye images might only increase prosocial behavior when the actor
perceives a real possibility of being observed by another individual (as suggested by(18,31)). Many collected using MTurk have consistently yielded no meaningful effect of eye images on Dictator
275
Game giving. Alternatively, eye images might only increase prosocial behavior when the actor
perceives a real possibility of being observed by another individual (as suggested by(18,31)). Many Dictator Games are conducted under conditions of anonymity but, in most cases, there is still an
experimenter in the room and even the presence of an uninvolved third-party (in the form of the
experimenter) has been shown to influence behavior in these types of game (1,32,33). By contrast,
280
studies conducted over the internet, or with manipulations that make participants feel unobserved
may reduce the feeling that behavior is being watched by others. A recent study conducted over the
internet found that eye images did increase Dictator Game giving (34). Discussion However, as the authors of
this study point out, subjects in their study may have felt less anonymous than MTurk workers
because (i) their identities were known to the experimenter and (ii) they also had to meet the
285
experimenter in person to collect their earnings from the game. The crucial question of whether eye
images only work when people feel their identity and behavior might really be revealed to others has
not been performed yet but may help to explain why there is apparently so much variation in the
effect of eye images on prosocial behavior. 280 290
By asking players about whether they had played a Dictator Game previously on MTurk, we were
also able to gain valuable information on whether Mturk workers are likely to have had experience
with this common game. We found that around a quarter of subjects had played a Dictator Game
before. This is consistent with a previous study which showed that, in a series of seven related
experiments conducted over the period of a few months, around 70 % of workers participated in only
295
one experiment (35). More importantly, we did not find any systematic effect of naïveté on subject
behavior in the Dictator Game. Again, this concurs with previous results (35), which show that
informed subjects' responses in traditional social psychology games are not qualitatively different to
those of naive subjects. Together, these results lend credibility to the assertions that data collected on 290 By asking players about whether they had played a Dictator Game previously on MTurk, we were
also able to gain valuable information on whether Mturk workers are likely to have had experience
with this common game. We found that around a quarter of subjects had played a Dictator Game
before. This is consistent with a previous study which showed that, in a series of seven related
experiments conducted over the period of a few months, around 70 % of workers participated in only
295
one experiment (35). More importantly, we did not find any systematic effect of naïveté on subject
behavior in the Dictator Game. Again, this concurs with previous results (35), which show that
informed subjects' responses in traditional social psychology games are not qualitatively different to
those of naive subjects. Discussion Together, these results lend credibility to the assertions that data collected on MTurk are comparable to those collected using traditional undergraduate samples and, in many cases,
300
may be more valuable since a more diverse slice of the true population can be sampled (Behrend et
al., 2011; Berinsky et al., 2012; Horton et al., 2010; Suri & Watts, 2011) (17,35). MTurk are comparable to those collected using traditional undergraduate samples and, in many cases,
300
may be more valuable since a more diverse slice of the true population can be sampled (Behrend et
al., 2011; Berinsky et al., 2012; Horton et al., 2010; Suri & Watts, 2011) (17,35). The evidence presented here is fairly unequivocal: in an online Dictator Game setting, eye images do
not decrease variance in donation amount; do not increase the likelihood of non-zero donations; and
305
do not increase compliance with descriptive norms. We therefore reiterate that the current evidence
supporting a positive effect of artificial surveillance cues on prosocial behaviour is consistent with
the hypothesis that the watching eye effect does not exist – or, if it does, it is fragile and highly
context-specific. 305 310 315 320 325 Parameter
Estimate
Unconditional
SE
Confidence Interval
Importance
Intercept
-0.46
0.13
(-0.71, -0.21)
Treatment (1 = norm)
0.05
0.14
(-0.22, 0.32)
0.32
Table 1. Full model-averaged estimates, unconditional standard errors, confidence intervals and
relative importance for variables included in the top model set for the model investigating whether
subjects complied with the “Give Half” norm. Image ID was included as a random term in the model. 330
Parameter
Estimate
Unconditional
SE
Confidence Interval
Importance
Intercept
0.40
0.19
(0.02, 0.77)
Treatment (1 = norm)
0.37
0.29
(-0.20, 0.93)
0.77
Image type (1 = eyes)
-0.05
0.14
(-0.31, 0.22)
0.26
Table 2. Full model-averaged estimates, unconditional standard errors, confidence intervals and
relative importance for variables included in the top model set for the model investigating whether
335
subjects complied with the “Give Something” norm. Image ID was included as a random term in the
model. Parameter
Estimate
Unconditional
SE
Confidence Interval
Importance
Intercept 1|2
-0.31
0.12
(-0.54, -0.08)
Intercept 2|3
3.45
0.30
(2.86, 4.03) Parameter
Estimate
Unconditional
SE
Confidence Interval
Importance
Intercept
-0.46
0.13
(-0.71, -0.21)
Treatment (1 = norm)
0.05
0.14
(-0.22, 0.32)
0.32 0.32 Table 1. Figure 2. Mean donation amounts ($, ± SEM) for eye and flower images, respectively, according to
whether participants saw the “Give Half” or the “Give Something” norm information. Discussion Full model-averaged estimates, unconditional standard errors, confidence intervals and
relative importance for variables included in the top model set for the model investigating whether
subjects complied with the “Give Half” norm. Image ID was included as a random term in the model. 330 Parameter
Estimate
Unconditional
SE
Confidence Interval
Importance
Intercept
0.40
0.19
(0.02, 0.77)
Treatment (1 = norm)
0.37
0.29
(-0.20, 0.93)
0.77
Image type (1 = eyes)
-0.05
0.14
(-0.31, 0.22)
0.26 Estimate
Unconditional
SE
Confidence Interval
Importance Table 2. Full model-averaged estimates, unconditional standard errors, confidence intervals and
relative importance for variables included in the top model set for the model investigating whether
335
subjects complied with the “Give Something” norm. Image ID was included as a random term in the
model. Parameter
Estimate
Unconditional
SE
Confidence Interval
Importance
Intercept 1|2
-0.31
0.12
(-0.54, -0.08)
Intercept 2|3
3.45
0.30
(2.86, 4.03)
Image type (1 = eyes)
-0.01
0.11
(0.20, -0.22)
0.27 Estimate
Unconditional
SE
Confidence Interval
Importance 0.27 Table 3. Full model-averaged estimates, unconditional standard errors, confidence intervals and
340
relative importance for variables included in the top model set for the model investigating donation
amount. Donation amount was converted into a 3-level ordered categorical variable, based on the
interquartile range of the distribution of the data. Image ID was included as a fixed term in the model. Table 3. Full model-averaged estimates, unconditional standard errors, confidence intervals and
340
relative importance for variables included in the top model set for the model investigating donation
amount. Donation amount was converted into a 3-level ordered categorical variable, based on the
interquartile range of the distribution of the data. Image ID was included as a fixed term in the model. 340 345 350
Figure 1. Images used in this study. 350 Figure 1. Images used in this study. Give Half
Give Something
Norm Information
donation Amount ($)
0.00
0.05
0.10
0.15
0.20
Eye Image
Flower Image Give Half
Give Something
Norm Information
donation Amount ($)
0.00
0.05
0.10
0.15
0.20
Eye Image
Flower Image Eye Image
Flower Image Norm Information 355 Figure 2. Mean donation amounts ($, ± SEM) for eye and flower images, respectively, according to
whether participants saw the “Give Half” or the “Give Something” norm information. Figure 2. Mean donation amounts ($, ± SEM) for eye and flower images, respectively, according to
whether participants saw the “Give Half” or the “Give Something” norm information. Figure 2. Discussion Mean donation amounts ($, ± SEM) for eye and flower images, respectively, according to
whether participants saw the “Give Half” or the “Give Something” norm information. 360 References References
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ASIANetwork Exchange: A Journal for Asian Studies in the Liberal Arts Full Issue PDF Spring 2014 Volume 21 Number 2
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ASIANetwork Exchange | Spring 2014 | volume 21 | 2 Editors Erin McCarthy, St. Lawrence University
Lisa Trivedi, Hamilton College Erin McCarthy, St. Lawrence University
Lisa Trivedi, Hamilton College Advisory Board Donald Clark, Trinity University
Lucien Ellington, University of Tennessee at Chattanooga
Carol Gluck, Columbia University
Rita Kipp, Georgian Court University
Barbara Metcalf, University of California, Davis
Henry Rosemont, Jr., Brown University
Harold Roth, Contemplative Studies Initiative, Brown University Editorial Board Timothy Cheek, University of British Columbia
Ben Dorman, Nanzan University
Steve Emmanuel, Virginia Wesleyan University
Pat Giersch, Wellesley College
Jih-Un Kim, Webster University
Siti Kusujiarti, Warren Wilson College
Darrin Magee, Hobart and William Smith Colleges
Mary Ann Milford, Mills College
Brandon Palmer, Coastal Carolina University
Marjorie Rhine, University of Wisconsin, Whitewater
Laura Ring, University of Chicago
Paul Watt, Waseda University ASIANetwork Exchange: A Journal for Asian Studies in the Liberal Arts gratefully
acknowledges the significant support it receives from Hamilton College and St. Lawrence University. 2 | ASIANetwork Exchange | Spring 2014 | volume 21 | 2 Articles C
Articles
Notes from the Editors
1
The Marianna McJimsey Award Winning Paper:
Countless Ramayanas: Language and Cosmopolitan Belonging in
a South Asian Epic
4
Rafadi Hakim
Continuity and Evolution: The Idea of “Co-creativity” in
Chinese Art
15
Jinli He
Re-Examining Extreme Violence:
Historical Reconstruction and Ethnic Consciousness in
Warriors of the Rainbow: Seediq Bale
24
Chia-rong Wu
Special Section Spring 2014: Teaching Modern Asian History Guest Edited by
Brian Caton
Introduction to ANE special issue
33
Teaching East and Southeast Asia through Asian Eyes
36
Tracy C. Barrett
Teaching South Asia beyond Colonial Boundaries
45
Brian Caton
Teaching Central Eurasia in Undergraduate Survey Courses:
Problems and Strategies
54
Amy Kardos
Teaching the History of Women in China and Japan: Challenges
and Sources
63
Danke Li
Book and Media Reviews
Book Review: A Guide to Asian Philosophy Classics
74 Contents
Articles
Notes from the Editors
1
The Marianna McJimsey Award Winning Paper:
Countless Ramayanas: Language and Cosmopolitan Belonging in
a South Asian Epic
4
Rafadi Hakim
Continuity and Evolution: The Idea of “Co-creativity” in
Chinese Art
15
Jinli He
Re-Examining Extreme Violence:
Historical Reconstruction and Ethnic Consciousness in
Warriors of the Rainbow: Seediq Bale
24
Chia-rong Wu
Special Section Spring 2014: Teaching Modern Asian History Guest Edited by
Brian Caton
Introduction to ANE special issue
33
Teaching East and Southeast Asia through Asian Eyes
36
Tracy C. Barrett
Teaching South Asia beyond Colonial Boundaries
45
Brian Caton
Teaching Central Eurasia in Undergraduate Survey Courses:
Problems and Strategies
54
Amy Kardos
Teaching the History of Women in China and Japan: Challenges
and Sources
63
Danke Li
Book and Media Reviews
Book Review: A Guide to Asian Philosophy Classics
74 Chia-rong Wu Chia-rong Wu Chia-rong Wu Contents
Special Section Spring 2014: Teaching Modern Asian History Guest Edited by
Brian Caton
Introduction to ANE special issue
33
Teaching East and Southeast Asia through Asian Eyes
36
Tracy C. Barrett
Teaching South Asia beyond Colonial Boundaries
45
Brian Caton
Teaching Central Eurasia in Undergraduate Survey Courses:
Problems and Strategies
54
Amy Kardos
Teaching the History of Women in China and Japan: Challenges
and Sources
63
Danke Li
Book and Media Reviews
Book Review: A Guide to Asian Philosophy Classics
74 Danke Li 74 Notes from the Editors The annual ASIANetwork conference, held this year in Bloomingdale, Illinois, was as
stimulating as it was affirming of our shared goals as educators in liberal arts institutions. The conference opened with a provocative Keynote Address by Peter Hershock (East-West
Center) titled “Valuing Diversity: Buddhist Reflections on Equity and Education,” which
will be featured in our Fall 2014 issue. Not only did Hershock’s address elicit lively discus
sion afterwards, it provided food for thought over the course of the entire conference. The
next morning attendees were treated by Robert Buswell, a past president of the AAS and
UCLA Professor of Buddhist Studies, to a Plenary Address, “Transplanting Buddhism to the
Korean Peninsula: Cultural Interchanges across Asia.” After a day of wide-ranging panels,
the conference’s formal evening featured a musical performance by Gao Hong and the
Carleton College Choral Ensemble, and a reception sponsored by the University of Macau
and the United Board. The membership recognized the extraordinary service of two of ASI
ANetwork’s most dedicated members: Teddy Amoloza, who steps down this year after serv
ing as executive director for three terms, and Van Symons, a founder, Executive Director
and, most recently, the point person for our Student-Faculty Fellows program. There were
moving tributes to both of our esteemed colleagues, and we returned from the conference
not only invigorated by the level of critical engagement, but also renewed by the extraordi
nary sense of community and common purpose we share.hii This is the final issue of our first three-year term as Editors. Having the opportunity
to work together at transitioning this publication through great change—from print to
electronic form—has been exceptionally rewarding for both of us. Having the support and
counsel of past Editors Tom Lutze and Irv Epstein, as well as Teddy Amoloza, enabled us
to successfully meet our goals for the publication thus far. The Board of ASIANetwork,
particularly its Publicity Committee, has also sustained our efforts to transition the journal
to support a blind peer-review process that has transformed the quality and quantity of the
work that we feature. We are grateful to the Board and incoming Executive Director Gary DeCoker for their
support as we continue our work for a second term as Editors. Now that the online system
is established and running, we have another set of goals for the journal that we would like
to share. Contents ASIANetwork Exchange | Spring 2014 | volume 21 | 2 1 | Notes from the Editors Notes from the Editors ASIANetwork is offering this service at cost, and so far (depending on the
length of the journal and the number of images) the price of each issue is averaging around
$20 (plus shipping). To order a copy, simply go to http://www.magcloud.com/browse/maga
zine/751856 and follow the instructions. This issue begins with the 2014 Marianna McJimsey Award winning essay, “Count
less Ramayanas: Language and Cosmopolitan Belonging in a South Asian Epic” by Rafadi
Hakim. A 2013 graduate of Carleton College’s Department of Sociology and Anthropol
ogy, Hakim closely explores the Kiski Kahani project located in Pune, India. Kiski Kahani
compiles fragmentary stories of the Ramayana, perhaps Hinduism’s most popular epic,
often told in Hindi or Marathi. Challenging Hindu-nationalist versions of the epic, Kiski
Kahani privileges improvised, local retellings, and publishes them in English so that they
are available to India’s large English-speaking population. Jinli He’s ““Continuity and Evolu
tion: The Idea of ‘Co-creativity’ in Chinese Art” explores the principle of “Co-creativity” in
both traditional and contemporary art. Drawing upon the recent work of feminist artists in
China, He’s article demonstrates this enduring characteristic of Chinese art. In “Re-exam
ining Extreme Violence: Historical Reconstruction and Ethnic Consciousness in Warriors
of the Rainbows: Seediq Bale,” Chia-rong Wu considers the cultural and political context of
beheading in Seediq culture and how filmmaker Wei Te-sheng depicts this in film. In doing
so, Wu explores how violence in the film Seediq Bale expresses both contemporary colonial
domination of Aboriginal peoples and the conflicts that exist within these communities
themselves. The issue continues with a special section on Teaching Modern Asian History, guest
edited by Brian Caton, Luther College. Developed from a pedagogical panel at the annual
meeting of the American Historical Association, this section features articles by four
historians whose expertise spans Chinese and Japanese history to Eurasia and from South
Asia to Southeast Asia. Each author carefully considers how to select appropriate primary
sources and develop exercises for students in introductory liberal arts college courses. Tracy
Barrett (North Dakota State University) recommends that scholar-teachers utilize a range
of sources on Southeast Asia, including films, memoirs, and fiction, so that students gain
exposure to Asian voices. Brian Caton suggests the use of longer extracts from primary
sources to aid in the understanding of material, social, and cultural life in South Asia that
often lay beyond the reach of colonial authority. Notes from the Editors First, we hope to focus on building a larger pool of outstanding reviewers. Provid
ing constructive criticism to our peers is a key professional responsibility. By agreeing to
review submissions for the journal, reviewers directly impact the quality of the pieces that
are selected for publication. If you are reading this and you have not registered as a reader
and as a reviewer in our system, please do so. With every submission that we receive, we
look for readers who have the skills and perspective to improve the work of our colleagues
and ready it for publication. Second, we hope to develop a broader active readership among
faculty within the ASIANetwork and beyond. Third, we are delighted to have seen such a
dramatic and steady increase in our submissions over the last two years. Our colleagues
in the China field in particular have embraced the new opportunities that the journal now
offers as a blind peer-reviewed publication. We want to call upon colleagues in other fields
to join them in this collective project, whether by submitting a research paper, a pedagogi 1 | Notes from the Editors ASIANetwork Exchange | Spring 2014 | volume 21 | 2 cal essay, a book/or media review, or by becoming a Guest Editor of a special section. It is
critical that ASIANetwork Exchange remain a journal for faculty engaged in research and
teaching in all fields of Asian Studies. With the publication of this issue, we are also launching two new features of the journal
for our readers. First, a “Books for Review” section will be accessible online through the
journal website. Here, readers will be able to browse the books we have available for review. If you wish to review a book, please send the editors an email (editors@asianetworkex
change.org) accompanied by a two-page CV. The second new feature is that members are now able to order bound, full color, print
copies of the journal. While it is already possible to print out high quality PDFs of indi
vidual articles as well as the full issue directly from the website at no charge, many mem
bers expressed a desire for a bound copy of the journal. As we announced at the Business
Meeting, we have found a print-on-demand service where members can order individual
bound copies. 3 | Notes from the Editors Erin McCarthy and Lisa Trivedi, Editors Rafadi Hakim graduated from
the Department of Sociology and
Anthropology at Carleton Col
lege, Minnesota, in 2013. Notes from the Editors Amy Kardos (University of Texas, San
Antonio) shows us how new scholarship on the silk road allows us to rethink the role,
influence, and initiative of Eurasian brokers in trade. And, finally, Danke Li (Fairfield
University) attempts to shake up conceptions of Chinese and Japanese women with primary 2 | Notes from the Editors ASIANetwork Exchange | Spring 2014 | volume 21 | 2 sources that are both textual and cinematic so that students hear the strong feminist voices
of women who have led many movements for social justice. Together, this special section
offers all Asianists important tools for approaching the broad content of Asia survey courses
that are the foundation of our Asian Studies programs. We are delighted to have had the
opportunity to work closely with Caton and his authors on this section. The issue concludes with Jason M. Wirth’s review of Puqun Li’s A Guide to Asian Philoso
phy Classics (with Arthur K. Ling), which provides a detailed overview of the many possi
bilities this important volume has to offer to courses on Asian Philosophy. Erin McCarthy and Lisa Trivedi, Editors ASIANetwork Exchange | Spring 2014 | volume 21 | 2 The Marianna McJimsey Award Winning Paper:
Countless Ramayanas: Language and Cosmopolitan Belonging in
a South Asian Epic Rafadi Hakim The Marianna McJimsey Award recognizes the best undergraduate student
paper dealing with Asia. The prize honors the outstanding service of Marianna
McJimsey, the first Executive Director of ASIANetwork and the first editor of
the ASIANetwork Exchange. For more information see http://asianetwork.org/
programs/the-marianna-mcjimsey-award/ Abstract: The Kiski Kahani project in Pune, India, is a not-for-profit program
that compiles stories of the Ramayana, a South Asian epic, and publishes them
in English. Kiski Kahani’s ideology rejects the Hindu nationalist master narrative
of the Ramayana, and privileges an emerging genre that consists of fragmentary,
improvised stories of the epics. As a socially-grounded language practice, Kiski
Kahani’s retellings are rooted in pan-Indian, cosmopolitan modalities that index a
sense of belonging to a pluricultural nation: the use of English rather than Hindi or
Marathi, and a curation of stories from diverse Indian regions and languages. Keywords India; Maharashtra; Ramayana; language; cosmopolitanism; genre and
intertextuality INTRODUCTION: WHOSE RAMAYANA?h The stories of the Ramayana are still alive in India. Although modern textual scholars
agree that the epic was first composed between 400 B.C.E. and 4th century C.E. (Brocking
ton 1998, 26-27), the Ramayana finds its way to countless channels of transmission even in
contemporary urban India: TV series, comic books, plays, novels, popular cinema, people’s
names, and figures of speech. Among their innumerable retellings, Ramayana stories are
alternately expressed as a battle between Rama, a god incarnated as a king on Earth, and
Ravana, the demon king; as a story of love and devotion between Rama and Sita, his wife;
and as a story of friendship between two brothers, Rama and Lakshmana, and Hanuman,
a simian god. Because Ramayana stories are told so variously and through so many chan
nels, people who grew up with these narratives modify, extend, interrupt, question, criticize,
forget, and remember them in countless different ways. Since the rise of Hindu nationalism in the 1980s, questioning and retelling the Rama
yana has become politically charged. Right wing, Hindu nationalist movements in India
evoke the Ramayana as an exclusively Hindu text and as a sacred, historical narrative in
religious and political debates. In 1992, Hindu nationalist activists demolished the Babri
Mosque, which was built over the supposed ruins of Lord Rama’s birthplace in Ayodhya,
Uttar Pradesh, and triggered communal riots that killed thousands of Muslims and Hin
dus across India. In 2011, an academic committee at Delhi University removed an essay
written by A.K. Ramanujan, a renowned Indian-American folklorist, poet, and intellectual, 4 | Countless Ramayanas ASIANetwork Exchange | Spring 2014 | volume 21 | 2 from the university’s undergraduate history syllabus. The Akhil Bharatiya Vishwa Parishad
(ABVP), a Hindu nationalist student organization, deemed Ramanujan’s “Three Hun
dred Ramayanas” (1991), which discusses the diversity of Ramayana stories in South and
Southeast Asia, offensive towards the divine characters of the epic.1 In the essay, Ramanujan
describes Hanuman, a deity worshipped throughout the subcontinent, as a “ladies’ man” in
the Thai story (44), while Sita, often the Hindu paragon of fidelity, is depicted among the
indigenous Santals of central India as Rama’s unfaithful wife (44). Hindu nationalists allow only a single version of the Ramayana stories to be discussed
in the public sphere: one that elevates the sacredness of Hindu divinities, especially Rama,
beyond questioning. INTRODUCTION: WHOSE RAMAYANA?h However, with all the debates surrounding the idea of a “proper” and
“true” Ramayana, which Ramayana is being talked about, and how is the Ramayana debated
in modern India? While I acknowledge that the Ramayana has been used to muster sectar
ian violence and to construct a vision of Hindu superiority by right wing, Hindu nationalist
interest groups, I emphasize that retellings of the epic can resist such hegemonic narra
tives. Kiski Kahani, a Hindi phrase which means “Whose story?,” is a not-for-profit public
program in Pune, Maharashtra, that gathered little-known and emerging retellings of the
Ramayana and circulated them through printed pamphlets and website posts throughout
2012 and the first half of 2013. This study, written after two months of ethnographic field
work, explains how Kiski Kahani’s retellings of the Ramayana illustrate ways of belonging to
a cosmopolitan, pluricultural Indian nation. LANGUAGE, INTERTEXTUALITY, AND PERFORMANCEi Understanding the cultural significance of Ramayana stories requires understanding
how a story’s meaning comes from a history of interrelated narratives, or, in Becker’s (1996)
words, “prior texts.” A retelling of the Ramayana, in other words, is not a genealogical
ancestor to subsequent retellings; rather, because of the fluidity of the epic genre, retellings
represent historically situated crystallizations of stories from a common pool of narratives
(Doniger 1988, Doniger O’Flaherty 1991, Narayan 1989, Ramanujan 1991, Richman 1991). The creation of relationships between texts, or intertextuality, explains how any particular
text is created from existing language practices and socially positioned (Bauman and Briggs
1992, Bauman 2004). Simply “counting” texts as discrete units does not allow us to see how
diversity across Ramayana stories illuminates their social contexts. One must pay attention
to the narratives in context. In this paper, I use concepts of “speech events” and “ethnography of speaking” from the
field of linguistic anthropology that emphasize socially grounded understandings of speech
and language (Bauman and Briggs 1992, Hymes 1964). The practice of using language
and language styles invites commentaries on the practice itself, or metapragmatic com
mentaries. I draw upon the concept of metapragmatic awareness, which is the “ability that
speakers have to talk about the pragmatics of their language use” (Silverstein 1981, as cited
in Duranti 2009, 16). In such “talk about talk,” we find socially situated understandings
about the use of language and language styles. Attention to collective beliefs about language
has given rise to contemporary scholarship on language ideology, which focuses on the
importance of bridging everyday speech-events with their macrosociological consequences
(Irvine and Gal 2009, Kroskrity 2000, Woolard and Schieffelin 1994). In this study, I draw
upon common sense ideas about Marathi, Hindi, and English as languages that my inter
locutors use in their daily speech and writing. The choice of language, language variety, or
language register is an element of communication that indexes social meaning. Indexical relationships between language use and communities of practice, however, do 5 | Countless Ramayanas ASIANetwork Exchange | Spring 2014 | volume 21 | 2 not form a linear correspondence. Against Anderson’s (1983) notion of organized linguistic
unity as the basis of imagined nationhood, scholarship on language ideology disputes a one-
to-one relationship between language and identity. Instead, language ideology scholarship
emphasizes the empirically multilingual and heteroglossic character of speech communi
ties (Irvine and Gal 2009). BELONGING AND COSMOPOLITANISM IN MAHARASHTRA In the Indian state of Maharashtra, where most of Kiski Kahani’s activities are con
ducted, Ramayana stories are situated at an intersection between ethnoregional and
national imaginings. Hindu nationalist and Maharashtrian, Marathi-speaking regionalist
movements, produce narratives of mytho-historical heroic figures, such as those of Rama
and Shivaji, which serve as a symbol of ethnolinguistic, caste, and religious allegiances
(Laine 2003; Hansen 2001, 20-22). Thus, the presence of caste, language, ethnic, and
religious diversities demands attention in the case of the heterogeneous audience who are
listeners of Kiski Kahani’s Ramayana stories. In mediating these tensions between the par
ticular against the universal, and the plural against the exclusivist, concepts of vernacular
cosmopolitanism accommodate culturally committed loyalties combined with translocal,
deterritorialized senses of belonging (Bhabha 1996; Pollock et al. 2000; Werbner 2008, 14). My argument contextualizes cosmopolitanism not as a “destination” for a cultural trajec
tory, but as a way of becoming, doing, rehearsing, and performing culture from a situated
historical conjuncture. LANGUAGE, INTERTEXTUALITY, AND PERFORMANCEi In South Asia, and in southern India in particular, the choice of
language and language registers plays a major role in forming ethnic identities and index
ing different audiences (Bate 2009, Mitchell 2009). Within a communicative practice, such
as the retelling of Ramayana stories, ethnolinguistic associations depend on relationships
between textual units, the performer of the text, the audience, and the “nonliterary” inter
pretive frames of performance surrounding the text (Bauman 1984, Bauman and Briggs
1992, Bauman 2004, Goffman 1974). However, because “texts” of the Ramayana stories are
interrelated, retellers and performers are not confined to producing new texts; rather, they
constitute the audience of retellings, or “consumers” of the stories, as well. In this study, critical attitudes towards the hegemonic, Hindu nationalist interpretations
of the Ramayana that govern the literary and performative elements of a retelling are key in
understanding the epic’s different audiences. Turner’s (1986) concept of performative reflex
ivity illustrates that performances of these narratives are not mere reflections of authority,
but are often commentaries on dominant sociopolitical ideologies. Performers of a story
do not merely reflect a text, but they “turn, bend, or reflect back upon themselves, upon the
relations, actions, symbols, meanings, codes, roles…and other sociocultural components”
(Turner 1986, 24). Therefore, to recognize the agency of retellers of the Ramayana stories as
performers and critics, I emphasize the life histories and ideological configurations of my
interlocutors that socially situate their textual productions. 6 | Countless Ramayanas PLACING LANGUAGES IN PUNE In October 2011, the Center for Open Society (COS), the public outreach section of a
nonprofit organization, received a grant from a European development agency for the Kiski
Kahani project to be conducted throughout 2012.2 COS operates out of a converted apart
ment in the Erandwane neighborhood in the western half of Pune, which is predominantly
upper-middle class and Marathi-speaking. The apartment complex is typical of the so-called
“housing societies” in major Indian cities, where two or three apartment buildings housing
about twelve nuclear families each are located in a walled compound. At COS’s compound, 6 | Countless Ramayanas ASIANetwork Exchange | Spring 2014 | volume 21 | 2 a guard, or “watchman,” is present throughout the day, paying attention to passersby and
cricket-playing children while reading a copy of Sakal, Pune’s most popular Marathi daily. Upstairs, in a small, two-room library, COS’s subscription to six English-language news
papers nourishes thirsty young minds from nearby universities, and watching jeans-clad
youth immersed in reading a Hindi or Marathi book is an exception rather than the norm. About 300 meters away towards the main road, a “thali” or full-service restaurant
describes its cuisine as authentically Maharashtrian and provides only menus printed in
Marathi, yet, just across the road, a stall that serves noodles and sandwiches for students
is run by a couple who always speaks in Hindi to their multiethnic customers. An article
in the Pune Mirror, an English-language daily newspaper, describes the rapid change in
Senapati Bapat Road, an adjacent neighborhood, from a residential district to a commercial
area in terms of a language shift: “Now, you hardly hear people speak Marathi around here”
(July 12, 2012). The city’s geography evokes ethnolinguistic associations, especially with the
other, eastern half of Pune housing an established population of Urdu-speaking Muslims,
Konkani-speaking Goan Catholics, and Gujarati-speaking Parsis in the neighborhoods sur
rounding the former British military cantonment area (Hansen 2005). Pune is home to approximately five million people, and is considered the cultural
heart of Maharashtrian, Marathi-speaking culture (Laine 2003). However, the geographi
cal boundaries of Maharashtra were defined only in 1960 after the state of Bombay was
divided along linguistic lines. Maharashtra, which contains a majority of Marathi speakers,
was separated from Gujarat, a state with a Gujarati-speaking majority, although linguistic
allegiances are fluid and diverse even within the present-day state boundaries (Laine 2003,
10). PLACING LANGUAGES IN PUNE Pune asserts itself as a city that speaks Marathi, a Maharashtrian language. Street signs,
local newspapers, and government documents are first published in Marathi instead of in
Hindi or English. Nonetheless, Pune is home to a sizable Muslim, Christian, and Zoroas
trian population, whose identification with different languages puts them in contrast to the
Marathi-speaking, Hindu majority (Hansen 2005). Because regionalist political movements
have built upon Marathi as an identity marker for local Maharashtrians, and because Hindi
is associated with the ethnic groups of northern India, English is seen as a more “politically
neutral” medium, although not without the connotations of British colonial legacy (Ramas
wamy 1999, 354) and of privileging the modes of education primarily accessible to the
urban upper and middle classes (Azam et al. 2013). In mediating this ethnolinguistic diversity, COS primarily uses English, and occasionally
Hindi, in its programs and publications despite its location in a predominantly Marathi-
speaking city. In Pune, those who are not Marathi speakers experience a sense of alienation
from belonging to the area, and lose the capability to become completely “local,” as Aditi
Kulkarni, a frequent visitor of COS’s library who grew up in Mumbai, explains: I don’t know Marathi because there [in Mumbai] you don’t feel the need for
Marathi. Even the local people will talk to you in Hindi…. You can converse with
them. Here [in Pune] you feel the necessity, because [when] you know Marathi…
they listen to you, otherwise they hardly entertain you.... I don’t know Marathi because there [in Mumbai] you don’t feel the need for
Marathi. Even the local people will talk to you in Hindi…. You can converse with
them. Here [in Pune] you feel the necessity, because [when] you know Marathi…
they listen to you, otherwise they hardly entertain you.... For Kulkarni, the rootedness of an individual in Pune is expressed in terms of Marathi as a
medium of speech, without which even an Indian person could experience a sense of being
an outsider, whom nobody “listens to” and “entertains.” While Mumbai and Pune both
house a slight majority of Marathi speakers and a vast array of other ethnolinguistic groups
bound together by the common use of Hindi, Pune’s local identity, unlike that of Mumbai,
uses Marathi as a language of vernacular belonging. PLACING LANGUAGES IN PUNE 7 | Countless Ramayanas ASIANetwork Exchange | Spring 2014 | volume 21 | 2 While being “a Maharashtrian local” and being a “cosmopolitan Pune urbanite” entail
different arrays of loyalties that are more than just linguistic and geographic, using a lan
guage performs a sense of belonging to either of these loosely compartmentalized cultural
modes. Yet, at the same time, these ways of speaking are not purely utilitarian; they are not
used simply as strategic self-presentation in front of potential evaluators (Bauman 1984,
Goffman 1959), but are also common sense ideas about how peoples, spaces, ideas, and
actions are tied to these “local” or “cosmopolitan” modes. Why, therefore, does COS choose to communicate in English more extensively than in
Hindi? Although Hindi might seem to be a more “Indian” language, the adoption of English
in different mediums of communication throughout India indicates that English has been
rooted in nationwide language practices. Anjali Pradhan, who writes for Kiski Kahani’s
upcoming anthology of Ramayana stories from throughout India in English, disagrees with
the notion of the language as foreign to India: I speak Hindi and Marathi, but…the language in my heart is English. I’ve grown
up with English, I think in English, I dream in English, I speak English.... That’s
my most...native language easily…. [Those who say that] you’re an English-speaker
[so] you can’t know the pulse of the country…I really think [that] is a lot of rub
bish.... My husband speaks Konkani.... His Hindi is appalling, but he communicates
with everybody…and almost all of his writing was with people who did not speak
English. If you have empathy with the people you’re talking to…you’ll always find
a way to communicate…. I’ve found a lot of people who are…fluent reading, writ
ing, speaking Hindi, and they know nothing about this country, and they just don’t
manage to connect with people on the street. As Pradhan explains, “empathy” is more important than fluency; both language and affec
tive attachments are key modalities in negotiating India’s ethnolinguistic and class diversity. For her, however, Hindi and English are not interchangeable, because they perform differ
ent roles. PLACING LANGUAGES IN PUNE English is an inward-looking medium of “thinking,” “dreaming,” and “speaking,
” while Hindi is a way of “knowing the country.” While Pradhan attaches affective ties to
English as her “most native” language, she does not disregard Indian languages as foreign or
unfamiliar, because no languages are exclusively “native” or exclusively “foreign” to her. f Pradhan explains Hindi as one way of establishing relations among people of different
subnational origins (“knowing the pulse of the country”) and different social classes (“peo
ple on the street”). I argue that it is not the simply the choice of a language that constitutes
her social relationships, but a consciousness of the common sense ideas and understandings
about language used in social interactions. Above all, Hindi and English carry intersecting
associations with the collective imagination of a multilingual Indian nation. Although it
is tempting to see English-speaking urban dwellers, such as Pradhan, as deterritorialized,
“floating” people with no allegiances to a locality and as less “authentically Indian,” her
comment on English as her “most native” language describes how simple divisions of lan
guages as local, rooted mother tongues and foreign languages are inaccurate in describing
COS’s and India’s polyglot identity. A Kiski Kahani pamphlet in English describes how the project is preoccupied with
imagining the Ramayana as a way of imagining India, the nation, as a place where a sense of
belonging is projected: 8 | Countless Ramayanas ASIANetwork Exchange | Spring 2014 | volume 21 | 2 The Ramayana was composed in Sanskrit some time between 200 BCE and 200
CE…. Over the centuries, the story has been re-told in almost all Indian lan
guages…. In fact, the Ramayana provides the metaphors through which Indians
understand themselves. Here are some of the many Ramayanas we know: Valmiki’s
Ramayana in Sanskrit, Tulsidas’s Ramcharitmanas in Awadhi Hindi, Kamban’s
Iramavataram in Tamil, the Adhyatma Ramayana in Malayalam, the Pothi
Ramayana in Urdu, the Krittibasi Ramayana in Bengali, Dastan-e-Ram-o-Sita in
Persian and Eknath’s Ramayana in Marathi called Bhavartha Ramayana. The writers, Haroon Sattar and Neha Madgulkar, conceptualize the pamphlet, “The
Ramayana Project: 300 Ramayanas & Counting,” as a nationwide project, and position
their writing as accommodating all of India’s plurality in interpreting and translating the
Ramayana stories. However, although this introduction highlights the Ramayana’s diversity
in terms of languages, the pamphlet is written entirely in English without any translation
into the Indian regional vernaculars of the individual stories. PLACING LANGUAGES IN PUNE Here, the Ramayana becomes
a translocal way of communication. Through these images of multiplicity, the publications
do not identify particular retellings as belonging to any specific social group. Instead, Kiski
Kahani advocates for transcending what appear to be ethnolinguistic and religious bounda
ries in the subcontinent. In conjunction with these common sense ideas about language, the life histories of the
two individuals who retell Ramayana stories at COS, Haroon Sattar and Neha Madgulkar,
are incorporated in how the stories are curated and produced. The Ramayana stories and,
more specifically, the Kiski Kahani retellings, are intricately tied to the personal journeys
and ideological articulations of its listeners and patrons. Eliciting life histories can illus
trate how individuals are agents that interact with and transform social structures, and
allow subjects to speak for themselves without completely surrendering narrative control
to the anthropologist (Fisher 1997, Marcus and Fischer 1986). Even commentaries on the
small sections and subplots of the Ramayana’s massive corpus are entangled in sociopoliti
cal debates about India-wide issues, such as the banning of Ramanujan’s essay. Drawing a
boundary between text and context, and, consequently, separating processes of contextu
alization and entextualization, underestimates the reflexivity between the two processes
(Bauman and Briggs 1990, 72-73). Therefore, in this study, I build upon the entanglements
of sociopolitical ideologies, life histories, and entextualization strategies. HAROON SATTAR: COMMUNICATING COSMOPOLITANISMS Haroon Sattar, Kiski Kahani’s project director, rewrites stories from different books and
anthologies published by Indian academic publishers. For Kiski Kahani programs, Haroon
gathers artists, writers, and theater directors to conduct workshops for the public. Among
fellow staff members at COS, Haroon is remarkable for maintaining his interest in the
“Hindu” epic in spite of his Muslim heritage, which is noticeable by the mere mentioning
of his name. Ashwini Parikh, a theatre specialist whom COS invites on a regular basis to
conduct workshops, once reminded me about how Kiski Kahani deals with imagining plu
ral religiosities: “After all, what is a Haroon Sattar doing with the Ramayana?” Haroon, who
grew up in Mumbai, Saudi Arabia, and Pune, classifies his own nuclear family’s linguistic
identity as English-speakers at home, and as Urdu- and Hindi-speakers outside. i Haroon, with his tightly-fitted jeans, checkered shirts, and bright red Hyundai hatchback
that he drives to the COS office, challenges those who classify the Ramayana as ancient, fos
silized, and trapped in the timeless realm of priests and academics. The Ramayana, accord 9 | Countless Ramayanas ASIANetwork Exchange | Spring 2014 | volume 21 | 2 ing to Haroon’s account, is part of growing up in India, of being exposed to a nationwide
literary culture, and of being able to appreciate a certain verbal artistry with language and
stories: All Indian kids grew up with Amar Chitra Katha [comic books]… It’s part of…
your… rite of passage….3 There was the Doordarshan Ramayana, which we all
knew about….4 And when this controversy happened, the [banning of the] A.K. Ramanujan essay, [those who banned the essay] were clearly saying that this text
does not belong to you anymore…. One of the big fears, for me, for any kind of oral
narrative is that it will get lost…. I’m really keen on…people talking and thinking
about…the diversity within the text. If you keep a text alive by reading and narrat
ing constantly, people will keep in touch with it…. For Haroon, the Ramayana is a set of texts that “all Indian kids” of his generation who are
within the reach of mass media encounter. Even into adulthood, the Ramayana stories
are narratives that Indians still “think” about and “keep in touch” with. What is politically
contentious, however, is identifying the “people” who belong to this tradition of Ramayana
stories and are legitimized to express ownership. HAROON SATTAR: COMMUNICATING COSMOPOLITANISMS As previously described in the opening of
the “300 Ramayanas” pamphlet, Haroon criticizes right wing interpretations, which advo
cate for a singular, authoritative Ramayana based on a conflation of Indian-nationalist and
Hindu allegiances. Nonetheless, Haroon argues for “keeping the text alive” by rethinking,
rewriting and retelling the Ramayana stories. For him, the Ramayana is an ever-growing
corpus of stories bound together by an embedded reflection of a cosmopolitan Indian iden
tity, and, concomitantly, a cosmopolitan way of communicating stories. How, therefore, do cosmopolitan aspirations become cosmopolitan modes of performing
culture without displacements, and without necessarily having the self-contained cultural
units that represent all the enumerated parts of the Indian nation? When Haroon describes
Ramayana stories as “mythology,” “metaphors,” and “language,” he works with an audience
aware of how the Ramayana is aesthetically evaluated through both literary and nonliter
ary framing devices. Both the holder of a master’s degree in English literature and a former
interior designer, Haroon not only archives and rewrites stories. He also spends many hours
on the colors and designs of COS publications, and strives to keep Kiski Kahani’s website
visually attractive. The Kiski Kahani pamphlet, on which he has worked intensively, shows
how graphic design and literary curation, which are key parts of Haroon’s own life history,
reframe Ramayana stories. A multitude of pictures are present on the cover: a classical, “calendar art” paint
ing of Rama, Sita, Lakshmana, and Hanuman in the middle; a Ramayana panel from the
English-language Amar Chitra Katha comic strip; a still from Ramanand Sagar’s (2001)
Hindi-language televised series; and another still picture from Nina Paley’s (2008) animated
retelling of the Ramayana through Sita’s story.5,6 This eclectic assemblage of images, which
spans the most “local” to the most “global” of Ramayana stories, mirrors the chaos of urban
India where the stereotypically old meets the stereotypically modern, and where stories are
decontextualized and recontextualized at a blinding pace as new forms of mass media are
invented. Through this mosaic-like construction, Haroon argues against a monolithic sense
of cultural ownership, and allows retellers to express their way of belonging to a cosmopoli
tan, pluricultural India. In line with his words, it is “reading” and “narrating”—the per
formative, audience-oriented aspects of the Ramayana—that characterize the making of a
Ramayana genre at COS’s Kiski Kahani. 10 | Countless Ramayanas ASIANetwork Exchange | Spring 2014 | volume 21 | 2 NEHA MADGULKAR: COMMUNICATING COSMOPOLITANISMS Neha Madgulkar, an undergraduate mass communications student and an intern for
Kiski Kahani, comes from a Hindu Maharashtrian family who moved around India con
stantly because of her father’s assignment as an air force officer. Having lived all over India,
Neha discovered different retellings of the Ramayana during her internship that she sees as
deviating from the Valmiki’s written, Sanskrit version, which is the authoritative text used to
retell the Ramayana in both Ramanand Sagar’s televised series (Lutgendorf 1995, 217) and
the Amar Chitra Katha comics (McLain 2009, 133-134). Neha frames the Ramayana as a
genre that accumulates new sets of stories throughout her life: When we were younger, we used to hear stories, because, we have…friends from
all over the country, and people from Tamil Nadu and they would say… ‘in my
home village they worship Ravana and I’ve seen this temple where they worship
Ravana….’ It’s typical of the epic, there are lots of stories running parallel to each
other…. By knowing what “deviates” from Valmiki’s text, Neha familiarizes herself with the eth
nic diversity of the Indian nation, as she explains that Tamil villages supposedly “worship
Ravana,” who is the archetypical, demonic character in many Ramayana stories. Neha’s
understanding of the epic through Kiski Kahani, with its capacity to accumulate stories,
often ones that are contradictory and conflicting on the surface, unites diverse ethnic and
religious strands under the umbrella of a common storytelling practice. Kiski Kahani,
therefore, allows these retellings, which diverge from the hegemonic, Hindu nationalist nar
ratives that center on devotion to Rama, to crystallize into a coherent, performable corpus. The Kiski Kahani stories, while fragmentary and evocative rather than continuous and
descriptive, represent a regime of cosmopolitanist ownership, where discourses of partial
senses of belonging override discourses of authenticity and cultural purity. THE MAKING OF A NEW GENRE: RAMAYANA STORIES WITHOUT AUTHORSf Kiski Kahani’s curation of Ramayana stories, which builds upon difference from Hindu
nationalist texts, is an attempt at widening intertextual gaps. The performances and the
publications are not projects to create a master narrative of the entire epic; rather, they play
upon the side stories and the characters of the Ramayana to entextualize cosmopolitan aspi
rations. As Ramanujan (1991) suggests, the structure of the Ramayana is such that stories
can always be inserted and crystallized within the epic, thereby interrupting authoritative
episodes with improvised episodes. In the following story, taken from the “300 Ramayanas” pamphlet, the notion of inde
pendent, singular authorship of any retelling of the Ramayana is challenged: After the epic battle between Rama and Ravana, Hanuman retreated to the moun
tains where he spent his time meditating and writing Rama’s illustrious deeds on a
rock, using his nails.7 In another part of the forest…Valmiki had just completed his own version of Rama’s
journey. Valmiki had been told that the mighty Hanuman would be the best critic
of his story, so he set out to climb the mountain…. As he reached the top of the
mountain, Valmiki saw, painstakingly etched on a rockface, a story so wondrous
that it made him weep. And weep, and weep and weep some more, partly because
the story was so beautiful but mostly because his own story was but a shadow of this 11 | Countless Ramayanas ASIANetwork Exchange | Spring 2014 | volume 21 | 2 one. Seeing the “rishi” [sage] reading his story with tears coursing down his cheeks,
Hanuman selflessly picked up the rock and with all his divine strength, and flung it
into the ocean. Taken aback, Valmiki swore to be reborn so that he may sing Hanuman’s
praises which he had understated…. Tulsidas, the great poet who wrote [the
Ramcharitmanas], is said to be an incarnation of Valmiki. Here, the god Hanuman, the sage Valmiki, and the poet-saint Tulsidas, are not divine fig
ures or “primordial” authors who wield authority over the “true” Ramayana. In fact, Valmiki
and Tulsidas are listeners who, despite being incapable of preserving the “entirety” of the
epic, become retellers whose names are embedded in iconic retellings of Ramayana stories. Even when there is a certain Ramayana attributed to Valmiki or Tulsidas, neither of them
are authoritative creators of the text. This passage illustrates that such stories are still imper
fect renditions of a mythical, inconceivable whole that nobody in particular owns. Here, the god Hanuman, the sage Valmiki, and the poet-saint Tulsidas, are not divine fig
ures or “primordial” authors who wield authority over the “true” Ramayana. In fact, Valmiki
and Tulsidas are listeners who, despite being incapable of preserving the “entirety” of the
epic, become retellers whose names are embedded in iconic retellings of Ramayana stories. Even when there is a certain Ramayana attributed to Valmiki or Tulsidas, neither of them
are authoritative creators of the text. This passage illustrates that such stories are still imper
fect renditions of a mythical, inconceivable whole that nobody in particular owns. By incorporating the “authors” themselves into a retelling and citing other retellings, this
episode demonstrates how intertextual relationships and genre formations are not simply
formal categories that are imposed by scholarly interpretations. Writers at Kiski Kahani
internalize previous retellings of the Ramayana and create a new array of interrelated stories
to comment on the sense of ownership of a text. This particular episode, as well as many
other stories that Kiski Kahani generates through Neha’s and Haroon’s literary curation,
question the notion of a text to which all other retellings are compared. Ramayana perfor
mances, therefore, do not start at the reteller and end at the listener. Listeners are, in fact,
prolific retellers as well. There is no single author who owns the text. CONCLUSION My study poses two main questions: How do retellings of the Ramayana stories reflect
a way of belonging to a cosmopolitan, pluricultural Indian nation? How do performance
modalities contextualize the text as a socially contested space? I found that interpreting how
a curation of Ramayana stories is discussed and debated does not continuously explain the
epic’s social position; rather, it suggests how the Ramayana’s retellers configure their sense
of belonging and identity through listening to and performing these stories. Admittedly, the
Ramayana might not be easily reclaimed from its history of appropriation by Hindu nation
alists during the Ayodhya incident in 1992 and its violent aftermath. Even today, retelling
the Ramayana stories is still a politically vexed project. Nonetheless, understanding how the
epic is retold in emerging and improvised forms allows us to see how the Ramayana stories
have not been surrendered to sectarian divisions. Ideological contestation within the formal, textual structure of the epic, however, is not
the core of my analysis. Instead, I am interested in how the Ramayana stories index a sense
of belonging to an India-wide literary practice. My study builds upon the ethnography of
speaking approach, where the socially patterned use of language depends on the “nonliter
ary” context of the performance, such as the life histories of the retellers. The concept of
performance illuminates broader social processes as well. My approach to cosmopolitan
belonging argues against enumerating Indian nationhood in terms of discrete cultural units. The formation of a Ramayana genre from fragmentary, incomplete narratives illustrates
that culture, like stories, is lived through debates and differences. Unfinished retellings and
authorless stories, which Kiski Kahani compiles and frames as representations of the nation,
are simultaneously a reflection and a critique of macrosocial discourses of culture. Retell 12 | Countless Ramayanas ASIANetwork Exchange | Spring 2014 | volume 21 | 2 ers of the Ramayana, therefore, are not only Indian citizens, but also innovators of a way of
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Anthropology 2, no. 2: 131-172. Bate, Bernard. 2009. Tamil Oratory and the Dravidian Aesthetic: Democratic Practice in South Asia. New York:
Columbia University Press, 2009. y
Becker, Alton Lee. 1996. “Silence Across Languages.” In Beyond Translation: Essays toward a Modern Philology,
283-294. Ann Arbor: University of Michigan Press.i 283-294. Ann Arbor: University of Michigan Press.i Bhabha, Homi. 1996. “Unsatisfied: Notes on Vernacular Cosmopolitanism.” In Text and Nation: Cross-Disciplinary
Essays on Cultural and National Identities, edited by Laura Garcia-Morena and Peter C. Pfeiffer, 191-207. Columbia, SC: Camden House. Brockington, John. 1998. The Sanskrit Epics. Leiden: Brill. gh
p
Doniger, Wendy. 1991. “Fluid and Fixed Texts in India.” In Boundaries of the Text: Epic Performances in South and
Southeast Asia, edited by Joyce Burkhalter Flueckiger and Laurie J. Sears, 31-42. Ann Arbor: Center for Southeast Asia, edited by Joyce Burkhalter Flueckiger and Laurie J. Sears, 31-42. Ann Arbor: Center for
South and Southeast Asian Studies at the University of Michigan. rEFERENCES “Ramayana Ruckus: Which Version of the ‘Ramayana’ would Ram Read?” Edited by Ar
pit Parashar and Viswajoy Mukherjee. October 24. http://archive.tehelka.com/story_main50. asp?filename=Ws241011RAMAYANA_RUCKUS.aspl Tehelka. 2011. “Ramayana Ruckus: Which Version of the ‘Rama
pit Parashar and Viswajoy Mukherjee. October 24. http:/
asp?filename=Ws241011RAMAYANA_RUCKUS.aspl i
Turner, Victor. 1986. “Images and Reflections: Ritual, Drama, Carnival, Film, and Spectacle in Cultural Perfor
mance.” In The Anthropology of Performance, 21-32. New York: PAJ Publications. h
Werbner, Pnina. 2008. Ed. of Anthropology and the New Cosmopolitanism. New York: Berg. f Woolard, Kathryn A. and Bambi B. Schieffelin. 1994. “Language Ideology.” Annual Review of Anthropology 23:
55-82. rEFERENCES New York: Oxford University Press. h h
Marcus, George E. and Michael M.K. Fischer. 1986. “Conveying Other Cultural Experience: The Person, Self, and
Emotions.” In Anthropology as Cultural Critique: An Experimental Moment in the Human Sciences, 45-77. Chicago: The University of Chicago Press. h
McLain, Karline. 2009. India’s Immortal Comic Books: Gods, Kings and Other Heroes. Bloomington, IN: Indiana
University Press. University Press. Mitchell, Lisa. 2009. Language, Emotion, and Politics in South India: the Making of a Mother Tongue. Bloomington,
IN: Indiana University Press. Mitchell, Lisa. 2009. Language, Emotion, and Politics in South India: the Making of a Mother Tongue. Bloomington,
IN: Indiana University Press. y
Narayan, Kirin. 1989. Storytellers, Saints, and Scoundrels: Folk Narrative in Hindu Religious Teaching. Philadelphia:
University of Pennsylvania Press. y
y
Paley, Nina. 2008. Sita Sings the Blues. DVD. Stanford, CA: Question Copyright. Pollock, Sheldon, Homi K. Bhabha, Carol Appadurai Breckenridge, and Dipesh Chakrabarty. 2000. “Cosmopoli
tanisms: Introduction.” Public Culture 12, no. 3: 577-589. Pune Mirror. 2012. “Now, You Hardly Hear Marathi Spoken around Here.” July 28. hhh ne Mirror. 2012. “Now, You Hardly Hear Marathi Spoken aroun Ramanujan, A.K. 1991. “Three Hundred Ramayanas: Five Examples and Three Thoughts on Translation.” In Many
Ramayanas: The Diversity of a Narrative Tradition in South Asia, edited by Paula Richman, 22-49. Berkeley:
University of California Press. y
Ramaswamy, Sumathi. 1999. “Sanskrit for the Nation.” Modern Asian Studies 33, no. 2: 339 381. hh y
Ramaswamy, Sumathi. 1999. “Sanskrit for the Nation.” Modern Asian Studies 33, no. 2: 339 381. Richman, Paula 1991 “Introduction: The Diversity of the Rāmāyaņa Tradition” In Many Ramayanas: The 13 | Countless Ramayanas ASIANetwork Exchange | Spring 2014 | volume 21 | 2 sity of a Narrative Tradition in South Asia, 3-21. Berkeley: University of California Press. y f
,
y
y
Sagar, Ramanand. 2001. Ramayan. DVD. Remastered, Mumbai: Sagar Art Enterprises.h y f
y
y
Sagar, Ramanand. 2001. Ramayan. DVD. Remastered, Mumbai: Sagar Art Enterprises.h y f
y
y
Sagar, Ramanand. 2001. Ramayan. DVD. Remastered, Mumbai: Sagar Art Enterprises.h Silverstein, Michael. 1981. The Limits of Awareness: Sociolinguistic Working Paper No. 84. Austin, TX: Southwest
Educational Development Laboratory. h Smith, H. Daniel. 1995. “Impact of ‘God Posters’ on Hindus and Their Devotional Traditions.” In Media and the
Transformation or Religion in South Asia, edited by Lawrence A. Babb and Susan S. Wadley, 24-50. Philadel
phia: University of Pennsylvania Press. Tehelka. 2011. 14 | Countless Ramayanas Notes 1. See Tehelka (October 24, 2011).h 2. The names of this organization and of individuals in this h
3. The Amar Chitra Katha (ACK) comic books are almost as popular today in urban India as they were in the
1980s. ACK, which is a colored, illustrated comic series, presents stories from the Ramayana in English. Although it has been criticized for using only “Hindu” texts, the ACK is a common way of experiencing
mythological texts among upper-middle class, urban Indians (McLain 2009). 4. Ramanand Sagar’s (2001 [1987]) Hindi-language television series of the R
through Doordarshan, the Indian central government’s official channel. 4. Ramanand Sagar’s (2001 [1987]) Hindi-language television series of the Ramayana was broadcasted
through Doordarshan, the Indian central government’s official channel. fi
5. “Calendar art” or “god poster” is a popular genre of illustrations on mythological themes based on deities,
saints, and sacred sites that are commercially produced, especially in major Indian urban centers. See Smith
(1995). 6. Paley, an American cartoonist, released “Sita Sings the Blues” in 2008. The feature-length animation com
bines selections from the Ramayana stories with her life history as an artist. 7. Hanuman, a simian god whose virility and courage is indispensable to Rama during the numerous battles
with Ravana, is said to be an ascetic that secluded himself in the mountains of the Himalaya after the battles
of the Ramayana (Lutgendorf 2007,173-174). 14 | Countless Ramayanas ASIANetwork Exchange | Spring 2014 | volume 21 | 2 Continuity and Evolution: The Idea of “Co-creativity” in Chinese
Art Jinli He Abstract: This paper seeks to explore an important characteristic of both traditional
and contemporary Chinese art, that is, co-creativity. The author believes that co-
creativity is a particular Chinese cultural sensibility that establishes the continu
ity of Chinese art and allows it to endure despite historical, societal, and political
changes throughout the centuries. This paper starts with an introduction of the
idea of co-creativity in Chinese culture. One of its embodiments is the relationship
between yin and yang. Yin and yang both engender and fulfill each other, which is a
co-relational and co-creative process. The paper then analyzes how the idea of co-
creativity is demonstrated in traditional landscape painting through the expression
of the oneness with nature and invitation to join a journey with the artist. Lastly,
it demonstrates this continious co-creative cultural sensibility through analyzing
two contemporary artists’ works. The author reads the submissive openness and
vulnerability in Chinese female artist Chen Lingyang’s works as a continuity of the
co-creative spirit of yin and yang, of nature and human. Chen’s work, rooted in her
cultural sensibility, expresses a totally different statement of women’s desires and
conditions than does that of American feminist artists Judy Chicago and Carolee
Schneemann. Likewise, performance artist Ma Liuming’s Fen-Ma Liuming in…
series seems inspired by nature’s image of co-creating the world. Different as these
works may be in their formal aspects—from painting to poetry, from photography
to performance— “co-creativity” is at the heart of Chinese cultural expression. Keywords co-creativity; cultural sensibility; landscape painting; Chen Lingyang;
Ma Liuming Introduction Jinli He is assistant professor in
the Department of Modern Lan
guages and Literatures at Trinity
University, teaching Chinese
art history, Chinese philosophy,
civilization and literature. Her
research interests include Chi
nese aesthetics and comparative
aesthetics, Buddhist art in China
and Contemporary Chinese art. Jinli He is assistant professor in
the Department of Modern Lan
guages and Literatures at Trinity
University, teaching Chinese
art history, Chinese philosophy,
civilization and literature. Her
research interests include Chi
nese aesthetics and comparative
aesthetics, Buddhist art in China
and Contemporary Chinese art. Why co-creativity? My choice of this term and concept to describe a Chinese cultural
sensibility that plays a role in the creation of art was inspired by a conversation I had with
Professor Roger Ames several years ago. In their book Focusing the Familiar: A Translation
and Philosophical Interpretation of the Zhongyong (2001), Roger Ames and David Hall use
“creativity” and “co-creativity” to translate the central concept cheng (誠) in the Zhongyong. As we know, cheng has been conventionally translated as sincerity or integrity. I raised the
question of this discrepancy with Professor Ames while conducting an interview with him
for a journal edited by a friend of mine in Shanghai. Ames’s answer began with a discussion
of the cosmological meaning of the concept cheng (Ames and Hall 2005, 438): Cosmology is the projection of the human world onto the cosmos—the shaping of
experience of the world with the lens of human feelings. This is certainly what we
see in early Greek philosophy where “love” and “strife” are the creative forces which
shape cosmic order. Cheng (誠) should be understood in a cosmological sense—
that is in a philosophical sense. For this reason, cheng is not used in the conven 15 | Continuity and Evolution ASIANetwork Exchange | Spring 2014 | volume 21 | 2 tional way, but with a philosophical import—the cosmos as a projection of human
feelings. I take this to be a distinctive characteristic of the Zhongyong. What the
Zhongyong does is to “cosmologize” human affect. The human being is co-creator
with the cosmos—this is the main theme of the Zhongyong. It is just as we can see in
the language of “on a par with tian” (peitian, 配天) and “the three powers”(sancai,
三才). The human being is the heartmind of the cosmos—its collaborator. Introduction With this
kind of understanding you become aware of the fact that feeling is the foundation
of creativity. Hence, translating cheng as “creativity” or “co-creativity” is not aban
doning its meaning of “sincerity” because we in fact create each other—we literally
“make” our friends. It is on this basis that we use this vocabulary. When you are in
the process of creating yourself together with others, you should have integrity in
the sense of “becoming one together.” You are not exclusively “yourself,” you are
your relationships, and it is in these relationships that you become one together. So
“integrity” too is the foundation of “creativity” and “co-creativity.” Hence, “creativ
ity” or “co-creativity” does not abandon the conventional meaning of cheng as “sin
cerity” or “integrity,” but just appeals to a different way in which they are expressed.1 Cultural translation is a charming and mysterious process of meaning aggrandizement. According to Ames, the reason for their use of “co-creativity” to translate the Chinese
philosophical concept cheng is that the word “co-creativity” bears the same Chinese cultural
sensibility that cheng implies (“cosomologizing human affect,” the continuity of tian and
human, the sensibility of “becoming one together,” and so forth). Those qualities are essen
tial for Chinese artists as their goal of creativity is an ongoing process of self-cultivation,
being one together with the cosmos, nature, society, family, friends, and their surroundings. As Ames and Hall describe in their distinction of the notions of “power” and “creativity”: Power relationships reduce creativity to modes of external causation…the world is
sharply divided into creators and created—that is, makers and made… Creativity is always reflexive and…with respect to “self”…is transactional and
multi-dimensional. Thus creativity is both self-creativity and co-creativity…. All
relations are transactional in the sense that they are reciprocal and mutually deter
minative. (Ames and Hall 2001,12-13) One of the conventional embodiments of co-creativity is the relationship between yin
and yang—they both engender each other and consummate each other, which is a co-rela
tional and co-creative process. The concept of yin and yang, and by extension co-creativity,
is an enduring theme of Chinese art. It is demonstrated in all aspects of artistic creativity:
First, as I mentioned earlier, for a Chinese artist, the significance of artistic creativity is that
consummating a work is a means by which to self-consummate. Introduction Secondly, co-creative sen
sibility is one of openness that is embodied in the Chinese artist’s attitudes toward nature,
history, the viewer, and so forth. Handscroll painting is a fine example because its unique
format often allows people other than the artist to become literally involved in the creation
of art. In one case, a 15 x 41 inch framed landscape masterpiece from the Song dynasty
was extended to twenty times its original length through the ages. Paintings, poetry, com
ments, and styled seals were added by friends, collectors, or viewers, and collectors from
later generations continuously participated in this creative process. The moment the viewer
unrolls the handscroll painting, he is also participating in this historical co-creative process. The masterpiece itself becomes an ongoing open “system” which creates art history and an 16 | Continuity and Evolution ASIANetwork Exchange | Spring 2014 | volume 21 | 2 enduring self-creating process. The co-creative sensibility is the key to this distinctive Chi
nese tradition of art appreciation and creation. Another example of this co-creative and open artistic relationship is the traditional
art gathering, where artists enjoy creating a work together. The famous example is a work
co-created by the Yuan masters Gu An (ca. 1289-1365), Yang Weizhen (1296-1370), Zhang
Shen (dates uncertain), and Ni Zan (1301-1374) called Old Tree, Bamboo and Rock (1370-
1373). The former three artists co-created the original work in 1370; the old tree was
painted by Zhang Shen and the bamboo by Gu An. The middle left inscription was writ
ten by Zhang Shen and the upper left by Yang Weizhen. Three years later, after Gu An and
Yang Weizhen had passed away, Ni Zan saw the painting and added the rock as well as a
poem (upper right) to memorialize the late masters. The spirit of artistic co-creativity is well
expressed in this single painting. Finally, one of the unique characteristics of traditional Chinese art theory is that litera
ture, painting, and other art forms share the same critical standards and aesthetic terms. For example, the famous poet and painter Wang Wei, who lived in the eighth century, was
viewed favorably by later artists and poets because of the convertibility of his arts: “painting
in poetry, poetry in painting” (Su Dongpo, 1981, 37). Introduction As the legend goes, the famous cal
ligrapher Zhang Xu (eighth century), considered to be the sage of cursive style (caoshu), was
inspired by the martial arts dancer Gongsun Daniang’s sword dance. The aesthetic terms
such as yun (韻, rhythm), shen (神, spirituality), and wen (味, taste) are applied to all kinds
of art styles, from calligraphy to painting, poetry, dance, sculpture and music. Yuan Dynasty (1370-1373), Taipei,
Palace Museum. Digitalarchivers. Web. 4 December, 2011. http://catalog.digitalarchives.tw/
item/00/03/fa/e7.htmlalarchive Now I will turn to landscape painting to demonstrate how this cultural sensibility plays a
role in the creation of that type of art. Fig. 1. Yang Weizhen, Nizan, Gu
An, Zhang Shen, Old Tree, Bam
boo and Rock. Fig. 1. Yang Weizhen, Nizan, Gu
An, Zhang Shen, Old Tree, Bam
boo and Rock.
Yuan Dynasty (1370-1373), Taipei,
Palace Museum. Digitalarchivers.
Web. 4 December, 2011.
http://catalog.digitalarchives.tw/
item/00/03/fa/e7.htmlalarchive Yuan Dynasty (1370-1373), Taipei,
Palace Museum. Digitalarchivers.
Web. 4 December, 2011. Co-creativity and Landscape Painting Landscape painting was a major art form in traditional Chinese art and achieved its full
development during the Song Dynasty (tenth-eleventh centuries). Jing Hao (tenth century),
one of the leading artists of the “monumental style,” theorized that successful landscape
painting was based on six essentials: qi (氣, spirit, energy, life breath), yun (韻, resonance
and elegance), si (思, ideas), jing (景, scenery, i.e. “nature”), bi (筆, brush work), and mo (墨,
ink wash) (Jing Hao 1963, 3). Jing’s goal was to make the internal nature (breath, resonance,
ideas) harmonize with the external nature (scenery, and brush and ink—the tools for artis
tic creation). The process went something like this: through the artist’s contemplation of the
natural world, the self-cultivated qi will find a way to resonate with the qi of the universe;
then the artist rationalizes the process and expresses the harmony with bi (brush work) and
mo (ink wash). This is not a one-way movement: to achieve and appreciate the resonance of
qi requires mutual openness, reciprocity and co-creativity between the artist and the natural
world. Jing Hao’s idea of essentials was greatly influenced by the Neo-Confucian understanding
of the relationship between the self and the outside world. Neo-Confucian thinking, which
can be traced back to the eighth century but primarily developed during the Song Dynasty,
was a new, synthesized ideology combining basic ideas of Confucianism, Daoism, and
Buddhism, but claiming to be loyal to the Confucian tradition of the Great Way (the way of
life, the cosmos) developed by Confucius (551-479 BCE), Zisi (481-402 BCE), and Mencius
(372-289 BCE). Its followers aspired to achieve the great way through “self-cultivation.”hi This self-cultivation starts with qi (spirit, energy, life, breath). Qi has always been the first
and most important concept in traditional Chinese philosophy and aesthetics. The Confu
cian philosopher Mencius, whom Neo-Confucian scholars often quote, claimed that he was 17 | Continuity and Evolution ASIANetwork Exchange | Spring 2014 | volume 21 | 2 good at cultivating his “flood-like qi ” (浩然之氣), describing it as “most vast” (至大) and
“most firm” (至剛) (Meng Tzu 1941), but he did not explain how to cultivate or achieve it. The Song dynasty Neo-Confucians explained that the utmost truth is cultivating your own
flood-like qi to be one with the flood-like qi of the cosmos. Co-creativity and Landscape Painting Zhu Xi (1130-1200), the famous
Song Neo-Confucian philosopher, later theorized and clarified: “Heaven and Earth (the cos
mos) has the heart and mind of creativity, and this same heart and mind is attained in the
creation of human and things” (Zhu Xi 1973, 4186; 天地以生物為心，而人物之生，又
各得夫天地之心以為心者). For Zhu Xi, in other words, the heart and mind of the cosmos
is united with the heart and mind of humankind. This is the ultimate truth of universe that
the Neo-Confucians called li (理, principle, truth). Applying this ideology to art, the great Chinese master tries to unify his mind with the
mind of heaven and earth in his artistic creativity. The master studies nature until he or
she reaches the point where he is able to capture nature’s heart and mind in his own heart
and mind. Then he/she concentrates this cultivated life breath (qi) in the brush, in order to
convey the qi of nature he achieved in his heart and mind. In Chinese art theory, there is no such concept as “perspective,” instead there is yuan
(遠, distance). The great Song landscape painter and theorist, Guo Xi (c.1020-c.1090),
described three distances in landscape painting: high distance (高遠) (viewing from the
bottom of a mountain to its peak); level distance (平遠) (viewing from a near mountain to a
far mountain); and deep distance (深遠) (viewing from the front of a mountain to its back)
(Guo Xi 1993, 500). “Scientific perspective involves a view from a determined position and includes only
what can be seen from that single point” (Sullivan 2008, 176). Chinese “distances,” on the
other hand, are inclined to view “the part from the angle of totality” (Sullivan 2008, 176). Therefore, sometimes artists apply more than one “distance” in a piece of work, which
requires shifting perspectives. Guo Xi’s work Early Spring (1072) has been viewed as a great
example for applying “three distances”: first you see a tiny human figure at the bottom of the
majestic mountain—this is called high distance. Then, changing your distance, your mind
travels up, and you now use your level distance—you are among the mountains, and you
see the beauty around you. As you travel deeper and higher, you “climb” the mountain and
“see” more and more mountains behind the first one. This is the deep distance. Fig. 2. Guo Xi, Early Spring,
1072, ink and color on
silk, Palace Museum, Tai
pei. Courtesy of ARTstor :
ARTSTOR_103_41822003468954 http://library.artstor.org/library/
secure/ViewImages?id=8CJGcz
I9NzldLS1WEDhzTnkrX3osf1N9
dSc%3D Co-creativity and Landscape Painting The total
izing angle of Chinese landscape painting suggests that the viewer is going on a tour with
the artist to participate in the journey of appreciation. This is a process of interaction. The
harmonized qi, received by the Chinese master from his ongoing encounter with nature,
is carried to the brush, and the brush becomes part of his body and of the harmonized qi. In his work there is always the resonance of his enjoyment of the harmony of qi, so that
the work is made up of two natures coming together in utmost harmony—that of the artist
and that of the cosmos. The artist uses his imagination to paint the harmony of a beautiful
journey rather than a particular, static scene (such as a photograph would encapsulate) and
he also invites the viewer to join him, thus extending the creativity outward, in an ongoing
movement. Co-creativity in Contemporary Chinese Arth This cultural sensibility of co-creativity even survives in some more radical contempo
rary art forms. Here I have chosen a young female artist who uses photography to make her
artwork and a second who is a performance artist to demonstrate what I mean. We shall first examine the work of Chen Lingyang.2 Chen Lingyang was born in Zheji 18 | Continuity and Evolution ASIANetwork Exchange | Spring 2014 | volume 21 | 2 ang Province (south part of China) in 1975, and she graduated from Central Academy of
Fine Arts in Beijing in 1999. She lives in Beijing and mainly works with installation and
photography. Her works usually explore the beauty of female bodies and their intrinsic
meanings. Twelve Flower Months is her most famous work. Chinese women’s art has received increasing international attention since the 1990s. More and more exhibitions have been held both in China and abroad—solo exhibitions,
group exhibitions, and international exhibitions.3 There are numerous articles employing
Western feminist movements as the standard to analyze Chinese women’s art, reading it
as a gesture of liberation of women. For example, in her article “Female Body: True and
Beautiful - Chen Lingyang’s Twelve Flower Months and Women’s Self-Consciousness,”
the Chinese art critic Liao Wen relates Chen’s work to American artists Judy Chicago and
Carolee Schneemann, reading them as one and the same “female body,” united in confront
ing “the truth of the female body.” She describes how the works destroy the exoticization
of the “female scent” (the “scent of a woman”), whose beauty is the object of male gaze and
consumption, using the most “true” things—genitalia and menstrual blood—which had
been taboo in traditional art, to show the real form of a woman’s body. From this perspec
tive, Wen concludes that “Chen uses the superficial traditional art form to form tradition,
and the actual ‘female scent’ to destroy the ‘(traditional) female scent’…. Those ‘new new’
generation girls who were born in 1970s…growing up in a relatively open-minded social
environment, and a gradually internationalizing age of information…inspire us with the
self-consciousness and earnestness of their ‘feminist’ expression!” (Liao 2001)4 Unlike in Western art, the naked body never appears in traditional Chinese art history. As the body is the symbol of privacy, it makes perfect sense to see contemporary Chinese
artists using it to express that historical repression. Indeed, I do not exclude a “feminist”
interpretation of Twelve Flower Months. Fig. 3. Judy Chicago, Red Flag
65/94, 1971, photo lithgraph,
Collection of Constance
Gee. Courtesy of ARTstor:
AJCHICAGOIG_10313027095 Fig. 4. Carolee Schnee
mann, Interior Scroll, per
formance, 1975, ©2007
Carolee Schneemann/ARS,
New York. Courtesy of ARTstor :
ARTSTOR_103_41822000275568 Fig. 4. Carolee Schnee
mann, Interior Scroll, per
formance, 1975, ©2007
Carolee Schneemann/ARS,
New York. Courtesy of ARTstor :
ARTSTOR_103_41822000275568
http://library.artstor.org/library/
secure/ViewImages?id=8CJGcz
I9NzldLS1WEDhzTnkrX3kpeF98
eig%3D Co-creativity in Contemporary Chinese Arth Such an analysis does have its insights, but it cannot
exhaust the rich meaning of this work. My understanding of this work is that the cultural
sensibility plays an even more important role than does a feminist one. Although all three artists use the female “personal, everyday functions” to create their
art, the visual expression of Chen’s work is quite different from Chicago and Schneemann. One of Judy Chicago’s (b. 1939) famous works is Red Flag (1971). Chicago interpreted
viewers’ failing to recognize the gesture in the photo as “removing a tampon” (some people
thought it was a bloody penis, according to her) as showing how unwilling many men (and
women!) are to look at women’s personal, but everyday functions. Carolee Schneemann’s
(b.1939) Interior Scroll was performed in East Hampton, NY and at the Telluride Film Festi
val in Colorado in 1975. Schneemann ritualistically stood naked on a table, and later slowly
extracted a paper scroll from her vagina and read a eulogy of a vagina from it: “…I saw the
vagina as a translucent chamber of which the serpent was an outward model.... This source
of interior knowledge would be symbolized as the primary index unifying spirit and flesh
in Goddess worship” (Shneemann 1975). Both Chicago and Scheneemann’s works convey
strong feminist messages. One criticizes the ignorance of a society full of both men and
women who are unwilling to face the historically established inferiority of women; the other
eulogizes the female sex. Those powerful gestures claim a war for the justice of women. I do
not think the cultural sensibility behind Chen Lingyang’s work Twelve Flower Months sup
ports the same kind of interpretation. http://library.artstor.org/library/
secure/ViewImages?id=8DpJfzQs
KDI7KiAiFTx5RnsqXXwsdFE%3D http://library.artstor.org/library/
secure/ViewImages?id=8CJGcz
I9NzldLS1WEDhzTnkrX3kpeF98
eig%3D I will start with a brief introduction of the background of Chen’s work. After graduat
ing from art school, Chen Lingyang lived in isolation. She did not work, seldom contacted
friends, and lost or cut connections with public life for several months. She writes: “In that
situation, the physical aspects of my identity became prominent: hunger, cold, and espe 19 | Continuity and Evolution ASIANetwork Exchange | Spring 2014 | volume 21 | 2 cially menstruation, and menstrual pain, emotional turbulence, and so on. I became aware
of the cycles of day and night, the slow growth of plants and the changing patterns of the
weather…” (Chen 2003). Fig. 5. Chen Lingyang, Twelve
Flower Months. photograph.
Nov.1999-Dec.2000. Xindong
Cheng Space For Contemporary
Art Website, 28 June. 2011. Co-creativity in Contemporary Chinese Arth This emotional experience inspired Chen to create Twelve Flower
Months (Nov.1999-Dec. 2000), a “real work” about menstruation. The work took twelve
months and consists of handmade enlarged color photos. Each picture uses the perennial
blossom of that month, as well as a mirror of a different classical shape reflecting the geni
talia and menstrual blood of Chen’s own body; the borders of the photographs imitate the
shapes of the windows and doors of traditional Chinese gardens; the flowers shown in each
photograph are the blossoms of each month of the traditional Chinese calendar. Drawn
from her year of self-imposed exile, Twelve Flower Months is extraordinarily private and
self-reflective, while at the same time expressive of Chen’s effort to emerge from isolation
and establish an intimate connection with the world. Because of its attempt to connect to
the viewer through culturally-recognized symbols, this work can be viewed as being created
in a co-creative spirit with nature and culture despite the fact that its visual language and
artistic expression differ from the traditional landscape painting I discussed above. http://www.chengxindong. com/index.php?option=com_
rsgallery2&gal_id=131&page=sho
wImages&id=1080&NewsID=319
&Itemid=5&lang=en The menstrual blood in this work reflects woman’s nature, echoing the nature of the
cosmos, and the blossoms of the monthly flowers. The mirrors and self-portraits draw the
viewer into the image. The classical shapes of the mirror and the light, the shapes of the
borders of the photographs, and even the placement of the flowers have cultural resonance. The open blossom and the opened body in the reflected mirror and the gaze of the camera
all emphasize the idea of openness. But this openness is not a strong, open statement as
is clearly expressed in Chicago and Schneemann’s works, rather it implies a continuity of
nature and culture. It is worth quoting the artist’s own interpretation of her work here. Chen Lingyang com
posed a “self-interview” between “Chen Lingyang No. 2” (C2) and herself (C1): C2: Your work (Twelve Flower Months) seems to have a very strong emotional
expression… C1: Yeah. But I don’t want to explain this. I want to leave the space of interpretation
to the viewers. Of course, I’m happy to hear the opinions and discussions from the
others’ own perspectives. C2: Some people say that your work expresses privacy. When a private work is
exhibited in a public space, it is hard to anticipate, or in other words, to control the
consequences… C1. http://www.chengxindong.
com/index.php?option=com_
rsgallery2&gal_id=131&page=sho
wImages&id=1080&NewsID=319
&Itemid=5&lang=en Co-creativity in Contemporary Chinese Arth In some sense, it is aimed to have an impact upon the public space. C1. In some sense, it is aimed to have an impact upon the public space. Let me start from the background of this work. We are all familiar with the idea of
“Tian ren he yi” (the oneness of tian [nature] and the human) in traditional Chinese
culture. It is said that the utmost self-cultivated person achieves the feeling of one
ness with the cosmos and the ten thousand things; she totally loses herself in the
mysterious aesthetic world, loses all human feelings, such as joy, anger, sorrow,
pleasure, right, wrong, good or evil. Tian is the rhythm of the cosmos (come back
and go, round, round again); women can feel this cosmic rhythm from her monthly
physical (consequently, psychological) changes. 20 | Continuity and Evolution ASIANetwork Exchange | Spring 2014 | volume 21 | 2 When people view this work in a public space, they may have various opinions. However, the work itself also offers the possibility to deconstruct those interpreta
tions. The traditional allusions make this deconstruction possible. Engendering and
deconstructing, like the cycles of Tian. It is this relation of constructing and deconstructing that gives birth to new
possibilities. C2. I notice that the duration of Twelve Flower Months is one year and it includes
twelve photographs. You seem to give prominence to this. C1. Yes. The twelve photographs appear together as a completed work. Just as I
mentioned above, this work is related to the pattern and rhythm of the circulation
of things. A year is a completely basic palingenesis of time. Where is the beginning
and where is the ending actually doesn’t matter, it just goes round and round, but
each moment is unrepeatable…In a word, apparently, it is a year, but actually it is an
infinite time conception.” (Chen 2001)5 I would like to introduce another of Chen’s works, The 25th hour (2002), to deepen the
contrast between Chen Lingyang’s work and that of Chicago and Schneemann. While the concepts of privacy and openness, the oneness of nature and culture informed
Twelve Flower Months, The 25th hour can be read as women’s dialogue and communication
with the real world. Yet there is continuity between the two works, especially concerning
Chinese women’s sensibilities, including the established cultural co-creative spirit and open
ness. The 25th hour was created in 2002 and is a digital photograph. A giant woman kneels
down, her face hidden, on a top of a dark warehouse; around her is the night sky and many
industrialized city buildings. Fig. 6. Chen Lingyang, 25:00
No.1, C- Print, 2002. Chinesis
che Gegenwartskunst. Web. 29,
June.2011. http://www.chinesische-gegen
wartskunst.de/pages/galerie.
php?catid=07-01-29&id=15 C1. In some sense, it is aimed to have an impact upon the public space. Chen’s work, rooted in her cultural
sensibility, expresses a totally different statement of women’s desires and conditions than do
Chicago and Schneemann. hl This sensibility is also reflected in performance artist Ma Liuming’s work. Ma Liuming
was born in 1969 in Huangshi, Hubei Province, China. He graduated from the Hubei Acad
emy of Fine Arts in 1991 with a focus on oil painting. After graduation, Ma Liuming moved
to Beijing and began making nude performance pieces. He wears his hair long and has
feminine features, and his androgynous look is an important part of his art. Makeup helps
to transform his face into his female alter ego, Fen (Fen, 芬is a very common female name
in China). The coexistent sex egos in this single artist remind us of the co-creative relation
ship between yin and yang. Ma Liuming’s Fen-Ma Liuming in… series displays the idea of openness and invitation. For this particular project, Ma Liuming took sleeping pills and placed a camera in front of
him to record the whole scene of his co-creativity. We can see from the photographs how
the audience reacted. Some people made the same gesture as him. A lady took off one of
her stockings and rolled it on to one of his legs. A young boy kissed him with a reverential
attitude. Two guys made some funny postures with him. The willingness of the artist to sur
render to his audience reminds us of the silence of nature. Nature is always there, inviting
us to enjoy the harmony of the universe. She gives the choice to you: you can choose to offer
respect, friendship, appreciation, or violence. I view Ma Liuming’s performance as a gesture
of nature’s openness and invitation. The work reminds us to ask ourselves: do we cultivate
ourselves as the self—the so-called master of the universe? Or can we be sensitive enough
to nature’s openness and invitation to participate in the great harmony—that is, to co-create
the world? http://www.artlinkart.com/cn/
artist/wrk_sr/ab2bsz/b5aa “Co-creativity” is an enduring theme in Chinese art. It can be traced to the artist’s goal
of harmonizing one’s “flood-like” qi with the qi of the cosmos. The artist is not just a creator,
but is also created, and in this spirit, he or she invites the viewer to be part of the creative
process. C1. In some sense, it is aimed to have an impact upon the public space. In the introduction to this work, Chen says, http://www.chinesische-gegen
wartskunst.de/pages/galerie. php?catid=07-01-29&id=15 This ‘giantess’ is not really very brave, and so she will only freely change her size and
make these kinds of gestures when the clock strikes 25:00. Very often, the real world and the male world get mixed up in my mind. They both
come from outside me; they both exist very forcefully, with initiative, power and
aggression. Facing these two worlds, I often feel that I am weak and helpless, and
don’t know what to do. But just being alive means that I cannot avoid them, not
even for one day. I wish that every day there could be a certain time like 25:00, when
I could become as large as I like, and do whatever I want. (Chen 2002) Reading this work from a feminist perspective, we will see how different are the visual
messages Chen and Chicago and Schneemann convey in their expressions of women’s
desire and transcendence. The giant, kneeling Chinese woman is so different from her
Western sister who stands up with her powerful body right in the center of the stage to
show how her strength and height—everything a patriarchal society takes to be symbolic of
power. But that stage is for monologues—it is not intended to arouse dialogue. Chen’s giant
Chinese woman, on the other hand, kneels down under the twilight sky, amid the steel and
concrete forest which is much more powerful, more “giant” than she is. There is no central
stage; the stage for her is unstable and dangerous. As she kneels down and bends her naked
body, she takes the risk of being exposed, of being taken advantage of. She waits there, her
head and eyes covered, and seems to totally relinquish her rights to the viewer. This silent, 21 | Continuity and Evolution ASIANetwork Exchange | Spring 2014 | volume 21 | 2 submissive gesture is a kind of openness, like the openness of nature. This gesture invites
communication, as nature invites the participation of humans. It might be too ideal, too
dangerous, and too hopeless. It certainly requires a most sensitive heart from the viewer
to appreciate this gesture, lift her head and remind herself that she is actually as “giant” as
anyone. I read the submissive openness and vulnerability in this work as a continuity of the
co-creative spirit of yin and yang, nature and human. Fig. 8. Ma Liuming, Fen-Ma Lium
ing in Munich, Germany, 1999.
ARTLINKART Data. Web. 29
June, 2011. Fig. 7. Ma Liuming, Fen-Ma Lium
ing in Lyon, 2001. ARTLINKART
Data. Web. 29 June, 2011. C1. In some sense, it is aimed to have an impact upon the public space. In landscape painting, it is the oneness with nature and invitation to “enter the
scene,” as well as a journey with the artist. In contemporary art, it is the expression of the
artist’s openness and vulnerability. Different as these works may be in their formal aspects—
from painting to poetry, from photography to performance—each one demonstrates that
“co-creativity” is the heart of Chinese cultural expression. http://www.artlinkart.com/cn/
artist/wrk_sr/ab2bsz/b5aa rEFERENCES
Ames, Roger T and David L. Hall. 2001. Focusing the Familiar - A Translation and Philosophical Interpretation of
the Zhongyong. Honolulu: University of Hawaii Press.
Chen, Lingyang陳羚羊. 2002. “Introduction of 25:00”. ARTLINKART Data. Web. 26 June, 2011. http://www.
artlinkart.com/cn/artist/wrk_sr/f8div.
English version: praguebiennale online photes. Web. 26 June, 2011. http://www.praguebiennale.org/artists/cina/
chenglingyang.php.
------2001. “The Interview between Chen Lingyang and Chen Lingyang No.2.” Yishu Daobao. Web. 4 December.
http://www.mahoo.com.cn/artists/infodetail.aspx?id=41
Guo, Xi 郭熙. 1993. “The Lofty Message of Forest and Streams” (“Linquan Gaozhi” 林泉高致), Complete Works of
Chinese Calligraphy and Painting (Zhongguo Shuhua Quanshu中國書畫全書), Vol.1, Shanghai: Shanghai
Shuhua Chubanshe. Guo, Xi 郭熙. 1993. “The Lofty Message of Forest and Streams” (“Linquan Gaozhi” 林泉高致), Complete Works of
Chinese Calligraphy and Painting (Zhongguo Shuhua Quanshu中國書畫全書), Vol.1, Shanghai: Shanghai
Shuhua Chubanshe. ------2001. “The Interview between Chen Lingyang and Chen Lingyang No.2.” Yishu Daobao. Web. 4 December.
http://www.mahoo.com.cn/artists/infodetail.aspx?id=41 Fig. 7. Ma Liuming, Fen-Ma Lium
ing in Lyon, 2001. ARTLINKART
Data. Web. 29 June, 2011.
http://www.artlinkart.com/cn/
artist/wrk_sr/ab2bsz/b5aa Ames, Roger T and David L. Hall. 2001. Focusing the Familiar - A Translation and Philosophical Interpretation of
the Zhongyong. Honolulu: University of Hawaii Press.
Chen, Lingyang陳羚羊. 2002. “Introduction of 25:00”. ARTLINKART Data. Web. 26 June, 2011. http://www.
artlinkart.com/cn/artist/wrk_sr/f8div.
English version: praguebiennale online photes. Web. 26 June, 2011. http://www.praguebiennale.org/artists/cina/
chenglingyang.php. rEFERENCES ------2001. “The Interview between Chen Lingyang and Chen Lingyang No.2.” Yishu Daobao. Web. 4 December. http://www.mahoo.com.cn/artists/infodetail.aspx?id=41 Guo, Xi 郭熙. 1993. “The Lofty Message of Forest and Streams” (“Linquan Gaozhi” 林泉高致), Complete Works of
Chinese Calligraphy and Painting (Zhongguo Shuhua Quanshu中國書畫全書), Vol.1, Shanghai: Shanghai
Shuhua Chubanshe. 22 | Continuity and Evolution ASIANetwork Exchange | Spring 2014 | volume 21 | 2 ing, Hao荊浩. 1963. Record of Brush Methods (Bi Fa Ji筆法記), Beijing: Renmin Chubanshe. Mencius (Meng Tzu 孟子). 1941. Harvard-Yeching Institute Sinological Index Series, Supp.17. Peking: Harvard-
Yenching Institute. Mencius (Meng Tzu 孟子). 1941. Harvard-Yeching Institute Sinological Index Series, Supp.17. Peking: Harvard-
Yenching Institute. Liao, Wen廖雯. 2001. “Female Body: True and Beautiful - Chen Lingyang’s Twelve Flower Months and Women’s
Self-Consciousness”(Nüxing shenti: Zhenshi er Meili—Chen Lingyang Shieryuehua he Nüxing Fagnshi
de Ziju女性身體：真實而美麗－陳羚羊的十二月花和女性方式的自覺”. 9 April, 2001. ARTLINKART
Data Web 4 December 2011 http://www artlinkart com/cn/artist/txt ab/e93aABnm/3e0gsut Liao, Wen廖雯. 2001. “Female Body: True and Beautiful - Chen Lingyang’s Twelve Flower Months and Women’s
Self-Consciousness”(Nüxing shenti: Zhenshi er Meili—Chen Lingyang Shieryuehua he Nüxing Fagnshi
de Ziju女性身體：真實而美麗－陳羚羊的十二月花和女性方式的自覺”. 9 April, 2001. ARTLINKART
Data. Web. 4 December 2011. http://www.artlinkart.com/cn/artist/txt ab/e93aABnm/3e0gsut p
_
g
Su, Dongpo蘇東坡. 1981. “Inscription for Wang Wei(Mojie)’s Painting of Misty and Rainy Lantian” (Shu Mojie
Lantian Yanyutu書摩詰藍田煙雨圖), in Selected Writings of Chinese Aesthetics History (Zhongguo Meix
ueshi Ziliao Huibian中國美學史資料選編), Beijing: Zhonghua Shuju. Su, Dongpo蘇東坡. 1981. “Inscription for Wang Wei(Mojie)’s Painting of Misty and Rainy Lantian” (Shu Mojie
Lantian Yanyutu書摩詰藍田煙雨圖), in Selected Writings of Chinese Aesthetics History (Zhongguo Meix
ueshi Ziliao Huibian中國美學史資料選編) Beijing: Zhonghua Shuju Su, Dongpo蘇東坡. 1981. “Inscription for Wang Wei(Mojie)’s Painting of Misty and Rainy Lantian” (Shu Mojie
Lantian Yanyutu書摩詰藍田煙雨圖), in Selected Writings of Chinese Aesthetics History (Zhongguo Meix
ueshi Ziliao Huibian中國美學史資料選編), Beijing: Zhonghua Shuju.hit Sullivan, Michael. 2008. The Arts of China (fifth edition), Berkeley: University of California Press. Zhu Xi 朱熹1973 “A treatise on Ren [humanity]”仁說Zhuzi Yulei (vol 7) Taipei: Zhongzheng Shu Sullivan, Michael. 2008. The Arts of China (fifth edition), Berkeley: University of California Press. 23 | Continuity and Evolution 1.
Ames and Jinli He (interview), “The Meaning of Cultural Dialogue,” June 2004. This interview was included
in the Chinese translation of Ames and Hall’s Thinking through Confucius (1987. Albany: State University of
New York Press. Chinese version: 2005. Beijing: Peking University Press). The paragraph quoted here was
revised by professor Roger Ames. 3.
To name just a few exhibitions inside of China: Female Artists’ world (I,1990, II,1995, III,2000, Beijing);
Century, Women (1998, Beijing); Women Declaration: Contemporary Women Artists’ Exhibition (2004,
Shanghai) SHE: Ten Female Artists’ Works, (2004, Beijing), Dream, Dilemma, Modern Feminine Nature
(2004, Beijing), Between Liberation and Bondage (2005, Beijing); Combination: Female Artists’ Exhibition
(2006, Beijing) [titles are all my translations]. 2.
I have used the case of Chen Lingyang in another article, “Second Sex and Contemporary Chinese Women’s
Art: A Case Study on Chen Lingyang’s Work” (in Subversive Strategies in Contemporary Chinese Art, edited
by Mary Bittner Wiseman and Liu Yuedi, 2011. Leiden: Brill Academic Publishers, 147-169), to discuss the
difference between the characters of contemporary Chinese women’s art and Western women’s art. I have
revised section of that work for the purpose of this paper. Chia-rong Wu Abstract: This article focuses on the ideological representation of extreme violence
in Wei Te-sheng’s Warriors of the Rainbow: Seediq Bale with an aim to explore the
cultural and political dimensions of the Wushe Incident and to restore the voice of
the oppressed and colonized. The first part of the article accounts for the histori
cal setting of the film and the trope of beheading in traditional Seediq culture. The
second part discusses how Wei’s film sheds a new light on localized spectacles of
decapitation and further addresses various forms of violence caused by inner and
outer conflicts within the Seediq tribes in the face of aboriginal traditions and colo
nial hegemony. The final section brings into focus the issue of how the extremism
of the film engages the global trend of violence in cinema and facilitates aboriginal
glory and consciousness in defiance of continued symbolic oppression in twenty-
first-century Taiwan. Keywords Seediq Bale; Wei Te-sheng; extreme violence; decapitation; ethnic con
sciousness; anti-colonialism Written and directed by Wei Te-sheng魏德聖 [Wei Desheng], Warriors of the Rainbow:
Seediq Bale 賽德克巴萊 [Saideke balai] (2011) has become the No.1 blockbuster in the
film history of Taiwan. This film is a compelling action epic that has drawn both praise and
controversy. The historical drama centers on the Wushe Incident霧社事件, which is one of
the most famous anti-Japanese revolts. It was also the bloodiest, characterized by exces
sive violence and decapitation. Led by tribal hero Mouna Rudo1 莫那魯道 [Mona Rudao],
three hundred Seediq2 warriors took part in the rebellion against the oppressive colonial
rule on October 27th, 1930, and brutally killed more than 130 Japanese settlers, including
women and children, at a sports event. The film begins with the early lives of the Seediq
people in the late nineteenth century, then shifts to the subjugation of Taiwanese aborigines
in the colonial period, and finally highlights the violent clash between primitive aboriginal
warriors and modern Japanese soldiers. In the film, these headhunters stage an appalling
bloodbath with direct visual impacts and profound ethnic complexities. This article focuses on the ideological representation of extreme violence in Wei’s
Warriors of the Rainbow: Seediq Bale with an aim to explore the cultural and political
dimensions of the Wushe Incident and to restore the voice of the oppressed and colonized. Notes 1. Ames and Jinli He (interview), “The Meaning of Cultural Dialogue,” June 2004. This interview was included
in the Chinese translation of Ames and Hall’s Thinking through Confucius (1987. Albany: State University of
New York Press. Chinese version: 2005. Beijing: Peking University Press). The paragraph quoted here was
revised by professor Roger Ames. 1. Ames and Jinli He (interview), “The Meaning of Cultural Dialogue,” June 2004. This interview was included
in the Chinese translation of Ames and Hall’s Thinking through Confucius (1987. Albany: State University of
New York Press. Chinese version: 2005. Beijing: Peking University Press). The paragraph quoted here was
revised by professor Roger Ames. 2. I have used the case of Chen Lingyang in another article, “Second Sex and Contemporary Chinese Women’s
Art: A Case Study on Chen Lingyang’s Work” (in Subversive Strategies in Contemporary Chinese Art, edited
by Mary Bittner Wiseman and Liu Yuedi, 2011. Leiden: Brill Academic Publishers, 147-169), to discuss the
difference between the characters of contemporary Chinese women’s art and Western women’s art. I have
revised section of that work for the purpose of this paper. 3. To name just a few exhibitions inside of China: Female Artists’ world (I,1990, II,1995, III,2000, Beijing);
Century, Women (1998, Beijing); Women Declaration: Contemporary Women Artists’ Exhibition (2004,
Shanghai) SHE: Ten Female Artists’ Works, (2004, Beijing), Dream, Dilemma, Modern Feminine Nature
(2004, Beijing), Between Liberation and Bondage (2005, Beijing); Combination: Female Artists’ Exhibition
(2006, Beijing) [titles are all my translations]. 4. My translation. ASIANetwork Exchange | Spring 2014 | volume 21 | 2 Chia-rong Wu is anAssistant
Professor of Chinese at Rhodes
College. 24 | Re-Examining Extreme Violence Chia-rong Wu The first part of the article accounts for the historical setting of the film and the trope of
beheading in traditional Seediq culture. The second part discusses how Wei’s film sheds a
new light on localized spectacles of decapitation and further addresses various forms of vio
lence caused by inner and outer conflicts within the Seediq tribes in the face of aboriginal
traditions and colonial hegemony. The final section brings into focus the issue of how the
extremism of the film engages the global trend of violence in cinema and facilitates aborigi 24 | Re-Examining Extreme Violence ASIANetwork Exchange | Spring 2014 | volume 21 | 2 nal glory and consciousness in defiance of the continued symbolic oppression in twenty-
first-century Taiwan. i
Director Wei Te-sheng has become one of the most significant cultural icons in Taiwan
since his hit movie Cape No.7 海角七號 [Haijiao qihao] was released in 2008. Three years
later, Wei’s Seediq Bale further leads audiences to look closely at the violent anti-Japanese
uprisings in colonial Taiwan. Despite their differences in tone and scale, both films are con
nected with the intercultural relationship between Taiwan and Japan. Actually, Wei had the
idea of filming Seediq Bale even before he produced Cape No.7. On several occasions, he
has made clear to the public media that he was first inspired by The Wushe Incident, a comic
book written by Chiu Row-Ling 邱若龍 [Qiu Ruolong] in the late 1990s. However, Wei did
not have enough funding to make such a big-budget movie until the domestic success of
Cape No. 7. Wei and Chiu later became close friends and shared the vision of producing a
film about the Wushe Incident. Chiu even served as one of the costume designers of Seediq
Bale. Before the popularity of Seediq Bale, representations of the history of aborigines in
Taiwan had usually been shown on TV alone, and had relatively limited impacts on the
Taiwanese psyche and market. It is Seediq Bale that helps to reunite Taiwanese aborigines as
a whole by formulating a collective, national Taiwanese identity for both the Han and the
indigenous peoples. Chia-rong Wu As the most expensive historical epic film ever produced in Taiwan, Seediq Bale runs for
four and a half hours in the full, two-part version (Part 1: The Flag of Sun 太陽旗 [Taiyang
qi] and Part 2: The Bridge of Rainbow 彩虹橋 [Caihong qiao]) and two and a half in its
international cut. Given that its international cut leaves out some details regarding the lives
of the Seediq people, the focus of this paper is on the full version. With a cast made up of
indigenous, Hans, and Japanese actors, the film clearly shows Wei’s ambition to display
an authentic picture of Seediq lives in terms of language, costumes, and weaponry. The
film starts with an action-packed sequence during a boar hunt and quickly shifts to more
violent man- and headhunting between two aboriginal tribes, Seediqs and Bununs. Young
Mouna Rudo (游大慶 Da-ching), son of the tribal chief Rudo Ruhei (曾秋勝 Pawan Nawi),
is figured as a tall and muscular Seediq hero. In the opening scene, young Mouna Rudo
beheads two Bunun hunters and escapes successfully with their heads as trophies, a victory
for which he receives a facial tattoo as a spiritual mark. In the Seediq tradition, a young
male can never become a true man “commemorated by a facial tattoo” unless he presents “a
human head to the tribe” (Berry 2008, 100). The process of transformation from boy to man
is also echoed by the film title Seediq Bale, which means “true man.” Seediq Bale then recounts the outcome of the first Sino-Japanese War in 1895. The
defeated Qing court signed the Treaty of Shimonoseki 馬關條約 [Maguan tiaoyue] and
ceded Taiwan and Pescadores to Japan. Under colonial rule, the islanders, including the
indigenous people, and the Hans, Chinese immigrants mainly from Fujian Province,
experienced “assimilation […] from 1895-1919” and “integration from 1919 to 1930” (Liao
2006, 2). During the “assimilation” period, the Japanese government’s attempt to make Tai
wan a colony was not as smooth and successful as expected due to constant riots organized
by the Hans. The “assimilation” process actually sped up in the “integration” period through
educational reform. After 1919, Japanese education was systematically implemented by the
colonial government, and it radically changed the lives of both the Hans and the aborigines. In the name of modernization, the Japanese colonial education involved “formal schooling”
and “[Japanese] language standardization” (Heylen 2004, 5). 25 | Re-Examining Extreme Violence Chia-rong Wu The devastating consequence
is the decline of indigenous culture, as shown in Seediq Bale. Some critics have argued that
Japan actually introduced modernity to Taiwan by developing its economy, education, and 25 | Re-Examining Extreme Violence ASIANetwork Exchange | Spring 2014 | volume 21 | 2 transportation. Nevertheless, it is also important to take into account the negative aspects of
colonialism, including high-handed control, violence, and exploitation. Throughout the colonial period, Japanese settlers were eager to exploit the island’s
resources, such as lumber and mines. The so-called “assimilation” and “integration” process
was simply a colonial means to complete the colonizers’ exploitative mission. Although the
Hans were generally subjugated and ill-treated as subhumans, they were still involved in
society, albeit with class divisions. Aborigines, on the other hand, were directly linked to
savagery and granted limited access to modernity. One major reason for this divide is that
bridging the cultural gap between Japanese and Hans is much easier than doing the same
thing between Japanese and aborigines. Back in 1895, the Japanese colonial government
in Taiwan started banning hunting and ritual headhunting by the aborigines. In order to
fully assimilate indigenous people into the Japanese colony, colonial officials even orga
nized seven “tours to Japan proper” between 1897 and 1925. During these trips, selected
aboriginal leaders, including Mouna Rudo, “were directed to and shown various industrial
and military facilities, the imperial palace, and Shinto shrines [in Japan]” (Ching 2000,
795). The colonizers later asked these “savages” to reveal to their tribesmen a grand modern
picture of the Japanese empire. Despite this and other colonial teachings on industrializa
tion, indigenous people still practiced hunting and headhunting in some regions until the
late 1920s, when the Japanese colonizers were finally determined to tighten up control over
the indigenous people. The enforced banning of traditional indigenous practices had a
huge impact on aborigines. Without their hunting rights and ground, aborigines gradually
became lumberjacks, miners, and maids. It is worth noting that Seediq Bale valorizes primitive violence in response to colonial
atrocity by sharply delineating the contrast between courageous aborigines in the moun
tains and weak Chinese immigrants in the plains. While the unorganized Han rebels are
easily conquered by the well-equipped Japanese army in the film, resilient aboriginal
warriors set up a series of successful ambushes against their foreign enemies. Actually, the
Hans’ military rebellion against the Japanese did not end until 1915. 26 | Re-Examining Extreme Violence Chia-rong Wu Still, some of
Chang’s films have a close engagement with the historical and cultural background of
China. The legendary cultural icon Bruce Lee 李小龍 [Li Xiaolong] further reversed the
weak, feminized Asian male image in the west through kung fu classics like Fist of Fury 精
武門 [Jing wu men] (1972) and Enter the Dragon 龍爭虎鬥 [Longzheng hudou] (1973). Bruce Lee’s success was later repeated by Jackie Chan 成龍 [Cheng Long] and Jet Li 李連杰
[Li Lianjie] in the 1980s and 1990s. In addition to these types of films, Chi-Yun Shih provides an intriguing analysis of the
Asian Extreme films by Tartan Films, such as Japan’s Battle Royale (2000) and South Korea’s
Oldboy (2003) in the essay “Art of Branding: Tartan ‘Asian Extreme’ Films.” While Tartan
Films employs “the visceral and hyper violent nature” of their films as a marketing strategy
(Shih 2008), there is much less cultural and historical context covered in Tartan’s Asian
Extreme series. We need to keep in mind that Seediq Bale is different from Tartan’s Asian
Extremism in that the extreme violence in the film does not exist for its own sake, but rather
as a means of regaining ethnic identity and cultural autonomy for Seediq people. In this
light, we might consider the film to be closer to the kung fu genre, which celebrates extreme
physicality, individual heroism, and sometimes national consciousness. By watching Seediq
Bale, viewers are given close-ups and slow-motion shots of beheading acts, but what really
matters lies behind severed and headless cadavers and beyond shocking effects and uncanny
feelings. Here, the cinematic representation of decapitation is realigned with its original
honor in tandem with the Seediqs’ ancestral spirits and cultural roots. Through the spectacles of violence, director Wei Te-sheng masterfully retells the epic
story and explores a disintegrating ethnic culture of Taiwan. The exceptional vividness of
combat engagement in Seediq Bale has garnered a lot of attention from the public. We can
even say that the director celebrates primitive violence not just as a visual shock, but as a
significant way for aboriginal men to complete their lives and souls. The aboriginal extrem
ism in the film is justified in several aspects. Each aboriginal tribe has its own hunting
ground, which represents natural resources, cultural basis, and spiritual home. According
to Omi Wilang, “Land is the medium through which culture is passed from one genera
tion to the next. Chia-rong Wu This part of the history,
of course, would not fit properly in this indigenous-centered film. In Seediq Bale, there are
only two noticeable Han figures, the nameless owner of the trade center (馬如龍 Ju-Lung
Ma) and Mr. Jin-Dun Wu (鄭志偉 Chi-Wei Cheng), a grocery store owner in the Wushe
district. Unlike robust aboriginal hunters, both Han Chinese men merely engage in trad
ing with aborigines. The passive tone expressed through these harmless Han characters is a
stark contrast to both indigenous extremism and imperial enormity. As a matter of fact, the violent scenes of Seediq Bale make it comparable to Mel Gibson’s
aboriginal epic—Apocalypto (2006). Some critics may argue that Apocalypto is produced
to represent Mayan culture in an authentic and unvarnished way—that is, a strategy also
used by Wei in filming Seediq Bale. However, Apocalypto and Seediq Bale differ in their
perspectives and receptions in domestic communities. The former film aims to represent
the cultural other, and it is perceived as such. The latter re-claims the right to represent the
self, and it greatly raises Taiwan’s indigenous consciousness to the level of national spirit. Moreover, the bloody massacres represented in Seediq Bale further correspond with the
New Extremism in European cinema in which “brutal and visceral images [are] designed
deliberately to shock or provoke the spectator” (Horeck and Kendall 2011, 1). Director
Wei Te-sheng’s handling of extreme violence has received a lot of negative feedback. For
instance, Stephen Holden claims that this film is a “bloodbath that fetishizes the machete
as the ultimate human slicing machine” (2012). Focusing on mechanical killing acts only,
Holden misses the cultural essence of the Seediq people that the director wants to retrieve 26 | Re-Examining Extreme Violence ASIANetwork Exchange | Spring 2014 | volume 21 | 2 through the repeated spectacles of violence. through the repeated spectacles of violence. To renarrate the bloody event from an aboriginal angle, the director carefully presents
the heroic image of Mouna Rudo, along with other Seediq warriors, and instills spirituality
into endless headhunting. By aestheticizing headhunting, the director successfully revives
Asian Extreme cinema outside of the framework of brutal martial arts and horror. Since the
1960s, kung fu has emerged as one of the most popular genres in Chinese film production. In the 1960s and 1970s, Shaw Brothers director Chang Cheh 張徹 [Zhang Che] pro
duced dozens of kung fu flicks that exploit violence primarily for profit. Chia-rong Wu In the field of sports, spectacular examples are Olympic medalists
Chuan-kuang Yang 楊傳廣 [Yang Chuanguang] and Chi Cheng 紀政 [Ji Zheng]. While
Yang is wholly aboriginal by birth, Chi is partially so. As for show business, it is impossible
to ignore the success of the queen of Chinese pop music A-Mei 張惠妹 [Zhang Huimei],
and pop idol Show Luo 羅志祥 [Luo Zhixiang]. Not to mention that the local professional
league of baseball, the national sport of Taiwan, is dominated by indigenous players. Given
his brilliant record against Japan in international baseball games, the most-loved local hero
Chin-Feng Chen 陳金鋒 [Chen Jinfeng] is a perfect case in point. Whenever an interna
tional baseball tournament comes up, the majority of Taiwanese people, baseball fans or
not, are obsessed with the performance of Taiwan’s team. Thus, Taiwanese people’s passion
for baseball in international competition is lifted to the level of national consciousness,
which is shared by all residents in Taiwan. Following the successful model of aboriginal
heroism described above, Seediq Bale showcases a revolutionary narrative of extreme vio
lence on a national level. To the above question, director Wei Te-sheng’s answer is yes. Despite some critiques
on its extreme violence and poor CGI (Computer Generated Imagery) effects, Seediq Bale
has been wellreceived in indigenous communities as an epic film bound up with ethnic
consciousness. For general Taiwanese viewers, other than aborigines, the film can even be
treated as a national calling and a collective endeavor for resistance against the traumatic
colonial past. Some critics may question how the film creates a collective vision against
Japanese colonizers and formulates a shared national identity while focusing on aborigines
and marginalizing the Han Chinese, the dominant ethnic group in Taiwan. At this point,
it is essential to take into account the enormous influence of aboriginal cultural icons, In presenting the primitive violence of the Seediqs, Wei Te-sheng implicitly criticizes
the ideological suppression and physical torture from which the Seediqs suffer. Therefore,
the film idealizes aboriginal uprising and celebrates the aesthetic of primitive violence. The
director uses “words” and “lyrical voiceovers set to battle scenes” to “enhance” the primi
tive “violence ”and “justify” aborigines’ “brutality” (Nordine 2012). Chia-rong Wu When people’s land rights are lost, their autonomy is also lost” (2011). In one scene, middle-aged Mouna Rudo (林慶台 Lin Ching-tai), now the tribal chief, is
confronted by the son of a Japanese officer. The Japanese boy emphasizes that all the hunt
ing grounds in Taiwan belong to imperial Japan. Without the rights to claim their hunt
ing ground, the Seediq men lose their glory as hunters and are mistreated as lumbermen,
while some Seediq women serve as maids in Japanese households and bar girls to entertain
Japanese men. In order to fully understand the Wushe Incident, we need to go back to the
historical fact that the Japanese government in the late 1920s coercively banned headhunt
ing acts widely conducted by the indigenous people in Taiwan. As Michael Berry cleverly
argues, The Japanese prohibition against headhunting, which resulted in an entire genera 27 | Re-Examining Extreme Violence ASIANetwork Exchange | Spring 2014 | volume 21 | 2 tion of [Seediq] males who were relegated to a state of perpetual adolescence and
psychological inferiority can be seen as an important factor that, combined with
exploitation, mistreatment, and other abuses, contributed to the explosion of vio
lence [in the Wushe Incident]. (2008, 100) A critical concern or question here is as follows: if we take a closer look at the extreme
violence of Seediq Bale vis-à-vis the ethnic rite of passage from adolescence to manhood,
can such primitive extremism be perceived and embraced despite its challenge to the rules
of civility in the (post)colonial period? y
p
p
To the above question, director Wei Te-sheng’s answer is yes. Despite some critiques
on its extreme violence and poor CGI (Computer Generated Imagery) effects, Seediq Bale
has been wellreceived in indigenous communities as an epic film bound up with ethnic
consciousness. For general Taiwanese viewers, other than aborigines, the film can even be
treated as a national calling and a collective endeavor for resistance against the traumatic
colonial past. Some critics may question how the film creates a collective vision against
Japanese colonizers and formulates a shared national identity while focusing on aborigines
and marginalizing the Han Chinese, the dominant ethnic group in Taiwan. At this point,
it is essential to take into account the enormous influence of aboriginal cultural icons,
even in modern Taiwan. Chia-rong Wu It is Wei’s belief that
the violent custom of headhunting has to be reenacted and represented from the Seediq
perspective: “Ultimately, I decided to keep those [violent] scenes because they reflected the
truth and I felt it was important to face such things head on, but rather encourage the audi
ence to consider how such brutality should be dealt with” (Wei 2011). In the film, Mouna
Rudo witnesses the declining morale and norms of the Seediq people. Once their hunting
grounds are taken away, their identity and culture gradually erode. After the law against
headhunting was put into practice, the life of the Seediq people “changed dramatically” and
many of them “quickly adapted to Japanese ways” (Balcom 2005, XVII). In this sense, the
Wushe Incident emerges as Seediq warriors’ last revolt against the cruelty and corruption
of Japanese colonists. In defiance of Japanese desecration of the Seediq society, such violent
performances are required and highlighted. 28 | Re-Examining Extreme Violence ASIANetwork Exchange | Spring 2014 | volume 21 | 2 To a great extent, the director follows the conventional revolutionary narrative between
the colonizers and the colonized. Still, some twists exist and are worthy of discussion. Besides antagonism against Japanese intruders, aborigines encounter domestic conflicts
and high tensions that result from headhunting among various tribes. The Seediq tribe of
Mehebo 馬赫坡 [Mahebo] led by Mouna Rudo, the Seediq tribe of Toda 道澤 [Daoze], and
the Bununs are cases in point. In the film, Mouna Rudo’s major opponents are the Japa
nese Major General Yahiko Kamada (Sabu Kawahara) and the tribal leader of Toda, Temu
Walis (馬志翔 Umin Boya). If the 300 Seediqs’ uprising against the Japanese is a wrenching
experience of resistance for film audiences, the internecine violence among the Seediq com
munities is no less unnerving. After the Japanese settlers in the Wushe area are slain and
decapitated by the Seediqs, the colonists soon strike back with force and gas bombs. The
colonists even incite the tribe of Toda to hunt the 300 Seediq warriors’ heads. In this case,
Mouna Rudo, Temu Walis, and Yahiko Kamada are cast as the ethnic hero, the tribal traitor,
and foreign villain, respectively. To a great extent, the director follows the conventional revolutionary narrative between
the colonizers and the colonized. Still, some twists exist and are worthy of discussion. More importantly, Seediq Bale stages varied forms of extreme violence in the colonial
context. Chia-rong Wu In addition to the visual and visceral shock of decapitation, the film portrays
some upsetting scenes about excessive violence performed by the Seediq adolescents and
imposed on innocent women and children. In order not to become tribal warriors’ burdens,
a number of the starving Seediq women decide to commit suicide by hanging themselves
in the forest after the rebellion begins. They die in peace, knowing that they will be waiting
on the other end of the rainbow bridge—that is, the final destination and spiritual haven
of the Seediqs. However, this tragic scene is overshadowed by the “simplified” relationship
between the passive aboriginal women and their aggressive counterparts, as if aboriginal
women can act only as obedient daughters, faithful wives, and dutiful mothers in Wei Te-
sheng’s male-dominated narrative, or kill themselves. As Ian Inkster points out, [I]t should at least be noted that there is much evidence of independence and the
high status of women in Atayal—whom the [Seediq] had previously been classified
as —and related Aboriginal cultures, and early evidence of women engaged in fight
ing to the death, and at times using firearms. (2012) The Seediq women, including Mouna Rudo’s daughter Mahung Rudo (溫嵐 Landy Wen),
who survive the incident, cannot but suffer psychological traumas. Undoubtedly, the direc
tor’s “simplified” version of the male-female relationship successfully projects a riveting per
formance of aboriginal masculinity. However, the gender dynamic poses a serious question
to this allegedly authentic historical drama. Unlike the passive Seediq women, the Seediq
boys stand as merciless warriors and are thus given opportunities of reaching manhood
through headhunting. That said, viewers witness how the Seediq adolescents led by Pawan
Nawi (林源傑 Umin Walis) participate in fierce battles and kill their Japanese enemies,
including unarmed women and children. The bloody violence conducted by Pawan Nawi
and his friends stirs up viewers’ feelings of distress in that the brutality of/on children has
gone beyond the limit of common audiences’ comprehension and sympathy. Through these
cases of Seediq women and children, the director creates some gray space within the frame
work of the male-centered, anti-colonial narrative. Furthermore, Seediq Bale also reexamines two ambiguous figures: Hanaoka Ichiro (徐
詣帆 Hsu Yi-Fan [Xu Yifan]) and Hanaoka Jiro (蘇達 Soda Voyu), whose original Seediq
names are Dakkis Nobin and Dakkis Nawi. Both Ichiro and Jiro receive Japanese educa
tion and later serve as policemen in the Wushe district. 29 | Re-Examining Extreme Violence Chia-rong Wu In the film, they straddle the line
between Seediq traditions and Japanese teachings. Though unwillingly, in the film they help 29 | Re-Examining Extreme Violence ASIANetwork Exchange | Spring 2014 | volume 21 | 2 their tribal fellows gain firearms from the Japanese. However, they never join the physical
combats against the Japanese during the incident. Instead, they commit suicide along with
their families. It is worth considering that both Ichiro and Jiro choose to wear traditional
Japanese costumes before committing suicide. Ichiro takes the Japanese way and commits
seppuku, or stomachcutting, whereas Jiro adheres to the tribal custom and hangs himself
from a tree. In the film, Ichiro even slits the throat of his wife and suffocates his newborn
son before committing suicide. In real life, their deaths have drawn much attention in his
torical research and literary studies. As Michael Berry claims, [Hanaoka Ichiro and Hanaoka Jiro’s] chosen methods of ending their lives—sep
puku and hanging—seem to hint at a cultural schizophrenia: even in death they
were haunted by the tensions between loyalty to their [Seediq] tribe and loyalty to
the colonial empire that reared them. (2008, 56) [Hanaoka Ichiro and Hanaoka Jiro’s] chosen methods of ending their lives—sep
puku and hanging—seem to hint at a cultural schizophrenia: even in death they
were haunted by the tensions between loyalty to their [Seediq] tribe and loyalty to
the colonial empire that reared them. (2008, 56) While the 300 Seediq warriors’ anticolonialism mission is clear and straightforward, the two
Hanaoka cases are ambiguous due to their cross-cultural identity. While the 300 Seediq warriors’ anticolonialism mission is clear and straightforward, the two
Hanaoka cases are ambiguous due to their cross-cultural identity. As mentioned earlier, this indigenous revolt is embraced by Taiwanese audiences as a
nationalist epic. Therefore, the 300 Seediqs’ “drive to kill” can further be expanded into
“the entitlement called narcissism in an individual but nationalism in a country” (Dut
ton 2007, x). Such double-edged symbolic violence proves too overwhelming for Ichiro
and Jiro to handle. Thus, suicide serves as the only relief from their symptoms of “cultural
schizophrenia” and identity crisis. In this sense, Ichiro and Jiro’s inner problem of inter
cultural positioning complicates the heroic act and singular wish of guarding the hunting
grounds shared by Mouna Rudo and his followers. While Ichiro and Jiro die in anguish and
bewilderment, the 300 Seediq warriors fight until the end. 30 | Re-Examining Extreme Violence Chia-rong Wu Most of them are either slain/
beheaded or hang themselves from the tree as many of their tribespeople did before them. Mouna Rudo disappears, and his dead body is found in the deep forest four years later. As
for the few Seediq survivors, their destiny is summarized in the following lines from the
film: “After the incident, Kojima Genji instigated the Seediqs from Toda to revenge the
death of their chief Temu Walis by killing all the remaining defenseless tribesmen staying
in the shelter during the night” (Wei 2012). The film concludes with the 300 Seediq hunt
ers crossing the rainbow bridge. Because they have guarded their land, these true Seediqs
finally reach heaven and join their ancestors there. Through its multiple standpoints and perspectives, Seediq Bale challenges the conven
tional paradigm between good and evil, and between colonizers and colonized. Without
any doubt, the Japanese rule shatters the peace of aboriginal villages, and there are indeed
many high-handed and unsympathetic Japanese characters in the film. Still, viewers get to
examine the relatively friendly police officer Kojima Genji, who befriends Temu Walis and
successfully persuades the tribesmen of Toda to be Japanese colonists’ loyal allies. Seediq
Bale is also loaded with tribe-to-tribe conflicts as well as Hanaoka Ichiro and Hanaoka Jiro’s
dilemma. These multiple facets make the film more complicated than other contemporary
Chinese anti-Japanese epics, such as City of Life and Death 南京！南京！ [Nanjing Nan
jing] (2009) and The Flowers of War 金陵十三釵 [Jinling shisan chai] (2011). To viewers not on Taiwanese soil, Seediq Bale may seem merely sensational with its
exotic settings and violent spectacles. If we take a closer look, however, we see that the
appeal of the film actually springs from the dynamic mix of valor, fear, love, and hatred in
response to cultural contacts and conflicts within human civilization on a universal level. While the colonial history and postcolonial experience have become significant topics in
the global network, Seediq Bale not only engages the assertion of local identity, but echoes 30 | Re-Examining Extreme Violence ASIANetwork Exchange | Spring 2014 | volume 21 | 2 collective remarks against oppressive colonial rule. Unfortunately, the film did not achieve
great success outside Taiwan, given that the mixture of minority discourse and extreme
violence is difficult for global audiences to grasp. The lack of a starry cast and poor CGI also
make it harder to hit foreign markets. Chia-rong Wu Moreover, the truncated version of the film screened
in foreign countries leaves out a lot of historical and cultural context that make it under
standable. It is indeed a pity that Seediq Bale, as the highest grossing film in Taiwanese his
tory, could not spread its influence across the sea to neighboring countries like South Korea,
Singapore, and Malaysia. Still, the film has reached a milestone of the local film industry in
the form of national cinema. Since the 1980s, Taiwan has gone through various aboriginal movements towards
localization, including a series of political claims to preserve the land and promote the
tribal languages of indigenous people. Yet, the declining indigenous cultures often end up
as “nothing more than an exotic cultural commodity to be sold to the tourists” (Balcom
2005, XXI). That said, it is extremely challenging for local ethnic minorities to resist the
impacts of the Han-Chinese culture in the process of urbanization and modernization. There is still a long way to go for aborigines to raise their self-awareness and to be under
stood and respected by other ethnic groups. In a public meeting with indigenous people in
2007, President Ma Ying-jeou 馬英九 [Ma Yingjiu] made an indiscreet and much criti
cized remark: “I see you [aborigines] as people and educate you properly.” No matter how
hard Ma tried to justify himself afterwards, his statement clearly shows the strong sense of
superiority the Han Chinese feel over the aborigines. In terms of the revival of indigenous
awareness, Seediq Bale can be taken as a successful cultural product, if not commodity, tar
geting the local audience. Through the film, the fading history and customs of the Seediqs
are once again renarrated and projected in celebration of the collective spirit and pride of
ethnic minorities. To conclude, the graphic bloody scenes of Seediq Bale deliver messages of ethnic
consciousness and exercise the insights of the director into historical trauma. The film has
been officially recognized by the Taiwan Intellectual Property Office (IPO) as “an important
agent for ethnic identification and a call for historical awareness” (Taipei Times 2010). Chia-rong Wu As
the director expresses his attempt “to promote Taiwan in the international community” and
“bring peace and harmony to all the ethnic groups in the country” (Taipei Times 2011), the
beautifully crafted film provides the audience with a new angle to look into the complex
Wushe Incident through varied forms of extreme violence. This film indeed achieves a sense
of sincerity and vividness in covering the glorious past of aboriginal culture. Although the
extremity of the film sweeps Taiwanese viewers up in its excessive exposure to violent spec
tacles, watching this film is like going through a profoundly cathartic and healing process. The film achieves this affect by reconstructing the collective memories of historical trauma
and producing a localized vision of ethnic consciousness in the postcolonial context of
Taiwan. Balcom, John. 2005. “Translator’s Introduction.” In Indigenous Writers of Taiwan: An Anthology of Stories, Essays,
and Poems, edited by John Balcom and Yingtsih Balcom, XI–XXIV. New York: Columbia University Press.
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University Press. Balcom, John. 2005. “Translator’s Introduction.” In Indigenous Writers of Taiwan: An Anthology of Stories, Essays,
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to Commit Atrocities. Westport: Praeger Security International.h Balcom, John. 2005. “Translator’s Introduction.” In Indigenous Writers of Taiwan: An Anthology of Stories, Essays,
and Poems, edited by John Balcom and Yingtsih Balcom, XI–XXIV. New York: Columbia University Press. 31 | Re-Examining Extreme Violence 32 | Re-Examining Extreme Violence rEFERENCES “‘Seediq Bale’ and Pride in Taiwan.” Taipei Times, March 8. Liao, Ping-hui. 2006. “[Introduction].” In Taiwan Under Japanese Colonial Rule, 1895-1945: History, Culture,
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by Jake Chung http://www taipeitimes com/News/taiwan/archives/2011/10/04/2003514896 Wei, Te-sheng. 2011. “Director Wei Talks About ‘Seediq Bale’”Interview by Tsu-wei Lan. Liberty Times. Translated
by Jake Chung. http://www.taipeitimes.com/News/taiwan/archives/2011/10/04/2003514896. Wei, Te sheng. 2011. Director Wei Talks About Seediq Bale Interview by Tsu wei Lan. Liberty Tim
by Jake Chung. http://www.taipeitimes.com/News/taiwan/archives/2011/10/04/2003514896. by Jake Chung. http://www.taipeitimes.com/News/taiwan/archives/2011/10/04/2003514896. —. 2012. Warriors of the Rainbow: Seediq Bale [Saideke Balai]. DVD. CMC Movie Corporation.h ———. 2012. Warriors of the Rainbow: Seediq Bale [Saideke Balai]. DVD. CMC Movie Corporation. Wil
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i 2011 “Thi L
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l t d b J li
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20 ———. 2012. Warriors of the Rainbow: Seediq Bale [Saideke Balai]. DVD. CMC Movie Corporation. Wilang, Omi. 2011. “This Land Has Long Been Our Home.” Translated by Julian Clegg. Taipei Times, October 20. rEFERENCES Balcom, John. 2005. “Translator’s Introduction.” In Indigenous Writers of Taiwan: An Anthology of Stories, Essays,
and Poems, edited by John Balcom and Yingtsih Balcom, XI–XXIV. New York: Columbia University Press. Berry, Michael. 2008. A History of Pain: Trauma in Modern Chinese Literature and Film. New York: Columbia
University Press. Ching, Leo T.S. 2000. “Savage Construction and Civility Making: Japanese Colonialism and Taiwanese Aborigina
Representation.” Positions: East Asia Cultures Critique 8 (3): 795–818. Dutton, Donald G. 2007. The Psychology of Genocide, Massacres, and Extreme Violence: Why “Norm
to Commit Atrocities. Westport: Praeger Security International.h Donald G. 2007. The Psychology of Genocide, Massacres, and Ext Heylen, Ann. 2004. “The Modernity of Japanese Colonial Education in Taiwan: Moving Beyond Formal School and
Literacy Campaigns.” Taiwan Journal of East Asian Studies 1 (2): 1–36. 31 | Re-Examining Extreme Violence ASIANetwork Exchange | Spring 2014 | volume 21 | 2 Holden, Stephen. 2012. “Machismo, Obtained via Machete.” The New York Times, April 26. http://movies.nytimes. com/2012/04/27/movies/warriors-of-the-rainbow-seediq-bale-by-wei-te-sheng.html. Horeck, Tanya, and Tina Kendall. 2011. “Introduction.” In The New Extremism in Cinema: From France to Europe,
edited by Tanya Horeck and Tina Kendall, 1–17. Edinburgh: Edinburgh University Press. Inkster, Ian. 2012. “‘Seediq Bale’ and Pride in Taiwan.” Taipei Times, March 8. Liao, Ping-hui. 2006. “[Introduction].” In Taiwan Under Japanese Colonial Rule, 1895-1945: History, Culture,
Memory, edited by Ping-hui Liao and David Der-wei Wang, 1–15. New York: Columbia University Press. Nordine, Michael. 2012. “Warriors of the Rainbow: Seediq Bale.” Slant Magazine, April 23. http://movies.nytimes. com/2012/04/27/movies/warriors-of-the-rainbow-seediq-bale-by-wei-te-sheng.html. Shih, Chi-Yun. 2008. “Art of Branding: Tartan ‘Asia Extreme’ Films.” Jump Cut: A Review of Contemporary Media
50. http://www.ejumpcut.org/archive/jc50.2008/TartanDist/index.html. Taipei Times. 2010. “Government to Withdraw Seediq Bale Trademark.” Taipei Times, September 17. ———. 2011. “‘Seediq Bale’ Addresses Harmony.” Taipei Times, September 3. Wei, Te-sheng. 2011. “Director Wei Talks About ‘Seediq Bale’”Interview by Tsu-wei Lan. Liberty Times. Translated
by Jake Chung. http://www.taipeitimes.com/News/taiwan/archives/2011/10/04/2003514896. ———. 2012. Warriors of the Rainbow: Seediq Bale [Saideke Balai]. DVD. CMC Movie Corporation. Wilang, Omi. 2011. “This Land Has Long Been Our Home.” Translated by Julian Clegg. Taipei Times, October 20. Holden, Stephen. 2012. “Machismo, Obtained via Machete.” The New York Times, April 26. http://movies.nytimes. com/2012/04/27/movies/warriors-of-the-rainbow-seediq-bale-by-wei-te-sheng.html.h q
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Inkster, Ian. 2012. Holden, Stephen. 2012. “Machismo, Obtained via Machete.” The New York Times, April 26. http://movies.nytimes.
com/2012/04/27/movies/warriors-of-the-rainbow-seediq-bale-by-wei-te-sheng.html.
Horeck, Tanya, and Tina Kendall. 2011. “Introduction.” In The New Extremism in Cinema: From France to Europe,
edited by Tanya Horeck and Tina Kendall, 1–17. Edinburgh: Edinburgh University Press.
Inkster, Ian. 2012. “‘Seediq Bale’ and Pride in Taiwan.” Taipei Times, March 8.
Liao, Ping-hui. 2006. “[Introduction].” In Taiwan Under Japanese Colonial Rule, 1895-1945: History, Culture,
Memory, edited by Ping-hui Liao and David Der-wei Wang, 1–15. New York: Columbia University Press.
Nordine, Michael. 2012. “Warriors of the Rainbow: Seediq Bale.” Slant Magazine, April 23. http://movies.nytimes.
com/2012/04/27/movies/warriors-of-the-rainbow-seediq-bale-by-wei-te-sheng.html.
Shih, Chi-Yun. 2008. “Art of Branding: Tartan ‘Asia Extreme’ Films.” Jump Cut: A Review of Contemporary Media
50. http://www.ejumpcut.org/archive/jc50.2008/TartanDist/index.html.
Taipei Times. 2010. “Government to Withdraw Seediq Bale Trademark.” Taipei Times, September 17.
———. 2011. “‘Seediq Bale’ Addresses Harmony.” Taipei Times, September 3.
Wei, Te-sheng. 2011. “Director Wei Talks About ‘Seediq Bale’”Interview by Tsu-wei Lan. Liberty Times. Translated
by Jake Chung. http://www.taipeitimes.com/News/taiwan/archives/2011/10/04/2003514896.
———. 2012. Warriors of the Rainbow: Seediq Bale [Saideke Balai]. DVD. CMC Movie Corporation.
Wilang, Omi. 2011. “This Land Has Long Been Our Home.” Translated by Julian Clegg. Taipei Times, October 20. Holden, Stephen. 2012. “Machismo, Obtained via Machete.” The New York Times, April 26. http://movies.nytimes.
com/2012/04/27/movies/warriors-of-the-rainbow-seediq-bale-by-wei-te-sheng.html.h Introduction to ANE special issue This special section on teaching modern Asian history developed from papers originally
presented as a panel at the 2012 Annual Meeting of the American Historical Association. Tracy Barrett (North Dakota St. U.) and Xiaojia Hou (U. Colorado-Denver) constituted the
panel with an eye to drawing together research expertise from each of the “subregions” of
Asia (Central Asia, East Asia, South Asia, and Southeast Asia). It was their hope to identify
new strategies and materials that would support rigorous teaching of Asia in liberal arts
institutions. When Alison Mackenzie Shah (U. Colorado-Denver) was unable to attend the
conference, she kindly recommended me to participate as the representative for South Asia. As a relative latecomer to the project, I hope to convey accurately the aims and concerns of
these essays, which represent the trajectory of our discussions thus far. Each of the contributors perceived the need to address two pressing concerns. First,
for over a decade we have seen the creation of new faculty positions in one field of Asian
history or another. In the United States, many of these new positions are conceived very
broadly as “Asian history.” Such a broad category presents a problem, as the demands of
this definition are often at odds with the area studies boundaries that continue to define
graduate-level training. This presents scholars in all fields of Asia Studies with both a chal
lenge and an opportunity. The challenge is that scholars with training in one area may be
asked to teach courses that lay well beyond the boundaries of their training. For example,
an East Asia specialist may be asked to teach courses on South Asia or on Asia at large,
for which they have little training. Faculty find themselves at a loss to identify appropriate
readings and craft assignments for their undergraduate students because they cannot draw
upon their formal preparation. How does one develop a familiarity with whole new fields of
study, or keep an eye open for new trends in fields to which they are not accustomed? The
opportunity lies in the potential for faculty to read and engage more widely in discussions
across area studies boundaries, and to bring to bear analytical concerns and techniques
from one field of scholarship upon another. This breadth can serve us as scholars and our
students as young adults living in a connected, interdependent world. Notesh 1. This article uses Chinese pinyin, but follows the translations of specific names provided in the English
subtitles of the movie DVD released by CMC Movie Corporation in April, 2012. 2. Seediq is originally part of the Atayal tribe and has been officially recognized as one of the fourteen aborigi
nal tribes of Taiwan since 2007. ASIANetwork Exchange | Spring 2014 | volume 21 | 2 Introduction to ANE special issue Second, the area studies framework of graduate training, grounded as it is in study of
particular languages, has created a problem when it comes to finding and evaluating pri
mary sources. To be sure, the vast linguistic diversity of Asia plays a role in the structure of
this problem. But it is difficult for a specialist in Southeast Asian history to know what sort
of primary sources can best represent, for example, Japanese thinkers and connoisseurs of
high culture in the Muromachi period, unless that person has built and maintained a net
work of colleagues who can make these recommendations. While such appeals have been
made through H-Asia, and contributors to that network do periodically announce the pub
lication of electronic and print resources that might suit the needs of instructors attempting
to cross the various area studies boundaries, H-Asia alone is not enough. A cursory search
for “Muromachi culture” carried out on 16 March 2014 yielded just seven hits, including
announcements of scholarly publications and academic conferences. With new resources
becoming available electronically, and older print sources becoming less easily available or
disappearing entirely, annotated lists of resources need to be made available and updated 33 | Introduction to ANE special issue ASIANetwork Exchange | Spring 2014 | volume 21 | 2 regularly. This is a particular area in which ASIANetwork can serve educators in many dif
ferent kinds of institutions. Traditional formulations of Asian history cohere in one or two regions, including East
Asia, Southeast Asia, South Asia and Central Asia; alternatively, Asia is presented as a col
lection of disparate nation-states. One may be tempted to substitute a nation-state approach
for the problematic area studies approach; after all, much scholarship and course design
limit themselves to a single nation-state. The nation-state approach offers a mixed blessing:
it provides massive quantities of data, yet the production of such data naturalizes the teleol
ogy of nation-state. And the populations and territories claimed by nation-states constantly
change, for political and environmental reasons. Further, forces with causative power have
acted and continue to act without reference to area studies or nation-state boundaries. How
could a specialist in only one region of Asia easily assess and teach the effects of the 2005
tsunami or the Tablighi Jama‘at with sophistication? Given the range of problems, how
might we organize courses, particularly those taught at the introductory level? Introduction to ANE special issue How can we
present distinctively Asian voices from the past, without succumbing to the latent oriental
ism of the area studies mode of organizing knowledge? The essays in this special section
of ASIANetwork Exchange seek to introduce students to new methods of understanding
the interconnectedness of Asian history in modern times and in world history. The articles
focus on the idea of avoiding the description of historical actors as objects by incorporating
their subjective experiences through the use of their photographs, art, primary texts, music,
and other media. Each article considers specific sources drawn from their field and provides
insight into how one might incorporate them into existing syllabi or shape a new syllabus
altogether. Tracy Barrett focuses her essay on the common historiographical problem of who gets
to write histories and the linked pedagogical problem of how to get primarily American
students to think about Asian history outside of fundamentally colonial mentalités. Barrett
argues forcefully that, in the case of Southeast Asia, the answer to both problems requires
the classroom use of films, memoirs, and fiction produced by Southeast Asians. While this
pedagogy may require interpretive practices that are not staples of undergraduate historical
training, such primary sources are much more widely available in English translation than
are collections of documents that might otherwise shed light on the economic, social, or
cultural history of Southeast Asia. In addition, their narrative structures make them more
compelling than analytic narratives and thus better able to evoke an empathetic under
standing of the past. In my article, I address the pedagogical problems associated with building a course
covering the full range of South Asian history with the available textbooks on the subject
and the absence of a primary source reader free of the political history and Indological
proclivities of the mid-twentieth century. I offer some possibilities for combining textbooks
with each other as well as with longer primary sources. The article makes an introductory
effort at listing primary sources, mainly written, with an eye to those that shed light on the
material, social, and cultural lives of South Asians. While there is no single solution to suit
all needs, I argue that some materials do permit instructors to organize teaching in ways
that move students beyond the narrow focus on the colonial popularized through readings
of Subaltern Studies. 35 | Introduction to ANE special issue Brian Caton, Luther College Tracy C. Barrett is an Assistant
Professor in the Department of
History, Philosophy, and Reli
gious Studies at North Dakota
State University Introduction to ANE special issue Amy Kardos grapples with the historiographical chestnut of how to recover histories of
central Eurasian people through the often pejorative textual sources produced by their liter
ate neighbors. She shows how using new scholarship can help specialists in South and East
Asia to conceptualize the “silk road” not as a path along which material objects, people, and 34 | Introduction to ANE special issue ASIANetwork Exchange | Spring 2014 | volume 21 | 2 therefore ideas traveled, but as a shorthand for material and intellectual exchanges, carried
out by central Eurasian brokers along shifting frontiers defined by the exchanges them
selves. These can be traced through, for example, variations on the spiked fiddle that extend
from Persia to China, and through modes of playing them, from early times to Yo-Yo Ma’s
“Silk Road Project.” Danke Li thinks through the old, yet persistent, problem of transforming students’
one-dimensional views of women in Chinese and Japanese history. Though some of the
challenges posed by student assumptions detailed in her article could apply to instructors
in courses pertaining to virtually any other part of Asia, Li argues that the most effective
solution is to expose students to feminist scholarship produced by Chinese and Japanese
scholars and to supplement it with textual and film primary sources that let Chinese and
Japanese women, as much as possible, express themselves and depict their own lifestyles. The approach has the additional benefit of giving students tools with which to challenge and
therefore engage intimately with gender and feminist theory, both as an academic enterprise
and as a strategy for seeking social justice. Through this collection of articles, the special section on teaching modern Asian history
aims to initiate what we hope will be an ongoing discussion about the theoretical underpin
nings and practical approaches to a pedagogy of modern Asian history. We pointedly invite
comment, either in ASIANetwork Exchange or in other venues. 35 | Introduction to ANE special issue ASIANetwork Exchange | Spring 2014 | volume 21 | 2 36 | Teaching East and Southeast Asia through Asian Eyes Teaching East and Southeast Asia through Asian Eyes Tracy C. Barrett Abstract: Colonialism and indigenous responses to its varied forms dominate
modern Asian historiography and imbue the history of the region with rich and
multifaceted connections to world history. As a result, the histories of East and
Southeast Asian nation-states since 1500 cannot be viewed outside of the context of
global affairs. Imagining Asian peoples and cultures during this time is problematic
for students, who typically approach colonialism from a western perspective. This
article explores various means of incorporating pedagogical materials and diverse
media sources into the classroom to facilitate a more grounded examination of East
and Southeast Asian colonies, peoples, and nation-states. It pays special attention to
teaching colonialism, anticolonialism, nationalism, and transnationalism from the
perspective of Southeast Asia’s indigenous peoples. Keywords History; Pedagogy; Film; Literature; Memoir; East Asia; Southeast Asia Such discussions have emphasized the many colonial, neocolonial, or Orien
talist perspectives displayed by Western media, and then discussed best practices for analyz
ing these biased sources in a way that broadens student understanding of Asian issues in a
global society.1 Insofar as these approaches help us (and our students) understand the racial
and ethnocentric preconceptions that Western students often bring into the Asian history
classroom, they can be useful; however, using them to the exclusion of other materials fails 36 | Teaching East and Southeast Asia through Asian Eyes ASIANetwork Exchange | Spring 2014 | volume 21 | 2 to expose students to the breadth and depth of Asian memoirs, fiction, and film available
for classroom use. The late Chinese historian Harold Isaacs once commented that “every ‘reality’ is made
up of somebody’s images; every ‘image’ is part of someone’s reality. Images, moreover,
appear in the eye of the beholder. Who, then, is beholding, and who is being beheld, when,
where, in what circumstances...?” (Isaacs 1975, 258) No group is more capable of expressing
who “Asians” are—whether historical or contemporary—than the writers and filmmak
ers of Asia itself. Given that indigenous filmmakers and authors may not be historical or
ethnographical experts, issues of historical authority and veracity provide additional fodder
for contextualization and group discussion in class. This essay offers some suggestions for
resources to use when teaching Asian history in general and Southeast Asian history in
particular. It also examines ways in which these sources might be used to speak to some of
the broader historical themes that are central to courses in Asian and global history. Before entering into a more exhaustive survey of specific current Southeast Asian
sources, it is important to look critically at methods of conveying “images” of Asia that
transcend the bounds of classroom lectures and recitations of historical fact. In particu
lar, the use of film as a medium for enhancing historical and ethnographic understanding
remains hotly contested in pedagogical scholarship. In the field of Anthropology, a mourn
ful discourse has emerged addressing the issues surrounding what are largely documen
tary films and students’ less than optimal responses to them. Keywords History; Pedagogy; Film; Literature; Memoir; East Asia; Southeast Asia Keywords History; Pedagogy; Film; Literature; Memoir; East Asia; Southeast Asia In 1960, renowned Southeast Asian historian D. G. E. Hall published a call to arms for
Asian historians, and particularly historians of Southeast Asia, writing, “The need therefore
for a big effort of expansion in the serious study of Southeast Asian history in the United
States has become very urgent” (Hall 1960, 281). Despite the passage of more than 50 years
since the penning of that article, Hall’s complaint still resonates with Asian historians today. Nowhere is the dearth of Asia-centered histories and sources felt more keenly than in the
classroom. A myriad of challenges confronts teachers of Asian history in the U.S., and irre
spective of the types of institutions at which Asian history is taught, these challenges more
or less remain the same. Broadly speaking, these issues are well known to historians who visit the various profes
sional forums frequented by teachers of Asian history. Most of them concern the general
lack of familiarity with Asian philosophies, culture, and languages exhibited by American
students. A recent thread on the H-Asia listserv, for example, questioned the value of teach
ing Asian history beyond the survey level to students unfamiliar with Asian languages. The
crux of this dilemma seemed to center upon two questions: can one effectively introduce
Asia to non-Asian students and, if so, how? I believe that a solution to this difficulty can be
found in a careful reevaluation of the types of sources used in the teaching of Asian history. Film, fiction, and memoir are essential ingredients in the successful development of cultural
understanding in the classroom. In the past, Western discussions of pedagogical approaches to diverse media having to
do with Asia have tended to focus upon the failure of these media to give true insight into
Asian history. 37 | Teaching East and Southeast Asia through Asian Eyes Keywords History; Pedagogy; Film; Literature; Memoir; East Asia; Southeast Asia Elizabeth Bird and Jonathan
Godwin criticize what they term the “unspoken assumptions…that films ‘speak for them
selves’ in a direct way that books may not, or that students in a media-saturated society
will naturally respond better to media than to lectures or reading” (Bird and Godwin 2006,
285). They highlight the realities of the modern freshman classroom, warning readers not
to show “classic” ethnographic films in their entirety: “Do not expect distracted, media-
saturated freshmen to appreciate the elegiac beauty of [indigenous peoples] – they are much
more likely to react to ‘disgusting’ habits and ‘primitive’ conditions” (Bird and Godwin
2006, 296). Bird and Godwin are certainly correct in cautioning teachers to contextualize
their selections of media sources carefully. Teachers of language, culture, and history, on the other hand, generally report more
satisfaction with media-based pedagogy. Historian Denise Blum speaks of the power of
film when she suggests that, when properly presented it, “is not just about the transfer of
information as film is traditionally used in classrooms. Rather, it engages memory, emo
tion, and the intellect in the production of knowledge and meaning” (Blum 2006, 301). In
writing about her experience screening a film about the Arab-Israeli conflict, Blum notes
that “identities are a key ingredient in the film experience. Depending on which political
identities are engaged and the form in which the film is produced, viewed, and facilitated,
the experience can vary tremendously” (Blum 2006, 303). This idea is reinforced by the
work of Janet Staiger, who highlights the multidimensionality of film reception and audi
ence response, a heterogeneity based largely upon the experiences and identities with which
viewers, or, in our case, students, approach the screen (Staiger 2000). Elissa Tognozzi, an
Italian professor, concludes that film-based assignments expand “students’ notions of how
to interpret other cultures and how to communicate within their social contexts, emerging
from the experience with a far deeper cultural awareness” (Tognozzi 2010, 81). hi These inconsistencies in evaluating the medium of film revolve largely around issues of
audience bias and receptivity: pedagogical success or failure seems to hinge upon appro
priate contextualization of the media by the instructor. Contextualization is an essential
element in the use of any medium, whether film or printed, fiction or nonfiction. Keywords History; Pedagogy; Film; Literature; Memoir; East Asia; Southeast Asia Many 37 | Teaching East and Southeast Asia through Asian Eyes ASIANetwork Exchange | Spring 2014 | volume 21 | 2 teachers, especially in secondary and community college education, cite “providing subject
matter content” as a rationale for using expanded media, but this reasoning has serious
flaws (Marcus and Stoddard 2007, 315). Levstik and Barton (2001, 9-12, 358) have already
established that rote memorization is not an effective method of fostering historic under
standing, but the unproblematized use of fiction, film, and other media is equally unpro
ductive. Adam Woelders (2007, 365) argues for a distinction between ineffective teaching
with film and the immensely useful process of “teaching about history with film,” which
“challenges students to construct their own historical knowledge based on inferences,
speculations, and conclusions drawn from evidence, not solely from any authoritative
source such as the teacher, the textbook, or the information” they glean from other course
materials like film, fiction, and memoir. In this paradigm, in the college classroom, the act
of assigning critical value to narrative sources based upon information already gleaned from
more didactic pedagogical methods such as lecture instructs students in historical relativity
and bias while simultaneously enabling them to hone their skills at historical interpretation. f Film as a means of increasing students’ historic empathy, while potentially quite effec
tive, can also be pedagogically suspect. After all, increased empathy is no guarantee of
improved understanding or knowledge of historic events. As so many of the scholars cited
in this article have agreed, the key to enhanced cultural awareness and historic understand
ing lies in the careful contextualization of prominent events and cultural realities of the past. With proper groundwork, however, fiction, memoir, and film become invaluable resources
for enabling students to understand what otherwise might seem, as Bird and Godwin put
it, “primitive and disgusting” cultures and events. To this end, the remainder of this article
shall address a variety of literary and film sources that offer particular insight into various
time periods and themes of Asian history. One of the more difficult tasks when teaching Asian survey courses is to sufficiently
explain numerous and complex Asian philosophies in order to contextualize the histories
I am trying to illuminate. In my classroom, I have begun experimenting with assignments
that combine primary source examples of historical events with the philosophical texts
themselves. 38 | Teaching East and Southeast Asia through Asian Eyes Keywords History; Pedagogy; Film; Literature; Memoir; East Asia; Southeast Asia For example, when discussing the Confucian value of filial piety in an East
Asian survey course, we read and discuss brief excerpts from the Confucian Analects, Men
cius, and the Classic of Filial Piety (Xiao Jing) (de Bary, Bloom and Adler 2000), but we also
read Lady Hyegyong’s 1805 memoir (Hyegyong 1996, 241-337). This classic and chilling
tale of the path of madness and murder taken by Lady Hyegyong’s husband, Korean Crown
Prince Sado (1735-1762), eventually ends with Sado’s public execution at the hands of his
father, King Yongjo. This tragedy, given perspective and emotion by Lady Hyegyong herself
as she grapples with her own Confucian responsibilities, creates the perfect backdrop for a
writing assignment dealing with the multidirectional obligations inherent in the Confucian
family structure. The pathos and horror of Lady Hyegyong’s tale bring 18th century Korea
to vivid life—even for students raised on Law & Order and CSI. Carefully selected combinations of primary texts also lend themselves to writing
exercises that employ methods of historical, cultural, or political role playing. Historian
Noel Miner has argued convincingly in favor of the employment of historical role-playing
techniques, including having students write and perform their own three-act plays distilled
from selected historical events and sources in class. Miner argues that while simulation and role-playing techniques cannot be considered panaceas to replace
the lecture and textbook in every history class, … the potential emotional impact
of simulated events and the opportunity to manipulate events which will become
history justifies use of these pedagogical methods. Students involved in these classes ASIANetwork Exchange | Spring 2014 | volume 21 | 2 have stated that the exercise had been both intellectually stimulating and enjoya
ble.... Especially among non-history majors there was genuine enthusiasm for a sub
ject which too often succumbed to the listing of arcane facts and forgettable names. For a brief moment, history had come to life in the classroom. (Miner 1977, 227-8) While Miner’s complex multi-act play assignment might be too time consuming for use in
a survey class, role playing in other forms can still involve students more personally in the
philosophies of a given place and time. Keywords History; Pedagogy; Film; Literature; Memoir; East Asia; Southeast Asia Students have responded very positively, for instance, to a writing assignment that has
become a staple of my pre-modern East Asian history class: a letter written to an imaginary
king of the Warring States period urging him to eschew the ideas of certain prominent
philosophers of the time while encouraging the wholesale adoption of the ideas of another. Each student is required to develop his or her own rationale for supporting the chosen
philosophy. Given all of the philosophers from this broadly-conceived period—Confucius,
Mencius, Han Fei Zi, Mo Zi, Lao Zi, and Zhuang Zi—this assignment encourages students
to delve into and engage with a selection of philosophical readings in a way that mere
description or recitation of facts would not.2 Similar writing assignments addressing early
confrontations between Asia and the West have also piqued student interest and received
favorable reviews in end-of-semester course evaluations. In upper-division classes, longer papers and a heavier reading load allow for more com
plex assignments. In a recent senior-level course on Japan, my students looked at a variety
of sources dealing with three prominent periods in Japanese history in order to envision
these eras more clearly and personally. For the Heian period, Murasaki Shikibu’s Tale of
Genji (1990) provided a fictional account of Heian society that was brilliantly reinforced
by an iconic Heian diary, The Pillow Book of Sei Shonagon (Shonagon 1991). Video clips of
modern kabuki performances and PowerPoint presentations emphasizing Heian art and
architecture breathed life into a period whose aesthetics might otherwise appear fussy and
effete in a modern classroom. Finally, with regard to Japan in the aftermath of war, Kenza
buro Oe’s short stories (for example, those found in the 1994 collection Teach us to Outgrow
our Madness) nicely complemented John Dower’s exemplary traditional history Embrac
ing Defeat (Dower 2000). In this modern era, films such as Ichikawa Kon’s 1956 gem The
Burmese Harp, Honda Ishiro’s 1954 film Gojira, or even the more recent 1988 anime film
directed by Takahata Isao, Grave of the Fireflies (based on Nosaka Akiyuki’s semi-autobi
ographical account of his childhood in wartime Kobe, Japan) could all be used as further
platforms for discussion of the impact of war upon Japanese society. The strength of all of these memoirs, novels, stories, and films rests in the vividness with
which they portray the culture, people, or zeitgeist of a different place and time. 39 | Teaching East and Southeast Asia through Asian Eyes Keywords History; Pedagogy; Film; Literature; Memoir; East Asia; Southeast Asia By ridiculing and caricaturing the hypocrisies abounding in the colonial
Philippines, Noli Me Tangere provides a peerless platform for evaluating Spanish colonial
ism and evolving nationalist sentiments from the inside out. The injustice of the racial hierarchy of Dutch colonialism is the focus of the first volume
in Pramoedya Ananta Toer’s Buru Quartet.3 This Earth of Mankind (Toer 1996) was nar
rated to Pramoedya’s fellow political prisoners on Buru Island beginning in the early 1970s. This novel, too, provides a scathing critique of colonialism, in this case as it operated under
Dutch rule in the Netherlands East Indies. In this tale, the main character, Minke, a Java
nese youth of uncommon intellect and promise, is scorned by his classmates and limited
in achieving his ambitions as a result of his race, while others of lesser ability find greater
opportunity for happiness and success because of their European blood. When read along
side other, earlier primary sources, such as Raden Ayu Kartini’s turn-of-the-nineteenth-
century writings about Javanese society and the social plight of Javanese women, student
understanding of the difficulties faced by the denizens of the Netherlands East Indies
resonates with an even deeper poignancy (Kartini 1992). Equally critical of French colonialism in Vietnam but far more satirical in their
approaches to it are Vu Trong Phung’s 1936 novel Dumb Luck (2002) and his 1934 report
age, The Industry of Marrying Europeans (2005). Dumb Luck, the story of the absurd ascent
of a mendicant named Red-Haired Xuan through the social strata of colonial Hanoi, sheds
light on the problems resulting from the abandonment of all things “traditional” in pursuit
of western-style modernization. By offering a powerful critique, less of colonialism than of
the people who fall prey to its influences, Dumb Luck preaches a subtle and nuanced mes
sage about the cultural and social threats made to Vietnamese society by French involve
ment in the region. The Industry of Marrying Europeans, using a combination of fiction
and non-fiction, examines a similar phenomenon: the lives of Indochinese women who
seek to improve their lots by marrying, even if temporarily, European men. The less farci
cal approach taken by The Industry of Marrying Europeans is particularly appropriate for
students in survey classes, as its critique leaves a bit less to the imagination. Keywords History; Pedagogy; Film; Literature; Memoir; East Asia; Southeast Asia When Sei
Shonagon writes lists of things she finds annoying, modern college students find that they
can relate because they find some of the listed things annoying too. When students weep
over the fate of Seita and Setsuko in Grave of the Fireflies (1988), they have transcended
cultural barriers to empathize personally with the film’s protagonists. When their hearts
break for The Burmese Harp’s (1956) main protagonist, Mizushima, as he wanders around
Burma to bury the untended piles of Japanese dead, the stereotype of the fearsome and
single-minded Japanese soldier is severely damaged. In the Western classroom, relating
to Asian characters on these levels transcends mere human empathy; in fact, it represents
the replacement of myth, stereotype, and fantastical imaginings with “images” of greater
authenticity. While The Tale of Genji may not be representative of the Heian-era Japanese
lifestyle in general, it still depicts with some significant fidelity the mores and aesthetics of a 39 | Teaching East and Southeast Asia through Asian Eyes ASIANetwork Exchange | Spring 2014 | volume 21 | 2 real time and place. Can the same be said of The Karate Kid’s (1984) Mr. Miyagi? h
The examples I’ve discussed so far have all involved the study of East Asian history,
which, while certainly exotic in the minds of most American students, is bolstered by a sub
stantial body of translated primary sources of all varieties and in all major media. Histori
ans of Southeast Asia, on the other hand, are not yet blessed with such a bounty of primary
source material in translation. But despite the paucity of major Southeast Asian works in
translation, many of the sources that do exist are ideal for “reimaging” Southeast Asia in the
eyes of American students. The remainder of this essay shall focus upon introducing some
of these Southeast Asian sources while, in the interest of brevity, limiting this discussion to
the central and overlapping themes of colonialism and war. By far the most iconic of Southeast Asia’s anticolonial novels, Jose Rizal’s Noli Me Tan
gere (2006), is stunning in its sweeping challenge to Spanish Colonialism. First published in
1887, this novel and its sequel, El Filibusterismo (2007), were originally written in Spanish
by Rizal, perhaps the most prominent of the Filipino nationalists. This story exposes the
corruption, ignorance, and venality of the Spanish colonial authorities and Spanish friars
in the Philippines. 40 | Teaching East and Southeast Asia through Asian Eyes Keywords History; Pedagogy; Film; Literature; Memoir; East Asia; Southeast Asia h For greater insight into the changing lives in colonial and postcolonial Thailand, Kukrit
Pramoj’s novel Four Reigns (1999) paints a sweeping portrait of the life of an upper-class
Siamese woman. Even though Siam itself was never colonized by a European power, this
novel, beginning in the 1890s and continuing through World War II, still depicts the dra
matic transitions of a single lifetime as the main character, Phloi, moves from her home to
the king’s palace, where she befriends the palace servants and courtiers, eventually marries
and begins a family of her own, and watches her children embark upon their own lives. This “story of a life” takes dramatic twists and turns due to the impacts of events happening
both in Asia and across the globe. Although the main character is a woman, and despite the
sensitive treatment of women’s powerlessness by the author, this novel is better used as a
fictionalized social history of fin de siècle and wartime Siam/Thailand than as a representa
tion of female voice. This “story of a life” takes dramatic twists and turns due to the impacts of events happening
both in Asia and across the globe. Although the main character is a woman, and despite the
sensitive treatment of women’s powerlessness by the author, this novel is better used as a
fictionalized social history of fin de siècle and wartime Siam/Thailand than as a representa
tion of female voice. Using a satirical approach more similar to Dumb Luck than to the more serious Four
Reigns, Y. B. Mangunwijaya’s novel Durga Umayi (2004) also tells the story of a Southeast
Asian woman through several decades of dramatic postcolonial change. This story begins
in the Netherlands East Indies in the 1930s and ends in Indonesia during the presidency
of Suharto. Set against the backdrop of the radical transformations wrought by Indonesian
nationalism, Durga Umayi faithfully, if surrealistically, depicts the chaos and capriciousness
of life in the new Indonesia while remaining faithful to the magical world of Indonesian
tradition. As an example of the postcolonial condition, this entertaining tale is well suited
to upper division Asian and World History classes, especially if students are encouraged to
persevere through the first chapter or two until they get a feel for the flow of the novel. 41 | Teaching East and Southeast Asia through Asian Eyes Keywords History; Pedagogy; Film; Literature; Memoir; East Asia; Southeast Asia These stories
provide a fascinating and personal narrative backdrop to more political sources, such as
those assembled in Truong Buu Lam’s exceptional collection, Colonialism Experienced: Viet
namese Writings on Colonialism, 1900-1931 (2000). In the category of cinema, when discussing the colonial era in Indochina, the main
pitfall is the proliferation of assorted French films of varying virtue and veracity that depict 40 | Teaching East and Southeast Asia through Asian Eyes ASIANetwork Exchange | Spring 2014 | volume 21 | 2 French recollections of the colonial. The most prominent of these is doubtless the 1992 film
Indochine, which tells the story of a plantation-owning mother and her adopted Vietnamese
daughter as they maneuver through a society being altered by rising anti-French sentiment
and a growing tide of Vietnamese nationalism. As a representation of French fascination
with the Indochinese exotic, this film can be quite useful in a classroom setting; it is a per
fect example of Orientalism in film form. This bias makes Indochine a helpful springboard
for upper-level discussions of the repeatedly-demonstrated French reluctance to relinquish
their Asian colonies. However, there is also a stellar Vietnamese film that depicts the lives
of Vietnamese peasants in the twilight of the colonial era, the 2004 production Buffalo Boy. This film, set in the late 1940s, tells the story of a teenage boy named Kim and his struggle
to save the water buffalo, which are instrumental to the family’s survival as subsistence
farmers. This coming-of-age story is set against a backdrop of water and reflects the relent
less impact of monsoonal floods upon a family terrorized by natural disaster and an oppres
sive French colonial government.h For greater insight into the changing lives in colonial and postcolonial Thailand, Kukrit
Pramoj’s novel Four Reigns (1999) paints a sweeping portrait of the life of an upper-class
Siamese woman. Even though Siam itself was never colonized by a European power, this
novel, beginning in the 1890s and continuing through World War II, still depicts the dra
matic transitions of a single lifetime as the main character, Phloi, moves from her home to
the king’s palace, where she befriends the palace servants and courtiers, eventually marries
and begins a family of her own, and watches her children embark upon their own lives. Keywords History; Pedagogy; Film; Literature; Memoir; East Asia; Southeast Asia Another interesting twentieth-century Southeast Asian source comes from Ba Maw, the
political leader and advocate for Burmese self-rule during the British colonial period. His
memoir, Breakthrough in Burma, depicts the chaos, violence, and betrayal that character
ized Burma during the late colonial era and the early years of World War II (Maw 1968). Although not explicitly an Asian voice, in his 1934 classic Burmese Days (2010), George
Orwell offers a blistering insider’s critique of British colonial rule that adroitly describes
not just the nature of British colonial society, but also the price of speaking out against it. When assigned consecutively, even if in excerpts, Breakthrough in Burma and Burmese Days
together provide an excellent framework for discussing the destabilizing influence of colo
nialism upon a colonized society. In any discussion of the overlapping colonialisms of Western nations and Imperial
Japan, Indonesian author Pramoedya Ananta Toer makes an excellent starting point. His 41 | Teaching East and Southeast Asia through Asian Eyes ASIANetwork Exchange | Spring 2014 | volume 21 | 2 first major novel, The Fugitive (2000), tells the story of Hardo, a young soldier who has been
involved in a failed revolt against the occupying Japanese. This story of betrayal and suffer
ing under a second round of foreign occupation is brilliantly complemented by two sections
of Pramoedya’s memoir, The Mute’s Soliloquy (1999). These chapters, entitled “Death in a
Time of Change” and “Working for the Japanese,” recall Pramoedya’s own experiences as
a young man living and working in an East Indies under Japanese occupation (Toer 1999,
153-91). Lee Kuan Yew, the first and longest-serving Prime Minister of the Republic of
Singapore, tells a similar story in his autobiography, The Singapore Story, reflecting upon the
arrival of the Japanese into the city-state and the impact that their presence had upon his
life as a young boy (Yew 1999). When addressing Southeast Asian history in America, it is impossible to discuss South
east Asian sources without talking about the wars in Indochina. When teaching upper-level
courses on Vietnam, I have had great success when presenting students with a combina
tion of war memoirs. Seemingly innumerable sources address the Indochina wars from all
aspects of the conflicts, but my favorite pairing is Bao Ninh’s The Sorrow of War (1996) and
Tim O’Brien’s The Things They Carried (2009). Keywords History; Pedagogy; Film; Literature; Memoir; East Asia; Southeast Asia In circles frequented by historians of Viet
nam, Bao Ninh often meets with mixed reviews, in large part because of the rambling and
disorganized nature of his story’s plot. That being said, both narratives, by eschewing efforts
to replicate faithfully the authors’ wartime experiences, manage to convey the essence of
that experience in a way that is far more powerful and affecting than a more dispassionate
account could hope to achieve. There are also more traditional accounts of the Vietnamese
experience during the wars of the 20th century. In particular, Nguyen Thi Dinh’s No Other
Road to Take (1976) is effective at helping American students understand why some Viet
namese civilians wanted to fight the French and the Americans. It is also quite useful as a
springboard for a discussion about biases in sources, in large part because Nguyen Thi Dinh
is so open and overt in her support for the communist cause in Vietnam. This memoir has
the added benefit of female authorship, providing an account of women’s wartime experi
ence actually written by a woman. As a result, among Southeast Asian sources available to
date, this memoir represents a unique voice. Finally, for history teachers, the Vietnam War was the catalyst for another human
catastrophe that is central to any discussion of modern global history. The Cambodian
genocide has been illuminated by a multitude of primary source accounts of its human toll
and tragedy. In particular, memoirs by survivors Chanrithy Him and Luong Ung tell equally
chilling stories of the horror of life under the Khmer Rouge. Both accounts, Him’s When
Broken Glass Floats (2001) and Ung’s First They Killed My Father (2006), provide an excel
lent counterpoint to class discussions about the political philosophies of the Khmer Rouge
and, in putting faces to the Cambodian tragedy, color the statistics of the tragedy in almost
unbearable ways. The widely acclaimed 1984 film The Killing Fields graphically depicts the experiences of
New York Times reporter Sidney Schanberg and his Cambodian translator Dith Pran during
and after the Khmer Rouge conquest of Phnom Penh, but what fewer people realize is that
the actor who played Dith Pran in the movie, a Cambodian doctor named Haing Ngor, was
himself a survivor of Cambodia’s Killing Fields. 42 | Teaching East and Southeast Asia through Asian Eyes Keywords History; Pedagogy; Film; Literature; Memoir; East Asia; Southeast Asia His memoir, Survival in the Killing Fields
(2003), is more than 500 pages long, but excerpts of it brilliantly complement the movie for
which its author won an Academy Award, due in part, no doubt, to his true understanding
of Cambodia’s tragedy. Two further films on Cambodia deserve special attention. The first, Rice People (1994), is
set in the crushing poverty of the rural countryside in post-Khmer Rouge Cambodia, where 42 | Teaching East and Southeast Asia through Asian Eyes ASIANetwork Exchange | Spring 2014 | volume 21 | 2 rice farmers must deal with natural disasters, deadly snakes, and relentless disease in order
to eke out a meager living in the rice paddies. The other film is a documentary in which two
of the seven known survivors of Tuol Sleng, the most notorious of the Khmer Rouge deten
tion facilities in which as many as 17,000 people were tortured and killed, return to the
prison with their captors to revisit the experiences of their imprisonment. Entitled S-21: The
Khmer Rouge Killing Machine (2003), the film is difficult to watch and even more difficult to
explain to undergraduates without significant historic preparation and contextualization. As discussed at the beginning of this essay, nowhere is the dearth of Asia-centered histo
ries and sources felt more keenly than in the classroom. As a result of this paucity, western
pedagogy over the past few decades has tended to fall back upon western accounts of Asia
as the lens through which to imagine Asian history and culture. Fortunately, even though
the list of accessible primary sources in translation has expanded at a glacial rate, the
number of Asian primary sources in translation that are accessible to history teachers grows
larger with every passing year and currently includes a wide variety of films, memoirs, and
works of fiction addressing virtually any significant period of the Asian past. This relatively
recent expansion in available sources represents a shifting of the historiographical tide, an
evolution of which all globally-inclined historians should take notice. rEFERENCES Bird, S. Elizabeth, and Jonathan P. Godwin. 2006. “Film in the Undergraduate Anthropology Classroom: Applying
Audience Response Research in Pedagogical Practice.” Anthropology and Education Quarterly 37, no. 3
(September): 285-99. Blum, Denise. 2006. “Expanding the Dialogue: A Response to Bird and Godwin’s ‘Film in the Undergraduate An
thropology Classroom.’” Anthropology and Education Quarterly 37, no. 3 (September): 300-6.h De Bary, William Theodore, Irene Bloom, and Joseph Adler. 2000. Sources of Chinese Tradition, Volume 1. New
York: Columbia University Press.hh Dinh, Nguyen Thi. 1976. No Other Road to Take: Memoir of Mrs. Nguyen Thi Dinh. Trans. Mai Elliott. Ithaca: Cor
nell University SEAP Publications. Dower, John. 2000. Embracing Defeat: Japan in the Wake of World War II. New York: W. W. Norton and Co.if Hall, D. G. E. 1960. On the Study of Southeast Asian History. Pacific Affairs 33, no. 3 (September): 268-81. y
y
fi
ff
Him, Chanrithy. 2001. When Broken Glass Floats: Growing Up Under the Khmer Rouge. New York: W. W. Norton. y
g
p
g
Hyegyong, Lady. 1996. The Memoirs of the Lady Hyegyong: The Autobiographical Writings of a Crowned Princess of
h
b
h
k l
f
l f Hyegyong, Lady. 1996. The Memoirs of the Lady Hyegyong: The Autobiographical Writings of a Crowned Princess of
18th Century Korea. Trans. JaHyun Kim Haboush. Berkeley, CA: University of California Press.h Isaacs, Harold R. 1975. “Some Concluding Remarks: The Turning Mirrors.” In Mutual Images: Essays in Ame
Japanese Relations, ed. Akira Iriye. Cambridge, MA: Harvard University Press. Isaacs, Harold R. 1975. “Some Concluding Remarks: The Turning Mirrors.” In Mutual Images: Essays in American- Japanese Relations, ed. Akira Iriye. Cambridge, MA: Harvard University Press. Kartini, R. A. 1992. Letters from Kartini. Trans. Joost Coté. Clayton, Victoria: Monash Asia Institute, Monash
University. Lam, Truong Buu. 2000. Colonialism Experienced: Vietnamese Writings on Colonialism, 1900-1931. Ann Arbor:
University of Michigan Press. y
g
Levstik, Linda, and Keith Barton. 2001. Doing History: Investigating with Children in Elementary and Middle
Schools 2nd ed Mahwah NJ: Lawrence Erlbaum Associates Publishers y
g
Levstik, Linda, and Keith Barton. 2001. Doing History: Investigating with Children in Elementary and Middle Levstik, Linda, and Keith Barton. 2001. Doing History: Investigating with Children i
Schools. 2nd ed. Mahwah, NJ: Lawrence Erlbaum Associates Publishers. ,
,
g
y
g
g
Schools. 2nd ed. Mahwah, NJ: Lawrence Erlbaum Associates Publishers. Mangunwijaya, Y. B. 2004. Durga Umayi: A Novel by Mangunwijaya. Trans. Keywords History; Pedagogy; Film; Literature; Memoir; East Asia; Southeast Asia If one of the most
difficult aspects of teaching Asian history to western students is students’ lack of familiarity
with the topics central to the course, then western students are best served by exposure to
Asian sources that help them “reimage” Asia: by putting a face to distant places, different
times, and foreign cultures; by replacing western stereotypes and political biases with Asian
voices; and by empathizing with the personal experiences of those who lived through the
events we study. This, I believe, is the future of successful Asian history pedagogy. Bird, S. Elizabeth, and Jonathan P. Godwin. 2006. “Film in the Undergraduate Anthropology Classroom: Applying
Audience Response Research in Pedagogical Practice.” Anthropology and Education Quarterly 37, no. 3
(September): 285-99. rEFERENCES This Earth of Mankind. Trans. M h
------. 1999. The Mute’s Soliloquy. New York: Hyperion East.h ------. 2000. The Fugitive. Trans. Willem Samuels. New York: Penguin (Non Classics).h Tognozzi, Elissa. 2010. “Teaching and Evaluating Language and Culture Through Film.” Italica 87, no. 1 (Spring):
69-91.h Ung, Luong. 2006. First They Killed My Father: A Daughter of Cambodia Remembers. New York, Harper Perennial. Woelders, Adam. 2007. “Using Film to Conduct Historical Inquiry with Middle School Students.” The History
Teacher 40, no. 3 (May): 363-95.h Yang, Anand A. 1980. “Images of Asia: A Passage through Fiction and Film.” The History Teacher 13, no. 3 (May):
351-69.h Yew, Lee Kuan. 1999. The Singapore Story: Memoirs of Lee Kuan Yew. New York: Prentice Hall College Division. Films Citedf Buffalo Boy. Directed by Minh Nguyen-Vo. 2004; Hanoi: Giai Phong Film Studio, 2006. DVD. h The Burmese Harp. Directed by Kon Ichikawa. 1956; Criterion, 2007. DVD. l h
Grave of the Fireflies. Directed by Isao Takahata. 1988; Tokyo: Studio Ghibli, 2002. DVD h
y
The Killing Fields. Directed by Roland Joffe. 1984; London: Enigma LTD, 2001. DVD. hf
Indochine. Directed by Regis Wargnier. 1992; Paris: Paradis Films, 1992. DVD.h The Rice People. Directed by Rithy Panh. 1994; Paris: La Sept Cinéma, 2006. DVD. The Rice People. Directed by Rithy Panh. 1994; Paris: La Sept Cinéma, 2006. DVD. S-21: The Khmer Rouge Killing Machine. Directed by Rithy Panh. 2003; Paris: Institut National de l’Audiovisuel
(INA), 2005. DVD. 44 | Teaching East and Southeast Asia through Asian Eyes rEFERENCES Ward Keeler. Seattle, WA: University of
Washington Press. Marcus, Alan S., and Jeremy D. Stoddard. 2007. “Tinsel Town as Teacher: Hollywood Film in the High School
Classroom.” The History Teacher 40, no. 3 (May): 303-30. h
Maw, Ba. 1968. Breakthrough in Burma: Memoirs of a Revolution, 1939-1946. New Haven, CT: Yale University
Press. Miner, Noel R. 1977. “Simulation and Role Playing in the Teaching of East Asian History.” The History Teacher 10,
no. 2 (February): 221-8. y
Murasaki, Shikibu. 1990. Tale of Genji. Trans. Edward G. Seidensticker. New York: Vintage.hh Murasaki, Shikibu. 1990. Tale of Genji. Trans. Edward G. Seidensticker. New York: Vintage. Ninh, Bao. 1996. The Sorrow of War: A Novel of North Vietnam. Trans. Phan Thanh Hao. New York: Riverhead
Books. Ninh, Bao. 1996. The Sorrow of War: A Novel of North Vietnam. Trans. Phan Thanh Hao. New York: Riverhead
Books. 43 | Teaching East and Southeast Asia through Asian Eyes ASIANetwork Exchange | Spring 2014 | volume 21 | 2 Ngor, Haing. 2003. Survival in the Killing Fields. New York: Basic Books.hhh Ngor, Haing. 2003. Survival in the Killing Fields. New York: Basic Books. g
g
g
O’Brien, Tim. 2009. The Things They Carried. New York: Mariner. hh
gh
y
Oe, Kenzaburo. 1994. Teach Us to Outgrow Our Madness. Trans. John Nathan. New York: Grove Press. hh
gh
y
Oe, Kenzaburo. 1994. Teach Us to Outgrow Our Madness. Trans. John Nat g
Orwell, George. 2010. Burmese Days. New York: Penguin Modern Classics. Phung, Vu Trong. 2002. Dumb Luck: A Novel by Vu Trong Phung. Trans. Nguyen Nguyet Cam. Ann Arbor, MI:
University of Michigan Press.h ------. 2005. The Industry of Marrying Europeans. Trans. Thuy Tranviet. Ithaca, NY: Cornell University Southeast
Asian Program Publications. Pramoj, Kukrit. 1999. Four Reigns. Trans. Tulachandra. Seattle, WA: University of Washington Press. Rizal, Jose. 2006. Noli Me Tangere. Trans. Harold Augenbraum. New York: Penguin Classics. ------. 2007. El Filibusterismo: Subversion: A Sequel to Noli Me Tangere. Trans. Soledad Lacson-Locsin. Honolulu:
University of Hawaii Press. Shonagon, Sei. 1991. The Pillow Book of Sei Shonagon. Trans. and ed. Ivan Morris. New York: Columbia University
Press. Staiger, Janet. 2000. Perverse Spectators: The Practices of Film Reception. New York: New York University Press.h h
Toer, Pramoedya Ananta. 1996. This Earth of Mankind. Trans. Max Lane. New York: Penguin Books.h h
1996. This Earth of Mankind. Trans. Max Lane. New York: Peng Toer, Pramoedya Ananta. 1996. Brian Caton is an Associate
Professor in the Department of
History at Luther College. Notesh 1. This issue is certainly not new to pedagogical forums. For a dated, but particularly exhaustive, examination
f i
f W t
ti
f A i
d
d
i
l
h
t A i
hi t
Y
(1980) 1. This issue is certainly not new to pedagogical forums. For a dated, but particularly exhaustive, examination
of issues of Western perceptions of Asia and pedagogical approaches to Asian history, see Yang (1980). 1. This issue is certainly not new to pedagogical forums. For a dated, but particularly exhaustive, examination
of issues of Western perceptions of Asia and pedagogical approaches to Asian history, see Yang (1980). p
p
p
g g
pp
y,
g (
)
2. Translations of assorted ancient Chinese philosophical writings can be found in De Bary, Bloom, and Adler
(2000).hh 3. The Buru Quartet consists of four books written by Indonesian author Pramoedya Ananta Toer: This Earth
of Mankind, Child of All Nations, Footsteps, and House of Glass. 44 | Teaching East and Southeast Asia through Asian Eyes ASIANetwork Exchange | Spring 2014 | volume 21 | 2 Teaching South Asia beyond Colonial Boundaries Brian Caton Abstract: Because of the methodological innovations of Subaltern Studies in the
1980s and 1990s, most historians’ familiarity with South Asian history is limited
to the colonial or modern period. While the subalternist view is undoubtedly use
ful, it does not provide much help in thinking about what came before or after the
colonial period. This limited context may prove to be a problem for a nonspecial
ist constructing a full course in South Asian history or adding South Asia content
to a course that seeks to break down area studies or nation-state boundaries. This
article provides a starting point for such an enterprise. It reviews the South Asian
history textbooks available in the market and identifies some of the scholarship that
would suit courses or units organized by theme or by a larger Asian geography. It
also reviews some of the collections of primary sources that could be used in such
coursework. Keywords Textbook; Primary sources; South Asia; History Historians of nearly every part of the world have acquainted themselves with histories of
colonial South Asia, primarily through the work of the Subaltern Studies group and the vari
ous discussions that have arisen from it (Ludden 2002b). For all the good that this reading
and discussion has done, few historians outside of those specializing in South Asia have
given much thought to locating the history of colonial South Asia within either a longer
chronological trajectory or a broader geographical context (beyond core-periphery dynam
ics). In this article, I do not intend to radically change this predicament, but this pattern of
limited reading does pose a problem when an East Asia specialist, for example, is asked to
teach a course in South Asian history or desires to add South Asia content to a thematically-
oriented course. There is no single solution to this sort of problem, but one or two good
textbooks and a carefully selected set of primary sources can do much to help situate colo
nial South Asian history in a broader context of time and space. 45 | Teaching South Asia beyond Colonial Boundaries Textbooks Crispin Bates’ Subalterns and Raj: South Asia since 1600 is probably the best book for its
stated time period, but it is too lengthy and detailed to be used with other texts in a one-
semester survey of South Asian history (Bates 2007). Ayesha Jalal and Sugata Bose’s Modern
South Asia, now in a third edition, could be combined with other texts covering periods
before 1500 (Bose and Jalal 2011). One such text could be Catherine Asher and Cynthia
Talbot’s India before Europe, perhaps the only excellent book for the period between 1000
and 1750 (Asher and Talbot 2006). Another text could be Burjor Avari’s India: the Ancient
Past, which covers the Neolithic era through the construction of Chola and Turkish states
up to the twelfth century (Avari 2007). Although it is not technically a textbook, because
its chronological scope is limited to the time period a few hundred years before and after
100CE, Richard Davis’ recent Global India makes important challenges to the standard
periodization of South Asian history and demonstrates South Asia’s linkages to the rest of
Eurasia. It is also short enough to be combined with a textbook covering the same time
frame (Davis 2009). Even if they do not cover all periods of South Asian history, most textbooks share a
remarkably durable periodization, although other possibilities are beginning to appear. Periodization has to some degree been standardized since the publication of James Mill’s
History of India, and affirmed in an important way by Jawaharlal Nehru’s Discovery of India
(Mill 1817, Nehru 1946). In defense of empire, Mill proposed that India had an “ancient”
period of cultural production and political stability, followed by a “medieval” period in
which Muslim forces plunged India into a period of political and cultural penury. This was
followed by a period of British tutelage, in which just administration and English knowl
edge would rebuild India from the wreckage of the medieval period. Nehru amended this
periodization by valuing the British period negatively and adding a “modern” or “national
ist” period, in which independent India would rebuild itself through political, economic,
and cultural self-sufficiency. The two-volume A History of India published by Penguin in
1965 was long the work that defined this periodization for a nationalist history of India
(Thapar 1965, Spear 1965). Textbooks While the market appears to offer several options for textbooks on the history of South
Asia, the offerings are limited in a variety of ways. Although many options are available for
East Asian history (e.g. Ebrey, Walthall, and Palais 2009), no major publisher of academic
textbooks has produced a narrative-plus-sources, magazine-stock textbook for South Asian
history. Most textbooks of South Asian history are single volumes, with a small number
of maps and plates, and average 300 pages in length (with a range of roughly 200 to 450),
excluding back matter. Most textbooks attempt a panoptic chronological scope, but the
great majority of those books do not allot equal time to the various periods of South Asian
history. For example, about half of Burton Stein’s posthumous History of India, recently
edited by David Arnold, deals with the period after the dissolution of the East India Com
pany in 1858, as does Peter Robb’s A History of India (Stein 2010, Robb 2002). Barbara Met 45 | Teaching South Asia beyond Colonial Boundaries ASIANetwork Exchange | Spring 2014 | volume 21 | 2 calf and Thomas Metcalf’s A Concise History of India spends a bit more than half its length
on this period (Metcalf and Metcalf 2002). Although it does not follow this same chrono
logical marker, David Ludden’s India and South Asia: A Short History spends about half its
length on the “modern” era (Ludden 2002a). Thomas Trautmann’s India: Brief History of a
Civilization achieves the reverse; about a quarter of its length deals with the colonial and
postcolonial periods combined (Trautmann 2010). Trautmann’s compression of modern
history might be a fair corrective to the general trend in textbook periodization, but the
emphasis on a “classical India” that produced “mentalities” prior to Islam risks providing
fuel to the flames of Hindu chauvinist historiography. In any case, a course that intends to
give equal time to South Asia’s historical periods will need to use multiple textbooks, or a
combination of a single textbook with other scholarly literature. Some instructors may not want a textbook attempting a full chronology, especially if the
South Asia content is to fit into a geographically broad or thematically-organized course. 46 | Teaching South Asia beyond Colonial Boundaries Textbooks Hermann Kulke and Dietmar Rothermund’s A History of
India, now in a venerable fifth edition, does probably the best job of balancing attention
among the many periods of South Asian history, but it is most closely the inheritor of a
nationalist historiography (Kulke and Rothermund 2010). Although they do not follow
this periodization strictly, their treatment of “the Freedom Movement” (note the singular)
and “the Republic” betrays an adherence to Nehruvian, if not Congressite, visions of India,
best captured in Rothermund’s close-up photograph of current Prime Minister Manmo
han Singh (Kulke and Rothermund 2010, 258). Pakistan appears only periodically, as an 46 | Teaching South Asia beyond Colonial Boundaries ASIANetwork Exchange | Spring 2014 | volume 21 | 2 irrational provocateur in India’s political history; the comparison of Jinnah to Shylock in the
narrative of Partition provides a tellingly uncharitable starting point (Kulke and Rother
mund 2010, 231-2). The most recent edition of Stanley Wolpert’s A New History of India
also cleaves closely to the Indian nationalist formula, and enjoys a long list of other faults,
including spending only one twenty-two-page chapter (of a nearly 500-page book) on “The
Impact of Islam (ca. 711-1556)”; a failure to recognize environmental change, particularly
in the well-documented period after 1860; and the stunningly Orientalist claim that “the
enervating effect of heat on Indian productivity is a factor that can hardly be underesti
mated” (Wolpert 2009, 4). For all of the complaints of historians about the Mill or Nehru
periodization of South Asian history, few textbook authors have done anything to generate a
new model. Ludden has done so in India and South Asia: two key innovations are, first, the
creation of a period defined by the formation of a political idiom that he calls “sultan” but
applies to regional powers from the post-Gupta successor states through the Mughals, and,
second, the imagination of a way to think about Partition as an event couched in political,
economic, social, cultural, and intellectual trends that neither ended nor began in 1947—in
other words, a historical event rather than a historiographical watershed. Textbooks tend to waver between using “India” and “South Asia,” although in many
cases the history of states other than the Republic of India tends to be pushed to the margin
or ignored altogether. 47 | Teaching South Asia beyond Colonial Boundaries Textbooks In part, this is a marketing decision: only Bates (2007) and Bose
and Jalal (2011) use “South Asia” rather than “India” in the title. Ludden’s (2002a) use of
both “India” and “South Asia” allows him to make a point about the intellectual tension
between the two terms. Constructions of “India” that use mountain ranges (Himalaya and
Sulayman) and the Indian Ocean as impregnable boundaries may be convenient but are
constantly problematized by the historical and continuing movement of people, material
objects, and ideas. We know a great deal about South Asian history from records left by
Chinese, Arab, and other intellectuals and travelers (Gordon 2008, Davis 2009), and many
groups of people, primarily from the Middle East and Central Asia, established expatriate
communities that became fully integrated into South Asian social and cultural relations. The smaller number of records of South Asians migrating or circulating to other places
in precolonial periods has given rise to past claims that South Asians did not or could not
make such long-distance migrations. However, Stephen Dale’s work on Multani traders
and David Rudner’s on Chettiar merchants, for example, suggest that migrations from the
geographically central parts of South Asia to distant destinations were not only common
but also relatively well documented (Dale 1994, Rudner 1994). Such narratives rarely if ever
make more than a fleeting appearance in panoptic textbooks. A more helpful approach to the problem of “India” and “South Asia” might be to think
in terms of mobility or circulation. Ludden (1994), in challenging the notion of “Indian
civilization” in South Asian historiography, set out an argument for the centrality of human
mobility to South Asian history. Markovits, Pouchepadass, and Subrahmanyam have
presented the concept of circulation as the movement of more than just people and mate
rial objects but also “information, knowledge, ideas, techniques, skills, cultural productions
(texts, songs), religious practices, even gods” (Markovits, Pouchepadass, and Subrahman
yam 2003, 2). Although no author has written a textbook with circulation as its organiz
ing principle for thinking about “India” or “South Asia,” circulation could provide a useful
pedagogical framework for discussing textbook reading assignments. Additionally, one
could use Stewart Gordon’s When Asia Was the World for getting students to think about
the connectedness of people, ideas, and material objects across Asia, even into its European
and African margins. Textbooks Its narrative style is very accessible to introductory-level students, 47 | Teaching South Asia beyond Colonial Boundaries ASIANetwork Exchange | Spring 2014 | volume 21 | 2 but its focus on travelogues puts a significant burden on the instructor to provide the South
Asian context (Gordon 2008). In the absence of or in conjunction with textbooks, other scholarly works may support
pedagogies based on theme. While one cannot adequately survey the state of scholarship
within the field of South Asian history in a short article, one can suggest that much of the
new scholarship tends to be focused on subjects or issues relating to certain themes or top
ics: Dalits, previously called Untouchables; gender; Maoists and Naxalites; science, technol
ogy, and medicine (STM); environment; and regions, typically within postcolonial India. Interest in religions or religious groups, including religious nationalisms, has declined
somewhat since the fall of the BJP government in the 2004 election, which also marked the
arrival of Dalit and lower-caste political parties on the national electoral scene. A unit on
caste, for example, in a thematically-organized introductory course could use Susan Bayly’s
contribution to the New Cambridge History of India, Nicholas Dirks’ Castes of Mind, or
Christophe Jaffrelot’s Religion, Caste, and Politics in India (Bayly 1999, Dirks 2001, Jaffrelot
2011), though Dirks and Jaffrelot may require some interpretation for novice readers. How
ever, the aforementioned shift in Indian politics—and the increasing prominence of Maoist
groups—has resulted in an increase of explicit attention paid in Subaltern Studies to Dalits
and by historians in general to Dalit questions (e.g. Mayaram, Pandian, and Skaria 2005,
and Rawat 2011). For a unit on gender, Geraldine Forbes’ NCHI volume or Radha Kumar’s
History of Doing are good starting points for thinking about women’s history but are per
haps less useful to get students to think critically about gender categories and the historical
processes of their construction over the very longue durée of South Asian history (Forbes
1996, Kumar 1993). They also don’t account well for the religious inflections of gender, for
example in the cases of female soldiers of the Liberation Tigers of Tamil Eelam in Sri Lanka,
women’s organizations of the Rashtriya Swayamsevak Sangh, or mothers of Lashkar-e-Tayy
iba recruits in Pakistan (de Mel 2007, Sarkar 2005, Haq 2007). Textbooks Perhaps the other aforementioned themes deserve a more detailed presentation, but
some themes that tend to be less popular recently may better serve the purpose of breaking
down the boundaries of South Asian history. While most readers of this journal are prob
ably familiar with the work on inter-Asian trade under the rubric of “Silk Road” or “Silk
Route,” some late twentieth century work on Indian Ocean trade can fill out the picture
(Chaudhuri 1985 and 1990, Das Gupta 2001). More recently, interest in this theme (over
land or seas) seems to be dwindling (despite some exceptions, e.g. Levi 2002, Hanifi 2011). Scholarly analysis of economic development and agriculture is available these days chiefly
through the periodical Economic and Political Weekly, environmental history work (e.g. Agrawal 2005, Saikia 2011), and the slightly problematic political science and economics
genre of “how to do business in India” (e.g. Kumar and Sethi 2005). 48 | Teaching South Asia beyond Colonial Boundaries Primary Sources i However, the scheme for selecting and organizing the extracts is arbitrary, and it would
require a fairly substantial investment of time to read through all of the entries to see what
would be most useful. Sadly, this seems to be the complete list of published primary source
readers. This was to have changed in 2013, when Routledge had scheduled to release a
reader edited by Bose and Jalal, as a companion to the third edition of Modern South Asia.3
However, there do not appear to be on the publishing horizon any collections that include
sources produced before 1600. Although published single items appear to be in the gradual process of being replaced by
digitized sources, they remain relatively easily accessible and provide an indispensable expe
rience as objects of analysis both inside and outside the classroom. Single items come in two
broad categories: sources that are stand-alone publications (usually available as new editions
or reprints) and those included in other kinds of publications. The first category includes
autobiographies. While many are likely to be familiar with Gandhi’s autobiography (Gandhi
1927-29), Stephen Dale reminds us that the Mughal emperor Babur’s was one of the earliest
“modern” autobiographies anywhere (Dale 1990).4 It also provides ample detail for thinking
about the material life and exchange between Central and South Asia, the cultural and intel
lectual habits of Central Asian Muslims, military strategies and administrative rewards—in
short, the mentalités of the Central Asian ruling classes. The Baburnama’s length requires
instructors to use excerpts, which also applies to many other single sources available in
print, such as the Kautilya Arthasastra and al-Biruni’s India (Kautilya 1992, Sachau 2000). Works of literature also permit an exploration of interpretive angles or themes that run
through the course. For example, Kalidasa’s Recognition of Sakuntala can sustain discussion
of gender and varna norms in ancient India, but it also allows for discussion of drama as a
literary genre and stage performance as a social event (Kalidasa 2001). In another example,
one could assign any of several poems, short stories, or novels written in the immediate
aftermath of Partition in order to gauge the effects of those events, at least on the literati. Primary Sources i As in most fields of history, one may obtain primary sources for pedagogical use
through published collections, published single items (such as autobiographies), web-based
resources, and materials drawn from one’s own research. By “published collections,” I
mean what are often called “document readers”—multiple sources written by a wide range
of authors, selected and annotated by one or more editors, linked to a long chronological
period—rather than the collected works of a single author, such as The Collected Works of
Mahatma Gandhi (Gandhi 2000-01).1 Because of the limitations of published collections,
and because nonspecialists are unlikely to have research materials relevant to South Asia
courses or units, published single items and particularly web-based resources are the most 48 | Teaching South Asia beyond Colonial Boundaries ASIANetwork Exchange | Spring 2014 | volume 21 | 2 promising avenues for finding good primary sources for classroom use. Published collections make certain kinds of primary sources available, but their modes
of selection and organization make them pedagogically problematic. The most well-estab
lished of this genre are the now two volumes of Sources of Indian Tradition. The subtitle of
the first volume, “From the beginning to 1800,” is a bit deceptive, since it is really organized
by religious tradition: Hindu, Jain, Buddhist, Muslim, and Sikh (Embree 1988). Many
interesting documents are available here, but they must be handled carefully in order to
avoid leading students to organize the South Asian past as the editor of this volume chose
to do. Volume Two, subtitled “Modern India and Pakistan,” is equally misleading, since it
consists of the writings of major Westernizing, reformist, and nationalist thinkers according
to canons defined by the nationalist histories of India and Pakistan (Hay 1988). Although
the second edition made some changes to the selection of documents (de Bary 1958), it
includes no sources that provide the intellectual argument for Bangladeshi nationalism, for
example; there are no thinkers from the other, smaller nation-states of the region, either.2 B. N. Pandey edited A Book of India, first published in 1965 and available in reprints (Pandey
[1965] 1977). His selections are all decidedly short and therefore suitable for introductory
level courses, some short enough to permit students to read and analyze them in class. 49 | Teaching South Asia beyond Colonial Boundaries Primary Sources i I
have used Khushwant Singh’s Train to Pakistan (1956), but a much superior novel is Amrita
Pritam’s Pinjar (1950).5 Finding primary sources published in other works requires a substantial investment
of time, including the arrangement of copyright clearances, but the pedagogical rewards
can be great. For example, I want students to get a sense of the physicality of writing, 49 | Teaching South Asia beyond Colonial Boundaries ASIANetwork Exchange | Spring 2014 | volume 21 | 2 even though few palm leaf or paper objects survive from the period before about 1000
CE. Edicts carved in stone in the Mauryan era (4th-3rd century BCE) are certainly avail
able in translation, but most surviving writing samples exist as etched into copper plates. I use a facsimile, Sanskrit transcription, and translation of a pair of copper-plate inscrip
tions from medieval Gujarat (late 6th century CE; Mirashi 1955, 38-44, 57-66). Obviously,
I don’t expect students to be able to read Sanskrit, but I want them to see how language
and script change over time. I also want students to think about the political relationships
between people and how objects like land and temples are used to mediate or express those
relationships. In later parts of the course, students read translations of paper documents
relating to the properties belonging to a religious institution in northern Punjab, for the
same thematic purpose (Goswamy and Grewal 1969, 83-99, 109-16, 125-47, 376-8, 381-2,
385-90). In another theme in my course, I want students to think about the why and how
of governmental information gathering by reading the documentary fruits of that process. I use a selection from the A’in-i-Akbari which shows production and price data (over time)
for one administrative unit of the Mughal Empire (Jarrett 1949, 68-93, 315-38). Later in the
course I ask students to search for specific pieces of information in the District Gazetteers
for Punjab, originally published between 1884 and about 1935. This gives students a sense
of the panoptical ambition of the colonial government as well as the gazetteer’s pragmatic
focus.6 These few examples can hardly begin to scratch the surface of the sources available
for teaching the material, political, and social history of South Asia; a flood of other options
are available for other themes one may wish to pursue. 50 | Teaching South Asia beyond Colonial Boundaries rEFERENCES Agrawal, Arun. 2005. Environmentality: Technologies of Government and the Making of Subjects. Durham: Duke
University Press. Asher, Catherine B., and Cynthia Talbot. 2006. India Before Europe. Cambridge: Cambridge University Press.h Avari, Burjor. 2007. India: The Ancient Past: A History of the Indian Sub-Continent from c.7000 BC to AD 1200. New
York: Routledge. g
Bates, Crispin. 2007. Subalterns and Raj: South Asia since 1600. London and New York: Routledge. Bayly, Susan. 1999. Caste, Society and Politics in India from the Eighteenth Century to the Modern Age. Vol. IV, part
3 of The New Cambridge History of India, ed. Gordon Johnson, C. A. Bayly, and John F. Richards. Cam
bridge: Cambridge University Press. Belcher, Wayne. Ancient Harappa in 3-D. http://www.harappa.com/3D/index.html (accessed August 31, 2012). Bose, Sugata, and Ayesha Jalal. 2011. Modern South Asia: History, Culture, Political Economy. 3d ed. New York:
Routledge. Chaudhuri, K. N. 1985. Trade and Civilisation in the Indian Ocean: an Economic History from the Rise of Islam to
1750. Cambridge: Cambridge University Press. ------. 1990. Asia before Europe: Economy and Civilisation of the Indian Ocean from the Rise of Islam to 1750. Cam
bridge: Cambridge University Press. g
g
y
Columbia University Libraries. South Asian Studies Virtual Library. http://library.columbia.edu/locations/global/
virtual-libraries/sarai.html (accessed April 2, 2014). Dale, Stephen Frederic. 1990. Steppe humanism: the autobiographical writings of Zahir al-Din Muhammad Babur,
1483-1530. International Journal of Middle East Studies 22, no. 1 (February): 37-58. ------. 1994. Indian Merchants and Eurasian Trade, 1600-1750. Cambridge Studies in Islamic Civilization, ed. David
Morgan. Cambridge: Cambridge University Press. g
g
g
y
Das Gupta, Ashin. 2001. The World of the Indian Ocean Merchant, 1500-1800. New Delhi: Oxford University Press. h
Davis, Richard H. 2009. Global India circa 100CE: South Asia in Early World History. Key Issues in Asian Studies, Davis, Richard H. 2009. Global India circa 100CE: South A
No. 5. Ann Arbor: Association for Asian Studies.h Davis, Richard H. 2009. Global India circa 100CE: South Asia in Early World History. Key Issues in Asian St
No. 5. Ann Arbor: Association for Asian Studies.h De Bary, Wm. Theodore, ed. 1958. Sources of Indian Tradition. New York: Columbia University Press. De Mel, Neloufer. 2007. Militarizing Sri Lanka: Popular Culture, Memory and Narrative in the Armed Conflict. New
Delhi: Sage Publications. Dirks, Nicholas B. 2001. Castes of Mind: Colonialism and the Making of Modern India. Princeton: Princeton Uni
versity Press. Ebrey, Patricia Buckley, Anne Walthall, and James B. Primary Sources i It may require talking with a South
Asia specialist to find the best source, and if a specialist is not at hand, a quite large group is
available through the H-Asia list.f Current websites vary in quality, but in total they offer a broader range of sources than
available in any single print collection. For discussions of the Indus Valley Civilization, the
Harappa.com site is indispensable (Khan). As one can tell from a few clicks, the site is full
of information, research reports, and other materials. The 3-D rendering of IVC sites allows
students to imagine more clearly what a town might have looked like during this period
in this region (Belcher). The site also has very short film clips from the early twentieth
century, which include professional newsreels and hobbyists’ works. The Fordham Inter
net History Sourcebook has some useful items, mainly for very old or very recent periods
(Halsall). South Asia Resource Access on the Internet (SARAI) has some useful items,
mainly for the most recent periods (Columbia University Libraries). The Digital South
Asia Library (DSAL), hosted by the University of Chicago, includes sources breathtaking
in their completeness—for example, the Imperial Gazetteer of India, which is fully search
able and includes atlases with their own zoomable interface (University of Chicago). But the
breadth of sources seems oddly limited, as though an ambitious project suddenly ran out of
institutional support. By far the most useful gateway site for primary sources is maintained
by Frances Pritchett at Columbia (Pritchett). Some of her items link to DSAL sources, but
most link to sites that are otherwise difficult to find. They cover all of the periods of South
Asian history, although more attention is devoted to precolonial periods and the territory
included in modern India, Pakistan, and Bangladesh. Some parts are organized alpha
betically rather than topically, so nonspecialists will get more out of this site after having
become familiar with the characters and plot of the history they want students to learn. Primary Sources i Most of the above-mentioned sites include some maps, but the Perry-Castañeda Library
Map Collection at the University of Texas has a particularly well-organized selection of
maps, which can also be used to help students think about shifting political boundaries
and the ways different mapmakers imagined the world included in “South Asia” (Univer 50 | Teaching South Asia beyond Colonial Boundaries ASIANetwork Exchange | Spring 2014 | volume 21 | 2 sity of Texas Libraries). Those interested in the lively visual culture of South Asia will find
a series of galleries accompanied by critical essays in the Tasveer Ghar site (Tasveer Ghar). Although one could ask students to analyze an image or two without reading the linked
essays, the authors of these pieces do a very good job of teaching readers how to look at
both single and sets of images. As libraries and archives obtain greater levels of funding for
digitizing their resources, web-based collections of primary sources are likely to increase in
number, scope, and depth. Conclusion No single primary source, and no single textbook, is sufficient to push the history of
South Asia beyond the geographical/political boundaries of colonial India (or the postcolo
nial Republic of India) or to situate the history of colonial India within a broader causative
framework that deemphasizes, if not effaces, the old boundaries of 1757 and 1947. Whether
in an entire course or in segments of a broader course, a judicious combination of textbooks
and primary sources can begin moving students toward a conceptualization of South Asia
that is not merely colonial—the immediate cause of the “now”—but that recognizes the
limits of colonial India as a product of a series of historiographical choices. 51 | Teaching South Asia beyond Colonial Boundaries rEFERENCES Palais. 2009. East Asia: A Cultural, Social, and Political His
tory. 2d ed. Boston: Houghton Mifflin. fl
Embree, Ainslee T., ed. 1988. Sources of Indian Tradition: From the Beginning to 1800. 2d ed. Vol. 1. New York:
Columbia University Press. y
Forbes, Geraldine. 1996. Women in Modern India. Vol. 4, part 2 of The New Cambridge History of India, ed. Gordon
Johnson, C. A. Bayly, and John F. Richards. Cambridge: Cambridge University Press. y y
g
g
y
Gandhi, Mohandas K. 1927-29. The Story of My Experiments with Truth. Trans. Mahadev Haribhai Desai and
Pyarelal Nair 2 vols Ahmedabad: Navajivan Publishing House Gandhi, Mohandas K. 1927-29. The Story of My Experiments with Truth. Trans. Mahadev Haribhai Desai and Mohandas K. 1927-29. The Story of My Experiments with Truth. Gandhi, Mohandas K. 1927-29. The Story of My Experiments with Truth. Pyarelal Nair. 2 vols. Ahmedabad: Navajivan Publishing House.h Gandhi, Mohandas K. 1927-29. The Story of My Experiments with Truth. Trans. Mahadev H
Pyarelal Nair. 2 vols. Ahmedabad: Navajivan Publishing House.h ------. 2000-01. The Collected Works of Mahatma Gandhi. 6th ed. 100 vols. New Delhi: Publications Division, Min
istry of Information and Broadcasting, Government of India. 51 | Teaching South Asia beyond Colonial Boundaries ASIANetwork Exchange | Spring 2014 | volume 21 | 2 Gordon, Stewart. 2008. When Asia was the World. Cambridge, MA: Da Capo Press.h Goswamy, B. N., and J. S. Grewal. 1969. The Mughal and Sikh Rulers and the Vaishnavas of Pindori. Simla: Indian
Institute of Advanced Study. Halsall, Paul. Internet Indian history sourcebook. http://www.fordham.edu/Halsall/india/indiasbook.asp (accessed
August 31, 2012).i Hanifi, Shah Mahmoud. 2011. Connecting Histories in Afghanistan: Market Relations and State Formation on a
Colonial Frontier. Stanford: Stanford University Press.h Haq, Farhat. 2007. “Militarism and motherhood: The women of the Lashkar-i-Tayyabia in Pakistan.” Signs 32, no. 4
(Summer): 1023-46. Hay, Stephen, ed. Sources of Indian Tradition: Modern India and Pakistan. 2d ed. Vol. 2. New York: Columbia
University Press. India, Pakistan and Sri Lanka movies 1929-1947. http://www.harappa.com/movies.html (accessed August 31,
2012). Jaffrelot, Christophe. 2011. Religion, Caste, and Politics in India. New York: Columbia University Press. Jarrett, H. S., trans. 1949. ‘Ain-i-Akbari of Abul Fazl-i-‘Allami. Vol. II. Ed. Jadunath Sarkar Calcutta: Royal Asiatic
Society of Bengal. Kalidasa. 2001. The Recognition of Sakuntala. Trans. W. J. Johnson. Oxford World’s Classics. Oxford: Oxford Univer
sity Press.h Kautilya. 1992. The Arthasastra. Trans. and ed. L. N. Rangarajan. New Delhi: Penguin Books.h Khan, Omar. rEFERENCES Inscriptions of the Kalachuri-Chedi Er
Indicarum. Ootacamund: Government Epigraphist for India. Mirashi, Vasudev Vishnu, ed. 1955. Inscriptions of the Kalachuri-Chedi Era. Vol. IV, part 1 of Corpus Inscriptionum
Indicarum. Ootacamund: Government Epigraphist for India. Mirashi, Vasudev Vishnu, ed. 1955. Inscriptions of the Kalachuri Chedi Era. Vol. IV, part 1 of Corpus Inscriptionum
Indicarum. Ootacamund: Government Epigraphist for India. hh ,
,
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Indicarum. Ootacamund: Government Epigraphist for India. hh Nehru, Jawaharlal. 1946. The Discovery of India. Calcutta: The Signet Press. hh
Pandey, B. N. 1977. A Book of India. New Delhi: Rupa & Co. (Orig. pub. 1965.) Pritam, Amrita. 1950. Pinjar. Amritsar: Sikh Publishing House, Ltd.h Pritam, Amrita. 1950. Pinjar. Amritsar: Sikh Publishing House, Ltd.h j
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------. 2009. Pinjar: The Skeleton and Other Stories. Trans. Khushwant Singh. New Delhi: Tara Press. --. 2009. Pinjar: The Skeleton and Other Stories. Trans. Khushw jh
g
Pritchett, Frances. Sources of Indian Traditions. Columbia University Press. http://www.columbia.edu/itc/mealac/
pritchett/00sources/index.html (accessed September 4, 2012). h
Pritchett, Frances. Sources of Indian Traditions. Columbia University Press. http://www.columbia.edu/itc/mealac/ Pritchett, Frances. Sources of Indian Traditions. Columbia University Pr
pritchett/00sources/index.html (accessed September 4, 2012). f
y
pritchett/00sources/index.html (accessed September 4, 2012). ------. South Asia study resources compiled by Frances Pritchett, Columbia University. http://www.columbia.edu/
itc/mealac/pritchett/00fwp/#fwp (accessed September 4, 2012). itc/mealac/pritchett/00fwp/#fwp (accessed September 4, 201 Rawat, Ramnarayan S. 2011. Reconsidering Untouchability: Chamars and Dalit History in North India. Blooming
ton: Indiana University Press. Robb, Peter. 2002. A History of India. Basingstoke: Palgrave. Robb, Peter. 2002. A History of India. Basingstoke: Palgrave. Rudner, David West. 1994. Caste and Capitalism in Colonial India: The Nattukottai Chettiars. Berkeley: University
of California Press. Sachau, Edward C., trans. and ed. 2000. Alberuni’s India. Vol. 1. (London: Routledge. (Orig. pub. 1910.) Saikia, Arupjyoti. 2011. Forests and Ecological History of Assam, 1826-2000. New Delhi: Oxford University Press.h Sarkar, Tanika. 2005. “Heroic women, mother goddesses: Family organization in Hindutva politics.” In The Sangh
Parivar: A Reader, ed. Christophe Jaffrelot, 148-77. Critical Issues in Indian Politics, ed. Francine R. Fran
kel, Zoya Hasan, and Kanti Bajpai. New Delhi: Oxford University Press. (Orig. pub. 1995) Singh, Khushwant. 1956. Train to Pakistan. London: Chatto & Windus. Singh, Khushwant. 1956. Train to Pakistan. London: Chatto & Windus. urton. 2010. A History of India. Ed. David Arnold. 2d ed. Chich Spear, Percival. 1965. A History of India. Vol. 2. Harmondsworth: Penguin Books. Tasveer Ghar: Digital archive of South Asian popular visual culture. rEFERENCES The ancient Indus Valley and the British Raj in India and Pakistan. http://www.harappa.com (ac
cessed September 13, 2012). p
Kulke, Hermann, and Dietmar Rothermund. 2010. A History of India. 5th ed. London and New York: Routledge.h Kulke, Hermann, and Dietmar Rothermund. 2010. A History of India. 5th ed. London and New York: Routledge. Kumar, Radha. 1993. The History of Doing: an Illustrated Account of Movements for Women’s Rights and Feminism
in India, 1800-1990. London and New York: Verso. Kumar, Radha. 1993. The History of Doing: an Illustrated Account of Movements for Women’s Rights and Feminism
in India, 1800-1990. London and New York: Verso. Kumar, Rajesh, and Anand Sethi. 2005. Doing Business in India: A Guide for Western Managers. New York: Palgrave
Macmillan.h Levi, Scott Cameron. 2002. The Indian Diaspora in Central Asia and its Trade, 1550-1900. Leiden: Brill. h
Ludden, David. 1994. “History outside civilisation and the mobility of South Asia.” South Asia 17, no. 1: 1-23. ------. 2002a. India and South Asia: a Short History. Oxford: Oneworld Publications. ------, ed. 2002b. Reading Subaltern Studies: Critical History, Contested Meaning and the Globalization of South Asia. London: Anthem Press. Markovits, Claude, Jacques Pouchepadass, and Sanjay Subrahmanyam, eds. 2003. Society and Circulation: Mobile
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People and Itinerant Cultures in South Asia, 1750-1950. Delhi: Permanent Black. p
Mayaram, Shail, M. S. S. Pandian, and Ajay Skaria, eds. 2005. Subaltern Studies XII: Muslims, Dalits, and the Fabri
cations of History. Delhi: Permanent Black and Ravi Dayal. Mayaram, Shail, M. S. S. Pandian, and Ajay Skaria, eds. 2005. Subaltern Studi
cations of History. Delhi: Permanent Black and Ravi Dayal. Mayaram, Shail, M. S. S. Pandian, and Ajay Skaria, eds. 2005. Subaltern Studies XII: Muslims, Dalits, and the Fabri
ti
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D lhi P
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Women. Metcalf, Barbara D., and Thomas R. Metcalf. 2002. A Concise History of India. Cambridge: Cambridge University
Press. Mill, James. 1817. The History of British India. 3 vols. London: Baldwin, Cradock and Joy. Mirashi, Vasudev Vishnu, ed. 1955. Inscriptions of the Kalachuri-Chedi Era. Vol. IV, part 1 of Corpus Inscriptionum
Indicarum Ootacamund: Government Epigraphist for India Mirashi, Vasudev Vishnu, ed. 1955. 52 | Teaching South Asia beyond Colonial Boundaries 53 | Teaching South Asia beyond Colonial Boundaries rEFERENCES http://tasveerghar.net (accessed September 13,
2012).h Thackston, Wheeler M., trans. and ed. 1996. The Baburnama: Memoirs of Babur, Prince and Emperor. Washington,
DC: Freer Gallery of Art, Arthur M. Sackler Gallery, Smithsonian Institution. Thackston, Wheeler M., trans. and ed. 1996. The Baburnama: Memoirs of Babur, Prince and Emperor. Washington,
DC: Freer Gallery of Art, Arthur M. Sackler Gallery, Smithsonian Institution. 52 | Teaching South Asia beyond Colonial Boundaries ASIANetwork Exchange | Spring 2014 | volume 21 | 2 Thapar, Romila. 1965. A History of India. Vol. 1. Harmondsworth: Penguin Books.h h
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Trautmann, Thomas R. 2010. India: Brief History of a Civilization. Oxford: Oxford University Press.h University of Chicago. The digital library of South Asia-Imperial Gazetteer of India. http://dsal.uchicago.edu/refer
ence/gazetteer/index.html (accessed August 31, 2012). University of Chicago. The digital library of South Asia-Imperial Gazetteer of India. http://dsal.uchicago.edu/refer
ence/gazetteer/index.html (accessed August 31, 2012). University of Texas Libraries. Asia Maps – Perry-Castañeda Map Collection – UT Library Online. http://www.lib. utexas.edu/maps/asia.html (accessed September 4, 2012). University of Texas Libraries. Asia Maps – Perry-Castañeda Map Collection – UT Library Online. http://www.lib. utexas.edu/maps/asia.html (accessed September 4, 2012). University of Texas Libraries. Asia Maps – Perry-Castañeda Map Collection – UT Library Online. http://www.lib. utexas.edu/maps/asia.html (accessed September 4, 2012). p
p
Wolpert, Stanley. 2009. A New History of India. 8th ed. New York and Oxford: Oxford University Press. Amy Kardos is a Senior Lecturer
in the History Department at
the University of Texas at San
Antonio Notes 1. I also exclude collections of primary sources, often digitized, meant for purchase by institutions, such as the
Foreign Office files published by Adam Matthew Digital, “FO India, Pakistan and Afghanistan,” accessed
September 5, 2012, http://www.amdigital.co.uk/Collections/FO-India.aspx. 1. I also exclude collections of primary sources, often digitized, meant for purchase by institutions, such as the
Foreign Office files published by Adam Matthew Digital, “FO India, Pakistan and Afghanistan,” accessed
September 5, 2012, http://www.amdigital.co.uk/Collections/FO-India.aspx. 1. I also exclude collections of primary sources, often digitized, meant for purchase by institutions, such as the
Foreign Office files published by Adam Matthew Digital, “FO India, Pakistan and Afghanistan,” accessed
September 5, 2012, http://www.amdigital.co.uk/Collections/FO-India.aspx. 2. Frances Pritchett, on her website, suggests that a major revision of both volumes of this work is forthcom
ing from Columbia University Press, although no target date is noted. If the tables of contents on the web
site will be retained in the print publication, then the revision will not answer some of the critiques I have
made above. See Pritchett (Sources of Indian Traditions, Columbia University Press).h 3. Routledge has not made this reader consistently searchable on its website. The release date was provided by
Pamela Moukrim, Administrative Support Clerk, Taylor & Francis, LLC, in a personal communication to
the author, March 20, 2012.hh 4. The best available edition of the original text is Thackston (1996). 5. Pinjar is available in English translation in Pritam (2009). Accounts of Partition given by those outside liter
ary circles tend to be embedded in secondary scholarship; see for example Menon and Bhasin (1998). 6. Sang-e-Meel Publishers (http://www.sangemeel.com) have reprinted the Punjab District Gazetteers (and
others) over the past fifteen to twenty years; digitized versions of the original gazetteers (and even the
Settlement Reports on which the gazetteers are based) are available through Google Books, although I think
students lose something of the materiality by reading this way. ASIANetwork Exchange | Spring 2014 | volume 21 | 2 Amy Kardos Abstract: Recent scholarship has challenged narratives of Central Eurasia’s relation
ships with its neighbors in East Asia, South Asia, and Southwest Asia. This scholar
ship describes the “silk road” as a cross-regional interconnected network of routes
that contributed to the development of diverse and dynamic civilizations, while also
functioning, from a Central Eurasian perspective, as the foreign trade component of
a complex internal Central Eurasian economy. Challenging long-standing narratives
of “needy” or “predatory” nomads that militarily overwhelm sedentary empires,
scholars of Central Eurasia have moved the region from its place as a distant fron
tier on the edge of civilization to one at the center of historical globalization. This
article discusses the importance of incorporating such ideas into world and Asian
history survey courses, which are often taught by nonspecialists who have only
encountered Central Eurasia in their respective fields as a periphery, and providing
opportunities for students to think critically about historical sources and move past
stereotypes of “barbarian” and “civilization.” Keywords Definitions; Misconceptions; Assignments; Transnational themes;
History 54 | Teaching Central Eurasia in Undergraduate Survey Courses Central Eurasia in World History Golden’s work, whose regional history is not dependent upon a
concept such as the silk road, which literally and figuratively moves over time. I hope that
this direction of scholarship continues—one that positions the silk road as a feature of the
continued global significance of Central Eurasia into the modern period. For pre-1500 silk
road studies, Hansen’s book contributes several ideas that reframe the role of the silk road
in world history. Hansen de-emphasizes the role of merchants while highlighting the long-
neglected role of differing states in creating and facilitating these networks (Hansen 2012, period and the advent of direct maritime trade, Millward suggests that significant exchanges
continued through the sixteenth, seventeenth, and eigtheenth centuries (Millward 2012,
111). In this case, the silk road should not be confined to the first half of a world history
survey course that ends in 1500, but should be part of the historical narrative of the early
modern and modern eras as well. This connection between the ancient and modern periods
is also a benefit of Peter B. Golden’s work, whose regional history is not dependent upon a
concept such as the silk road, which literally and figuratively moves over time. I hope that
this direction of scholarship continues—one that positions the silk road as a feature of the
continued global significance of Central Eurasia into the modern period. For pre-1500 silk
road studies, Hansen’s book contributes several ideas that reframe the role of the silk road
in world history. Hansen de-emphasizes the role of merchants while highlighting the long-
neglected role of differing states in creating and facilitating these networks (Hansen 2012,
235). Along the way she also critically discusses the evidence available to historians writing
about the silk road by explaining and including in each chapter samples of documents such
as labor contracts and medical prescriptions. The benefit of these documents, she convinc
ingly argues, is that they were meant to be thrown away as trash and were not compiled into
official histories. As such, “they offer a glimpse into the past that is often refreshing, per
sonal, factual, anecdotal, and random” (Hansen 2012, 5). Central Eurasia in World History Hansen uses these documents to
argue against the prevailing view of the silk road as a series of trade routes, instead showing
how the silk road was a superhighway of ideas and technology that was created by a diverse
group of immigrants from different cultures, occupations, and classes (Hansen, 2012, 5). As the author of a well-received earlier study, The Silk Road in World History, Xinru Liu has
also written a teaching supplement for pre-1500 survey courses, entitled The Silk Road: A
History With Documents. Both Hansen and Liu’s recent works offer excellent opportunities
to discuss the silk road through a study and analysis of primary sources. h
l
f
f h
h h h
h l
h
f In this essay, I outline a few of the ways in which this recent scholarship can inform
teaching. I discuss broad themes, specific historical examples, and possible assignments. These teaching strategies are only a small sample of this recently published rich material
that can be easily mined by nonspecialists. The first section discusses geographical termi
nology and how historians locate and define Central Eurasia. The second outlines strategies
for moving students beyond stereotypes of Central Eurasians and highlighting the cultural
diversity of the region. The third section takes the investigation of Central Eurasian culture
deeper into a study of its production through cultural blending. And, finally, the fourth
section makes the case for using the idea of silk road and Central Eurasian exchanges in
historical narratives of the twentieth century. 55 | Teaching Central Eurasia in Undergraduate Survey Courses Central Eurasia in World History Scholarship on Central Eurasia seeks to reframe the region as an integral and connected
current in the larger flow of world history. David Christian wrote in the Journal of World
History in an article published in 2000 that a study of the “silk road” indicates that scholars
need to take seriously the underlying unity of Afro-Eurasian history. He suggests that we
should regard modernity itself as an indirect product of the rich synergy created by systems
of exchanges rather than one particular regional culture (Christian 2000, 25-6). Four short
and accessible books published in the last three years, all by Oxford University Press, allow
world history scholars easy access to specific case studies that illustrate Christian’s argu
ment and also fit neatly into the organization of undergraduate survey courses: James A. Millward, The Silk Road: A Very Short Introduction (2013); Valerie Hansen, Silk Road: A
New History (2012); Peter B. Golden, Central Asia in World History (2011); and Xinru Liu,
The Silk Road in World History (2010). Even if these books are not assigned to undergradu
ates, the information they contain can be used by instructors to add depth to lectures and
as a springboard for creative assignments that highlight cross-regional connections. James
Millward’s appropriately titled very short introduction has an organization best suited for
incorporating discussions of the silk road into a thematic world history course. The book
contains chapters such as the “biological silk road” and the “technological silk road” that
offer examples of exchanges across Eurasia from ancient to modern times. He incorporates
some of the major contributions of Valerie Hansen’s 2012 research on the silk road before
1500, while also discussing the usefulness of the term in understanding Eurasian exchanges
into the modern era. While most silk road histories end with its decline after the Mongol 54 | Teaching Central Eurasia in Undergraduate Survey Courses ASIANetwork Exchange | Spring 2014 | volume 21 | 2 period and the advent of direct maritime trade, Millward suggests that significant exchanges
continued through the sixteenth, seventeenth, and eigtheenth centuries (Millward 2012,
111). In this case, the silk road should not be confined to the first half of a world history
survey course that ends in 1500, but should be part of the historical narrative of the early
modern and modern eras as well. This connection between the ancient and modern periods
is also a benefit of Peter B. Geography: Locating Central Eurasia and the “Silk Road” In my experience, students’ lack of familiarity with the geography of Central Eurasia and
its political boundaries is complicated even further by the region’s multiple names. In intro
ducing students to the distinctions between “Central Asia,” “Inner Asia,” and “Central Eur
asia,” I suggest using the resources available on three websites. The website of the Research
Institute for Inner Asian Studies at the University of Indiana, Bloomington1 explains the
origins of the term “Inner Asia” and the history of the discipline of Inner Asian studies and
Central Eurasian studies. As it notes, “Central Eurasia” can be used interchangeably with
“Inner Asia” to designate the homeland of the Altaic peoples and the Uralic peoples. Today
these peoples can be found in the five independent Central Asian republics of Uzbeki
stan, Turkmenistan, Tajikistan, Kyrgyzstan, and Kazakhstan; the republic of Mongolia; the
Xinjiang Uygur, Inner Mongolia, and Tibet Autonomous Regions of the People’s Republic 55 | Teaching Central Eurasia in Undergraduate Survey Courses ASIANetwork Exchange | Spring 2014 | volume 21 | 2 of China, as well as the Manchu lands. The term “Central Asia” is often used to refer to
the Islamic part of Inner Asia, meaning the Central Asian republics and Xinjiang.2 I adopt
the designation “Central Eurasia” in teaching world and Asian history survey classes and
illustrate its boundaries using several maps available at the website of The Silk Road Project
(www.silkroadproject.org). The section on “Maps of the Silk Road” is part of a set of cur
ricula designed for middle and secondary students co-developed by the Silk Road Project
and the Stanford Program on International and Cross-Cultural Education. The maps and
images available on the website are a helpful introductory tool for locating and defining the
boundaries of Central Eurasia. The third website I use to introduce and give depth to some
of the varieties of Central Eurasian societies is that of the Mongolia and Inner Asia Studies
Unit at the University of Cambridge (http://innerasiaresearch.org). This website has a link to
albums that show a wide range of images taken by scholars on their various research trips in
Inner Asia. Current albums include photos of Tuvans, the Altai Republic, Inner Mongolia,
and Xinjiang. Taken together, these three websites provide a much-needed supplement to
any world history or Asian history survey text. 56 | Teaching Central Eurasia in Undergraduate Survey Courses Geography: Locating Central Eurasia and the “Silk Road” When discussing Central Eurasia, instructors will also be forced to define the term
“silk road.” They should make explicit to students that the concept is a nineteenth century
European historiographical construct. The term did not exist before 1877 when the Baron
Ferdinand von Richthofen first used it on a map (Hansen 2012, 235; Millward 2013, 4-5). As
Hansen notes, no individual flying over the silk road at any point in history would be able
to identify its geographical boundaries. Hansen’s approach is to choose eight silk road cen
ters to organize her study, which allows her to examine and analyze the source base for the
historical record on the silk road. She concludes that the silk road as a network of exchanges
was not significant in terms of international commerce or the exchange of goods. Instead,
its main historical contribution was as a “cultural artery” (Hansen 2012, 235). She states,
“Refugees, artists, craftsmen, missionaries, robbers, and envoys all made their way along
these routes. Sometimes they resorted to trade, but that was not their primary purpose for
travelling” (Hansen 2012, 238). In survey courses, I use this concept of a cultural artery not
just when discussing the historical process of the silk road trade networks, but also when
teaching the history of Central Eurasian cultures into the modern period. How I do so is the
subject of the next section. Eurasia So, Central Eurasia was not a stop
or a transition between different places that produced and purchased goods, such as Rome
and Han China, it was its own integrated economy that had both foreign and domestic
components (Beckwith 2009, 328). In a different yet complementary critique of the term, Millward mentions that the “silk
road” would be better renamed the much-less-romantic “Soghdian Network” (Millward
2007, 29). Not only did these Iranian merchants dominate east-west trade, but Soghdian
became the lingua franca of the silk road in farming, artisan, commercial, and diplomatic
circles. Millward also notes that the term is misleading for other reasons: 1) silk was only
one of many products exchanged, 2) merchants used multiple routes instead of a single one,
and 3) Western imports to China were as important as Chinese exports (Millward 2007,
29). In addition, the focus on the east-west direction of the exchanges and the states on the
“ends” of the silk road obscures the importance of Indian and Persian empires (Millward
2013, 6-7). This expanded definition of the silk road also requires a reconsideration of Central Eur
asian culture. Beckwith notes that Central Eurasian societies were not composed of “needy”
or “predatory” nomads, but rather people who practiced different modes of production
and relied mostly on trade in order to obtain desired products. Beckwith argues that steppe
people mostly fought amongst themselves and went out of their way to avoid conflict with
peripheral states such as China, preferring to maintain a trade relationship. In fact, many
times Central Eurasians offended the peripheral states, such as Han China, not through
invasion but merely the insistence of remaining independent. Golden’s recent work comple
ments this characterization of the nomadic/sedentary relationship. He also notes that urban
Central Asia had a rich and cosmopolitan culture that had a symbiotic relationship with its
nomadic neighbors, and that the nomads themselves were “no more blood thirsty or covet
ous of gold or silks than their ‘civilized’ neighbors” (Golden 2011, 6). This idea challenges stereotypes of Central Eurasians as “barbarians.” Contrary to com
mon misconceptions, Central Eurasians, such as the Huns, the Xiongnu (Hsiung-nu), and
the Mongols, were no more cruel or aggressive than their contemporary large conquerors,
the Romans, the Persians, or the Chinese. Beckwith notes that all empires throughout
history were possessed of multiple personalities. Eurasia Survey courses on Asian history cannot avoid discussing the region of Central Eurasia
and its peoples. Yet, instead of understanding the contributions Central Eurasian societies
have made in world history, they are often portrayed in history classes as an anticiviliza
tional force due to unrecognized cultural misperceptions and biases (Beckwith 2009, xxi). I
ask students to rethink their idea of Central Eurasian nomads as barbarians that disrupt the
development of “civilization” instead of contributing to it. Christopher Beckwith’s ambitious
2009 text on the history of Central Eurasia from the Bronze Age to the present, while too
fragmented to be useful as a textbook for undergraduate students, offers an interesting way
of thinking about the history of the silk road that is missing from world history or Asian
history textbooks (Beckwith 2009). Beckwith defines the silk road as synonymous with the Central Eurasian economy. Central Eurasian peoples lived in three different ecological-cultural zones and practiced
three different modes of life that were tightly interconnected in a single economy. It was this
economy that created the commercial networks referred to by outsiders as the silk road. 56 | Teaching Central Eurasia in Undergraduate Survey Courses ASIANetwork Exchange | Spring 2014 | volume 21 | 2 Therefore, what is commonly referred to as the silk road was the international compo
nent of that Central Eurasian economy. In Beckwith’s definition, world history textbooks
that now progressively use the plural term “silk roads” to acknowledge the multiple trade
networks are still missing an important element since the term only encompasses one side
of the economy. According to Beckwith, the silk road was not a network of trade routes
or even a system of cultural exchange but rather created an entire local system of Central
Eurasia in which commerce, both internal and external, was highly valued and energetically
pursued, and reflected in cultural norms and political organization (Beckwith, 2009, 328). To get at the domestic component, Beckwith uses the Central Eurasian culture complex of
comitatus. In the Central Eurasia culture complex of comitatus, the primary mode of politi
cal organization was centered on a select group of elite warriors, who also acted as admin
istrators, who pledged personal loyalty to the ruler but in turn were rewarded with goods,
some of which were silk. Thriving commerce and the accumulation of foreign luxury goods
were required to meet the demand of the political system. 57 | Teaching Central Eurasia in Undergraduate Survey Courses Eurasia By turn, empires were destructive and con
structive, brutal and paternal, exploitative and beneficent, coercive and attractive, conserva
tive and innovative. (Beckwith 2009, 341-355). In a writing assignment meant to stimulate
in-class discussion, I ask students to think about the ways in which each of those adjectives
could be applied to different world empires throughout history, and more importantly, from 57 | Teaching Central Eurasia in Undergraduate Survey Courses ASIANetwork Exchange | Spring 2014 | volume 21 | 2 whose perspective would they be used. With Central Eurasians it is often the negative char
acteristics, such as destructiveness and brutality, that get molded onto some sort of homog
enous barbaric Central Eurasian culture and viewed as static and unchanging over many
different political systems over centuries (Beckwith 2009, 341-355). Millward also suggests
that, contrary to being an anticivilizational force, Central Eurasian could be thought of as
“proto-globalizers” who facilitated the cultural, technological, and political advancement of
neighboring states (Millward 2013). The roots of these misconceptions can be traced to problems with historical sources
about Central Eurasians—English language textbooks use the term “barbarian” to refer
to Chinese descriptions of Central Eurasians. Even using the term “barbarian” in quota
tion marks is inaccurate in the case of Chinese history, in which the language contained
a variety of different designations for foreigners. The English word “barbarian” embodies
a complex European cultural construct and was a generic pejorative term for a “power
ful foreigner with uncouth, uncivilized, nonurban culture who was militarily skilled and
somewhat heroic, but inclined to violence and cruelty, --yet not a savage or wild man. In
this case, the English term ‘barbarian’ does not have a single Chinese equivalent” (Beckwith
2009, 356-359). After discussing this problem of translation, I ask students to think about
other narratives of “civilization” and “barbarian” that they have encountered in their history
classes. How “civilized” was Rome, and how “barbaric” were the Mongols? In which ways
could Rome be viewed as barbaric and the Mongols as a civilizing society? I emphasize the
multiple personalities of empires and also the multiple sides of cultures. To further illustrate
the diversity of cultures and polities engaged in the network of exchanges throughout Cen
tral Eurasia, I organize the students into groups and assign chapters from Susan Whitfield’s
Life Along the Silk Road. Eurasia Using primary sources, Whitfield reconstructs the lives of a color
ful cast of historical actors, from an opening scene with an inebriated Soghdian merchant to
a painter of Buddhist art working in the Dunhuang caves. I ask my students to do addi
tional reading on different aspects of the person’s life, such as providing an overview of the
history of their hometown or explaining cultural and religious practices. Some of my most
interesting presentations have focused on fashion, with students researching and analyzing
the various functions and meanings of dress and how those changed over time. All of this
additional reading is incorporated into an oral presentation given to the class that provides
the backdrop to the personal narrative of a silk road traveler. 58 | Teaching Central Eurasia in Undergraduate Survey Courses Cross-regional Interactions and Cultural Blendingh His description of the Xiongnu mentions their lack
of family names, lack of respect for the elderly, and lack of a written language, all elements
that were an important part of Chinese culture. These biases about Central Eurasian peoples
as pure nomads who were oppositional in culture to the Chinese were written into Chinese
historiography; Sima Qian’s phrasing for describing the Xiongnu gets reproduced in later
descriptions of northern nomads (Millward 2009, 19). Therefore, I have students read the
description of the Xiongnu not as an accurate representation of Xiongnu societies, but as
a window into Chinese self-definition. Then, for understanding the Xiongnu and other
Central Eurasian societies, I draw on recent scholarship of Central Eurasianists, such as that
mentioned above. As another example of cultural fluidity, I use the history of Central Eurasian music. Nathan Light, who has a book on the process of standardizing the Twelve Uyghur Muqam,
wrote an article recently about the connection between premodern cultural forms and the
process in which modernizing states seek to reinforce and control populations by standard
izing these premodern forms and presenting them as static historical representations of a
people. The Twelve Muqams is itself a cultural representation that claims premodern origins
but could only have been produced in its current form by a modern state. In this sense,
modern Central Eurasian cultural production is part of the larger process of modernization
(Light 2008; Harris 2008). However, for undergraduate survey classes, I use more general
examples to show cross-cultural exchange and interaction. For undergraduate students,
music can be an effective tool in identifying and connecting with people of different
cultures. Yo-Yo Ma heads such a project. Ma’s Silk Road Project does not focus on Central
Eurasia, but rather takes the idea of the Silk Road as a metaphor for creating cross-cultural
connections through music. And he does not just reproduce these forms, playing standard
ized historical pieces, but seeks to reinvent and rethink them. Below, I link together several
websites that show such a dynamic in Central Eurasia—the cross-cultural connections that
have been made and the ways in which outside influences have been reinvented and reused
in new ways to form modern identity. Cross-regional Interactions and Cultural Blendingh I ask the students what value
they can get out of Sima Qian’s description and what Sima Qian’s document says about the
Chinese worldview. If people define themselves by what they are not, then the qualities that
Sima Qian emphasizes about the Xiongnu are less historically-accurate ethnography than
a glimpse into Chinese self-definition. His description of the Xiongnu mentions their lack
of family names, lack of respect for the elderly, and lack of a written language, all elements
that were an important part of Chinese culture. These biases about Central Eurasian peoples
as pure nomads who were oppositional in culture to the Chinese were written into Chinese
historiography; Sima Qian’s phrasing for describing the Xiongnu gets reproduced in later
descriptions of northern nomads (Millward 2009, 19). Therefore, I have students read the
description of the Xiongnu not as an accurate representation of Xiongnu societies, but as
a window into Chinese self-definition. Then, for understanding the Xiongnu and other
Central Eurasian societies, I draw on recent scholarship of Central Eurasianists, such as that
mentioned above. l Zhao, and Qing expanded northward, Chinese states began to build long walls to secure
newly conquered territories and gain pastureland on which to raise horses for chariots and
cavalry units. As a result, various northern tribes who lost land formed a powerful coalition
in response to the Chinese threat, a process that accelerated after unification under the Qin
in 221 BCE. The Xiongnu confederation that later threatened the Han empire, in particular
under Emperor Wu (r. 140-87 BCE), arose out of the crisis caused by this earlier northward
expansion of Chinese states (Di Cosmo 2002). After discussing this conflict, I have students
read a brief selection from the Records of the Grand Historian (Shiji) by Sima Qian (ca. 145-
ca. 90 BCE) describing the characteristics of the Xiongnu (Ebrey 1993, 54-56). His descrip
tion of the Xiongnu as pure nomads whose young boys are able to shoot mice with bows
and arrows does not hold up against the archeological evidence of Xiongnu houses, forti
fications, and agricultural settlements (Millward 2009, 19). I ask the students what value
they can get out of Sima Qian’s description and what Sima Qian’s document says about the
Chinese worldview. If people define themselves by what they are not, then the qualities that
Sima Qian emphasizes about the Xiongnu are less historically-accurate ethnography than
a glimpse into Chinese self-definition. Cross-regional Interactions and Cultural Blendingh This section discusses strategies for demystifying Central Eurasia and placing it within
the larger context of world history, while also illustrating the region’s remarkable ability
to create cultural hybridity. The idea of Central Eurasia as a cultural crossroads in which
identity is fluid and changing is widely accepted in the field of Central Eurasian history. Yet,
historical sources often have highlighted the differences between the steppe peoples of Cen
tral Eurasia and surrounding sedentary societies. Students should recognize that though it
is important to understand the significance of societies defining themselves in opposition
to Central Eurasians, they should not take that oppositional relationship as authoritative
and adopt the same framework for understanding cross-regional interactions. The historical
example that I use to illustrate potential biases in historical sources is the conflict between
the Han and the Xiongnu, which is already incorporated into most world history and
Asian history survey texts. This example not only shows students the complicated nature of
Central Eurasia’s relationship with its neighbors but also requires them to think about how
and why stereotypes became part of the historical record. As the early Chinese states of Yan, 58 | Teaching Central Eurasia in Undergraduate Survey Courses ASIANetwork Exchange | Spring 2014 | volume 21 | 2 Zhao, and Qing expanded northward, Chinese states began to build long walls to secure
newly conquered territories and gain pastureland on which to raise horses for chariots and
cavalry units. As a result, various northern tribes who lost land formed a powerful coalition
in response to the Chinese threat, a process that accelerated after unification under the Qin
in 221 BCE. The Xiongnu confederation that later threatened the Han empire, in particular
under Emperor Wu (r. 140-87 BCE), arose out of the crisis caused by this earlier northward
expansion of Chinese states (Di Cosmo 2002). After discussing this conflict, I have students
read a brief selection from the Records of the Grand Historian (Shiji) by Sima Qian (ca. 145-
ca. 90 BCE) describing the characteristics of the Xiongnu (Ebrey 1993, 54-56). His descrip
tion of the Xiongnu as pure nomads whose young boys are able to shoot mice with bows
and arrows does not hold up against the archeological evidence of Xiongnu houses, forti
fications, and agricultural settlements (Millward 2009, 19). 59 | Teaching Central Eurasia in Undergraduate Survey Courses UNESCO: The Uyghur Muqam of Xinjiang http://unesco.org/culture/intangible-heritage/10apa_uk.htm James Millward’s short history also has a section on musical exchanges in the chapter,
“Arts on the Silk Road.” He discusses the muqams while providing an impressively wide
survey of the origins and spread of different versions of lutes from Mesopotamia, Persia,
Central Asia and East Asia (Millward 2013, 91-98). 60 | Teaching Central Eurasia in Undergraduate Survey Courses Stanford Interactive Website Stanford Interactive Website
http://virtuallabs.stanford.edu/silkroad/SilkRoad.html http://virtuallabs.stanford.edu/silkroad/SilkRoad.html Aga Khan Music Initiative
http://www.akdn.org/aktc_music.asp Aga Khan Music Initiative
http://www.akdn.org/aktc_music.asp Smithsonian Folkways
http://www.folkways.si.edu Smithsonian Folkways
http://www.folkways.si.edu London Uyghur Ensemble http://uyghurensemble.co.uk/en-html/nf-research-article1.htmlh http://uyghurensemble.co.uk/en-html/nf-research-article1.html
This website describes the origins of muqam. A muqam is the melody type used in the
Uyghur system that developed over centuries from the Arabic maqam modal system. Large- This website describes the origins of muqam. A muqam is the melody type used in the
Uyghur system that developed over centuries from the Arabic maqam modal system. Large- 59 | Teaching Central Eurasia in Undergraduate Survey Courses ASIANetwork Exchange | Spring 2014 | volume 21 | 2 scale suites of sung, instrumental and dance music are called muqam. Music in Uyghur
culture also has religious significance because of popular Islam. Sufis used music to express
and promote their faith. Music also serves central roles in social gatherings. The instru
ments used in muqam indicate both the particular Uyghur interpretation of the music as
well as its outside influences. l
For example, the Chinese instrument erhu is thought to have developed from Chinese
contact with Central Asia, particularly popularized at Tang court. One of the instruments
used in muqam is the ghijek, which developed as a relative of the Persian spiked fiddle—an
image of which can be accessed on the Silk Road Project website. According to the London
Ensemble, the current form reflects the influence of the Chinese erhu. This example shows
that local identity in Central Eurasia is both very specific, most notably that local materi
als are used to make the instruments, as well as connected to outside cultural influences,
including Chinese, Turkish, and Persian. Uzbekistan also has its own versions, the shesh
maqam, videos of which can be viewed at the Smithsonian folkways website. One common
instrument is the dutar, used in both Uyghur muqam and the Uzbek Shashmaqam. Music
brings Central Eurasian culture alive for students and helps teach them about cultural
exchange and cultural production in a memorable and engaging way. I illustrate the above
relationships through recordings, images, and videos from the websites listed below. Bringing the Silk Road into the Modern Era Central Eurasia has also made significant contributions to world history in the modern
period. Asian modernization is one of the themes that can be used to link Central Eurasia
to its neighbors. One of the defining aspects of modernization is the emergence of active
states in studying, categorizing, standardizing, and then institutionalizing culture. In the
late seventeeth century the Russian and Qing empires partitioned Central Eurasia between
themselves (Beckwith 2009, 321). Central Eurasian culture and political systems did not
become flattened and absorbed after Russian and Qing conquest, but rather reconfigured in
ways that show continuity with the past while also reflecting global trends in modernizing
states. One example of Central Eurasia’s impact on modern Asia is the way the Soviet Union 60 | Teaching Central Eurasia in Undergraduate Survey Courses ASIANetwork Exchange | Spring 2014 | volume 21 | 2 reshaped and exported the Uzbek SSR’s capital of Tashkent as a model for Asian social
ism. In the late 1950s, Chinese Premier Zhou Enlai visited Tashkent, which Soviet leaders
viewed as the ideal representation of successful Asian socialism. During this time agricul
tural specialists from the Soviet Union also used cotton seeds imported from Uzbekistan
to oversee the new construction of cotton farms and factories in China’s northwest border
region of Xinjiang. Within China, several newspaper articles discussed Uzbekistan as the
model for Xinjiang’s post-1949 economic development. Outside of the People’s Republic of
China, Soviet leaders sought to export the Tashkent model of Asian socialism to South Asia
and Southeast Asia. They invited delegations and sent teams to South Asian and Southeast
Asian countries from the 1930s on to show other Asian countries an ideal example of Asian
socialism (Stronski 2010). This model of Asian modernization was meant to tie East Asia,
Central Asia, South Asia, and Southeast Asia together. After the 1950s, China moved away
from Soviet oversight of socialist economic development in Xinjiang and began seeking to
export itself as a model of successful Asian socialism to the Third World. I use this example
in class in discussing twentieth-century modernization movements to illustrate how Soviet
and Chinese leaders sought to reconfigure Central Eurasia by recreating a “Red Silk Road.”
This relationship was terminated shortly after its formation with the closing of the border
and the decline of Sino-Soviet relations. Bringing the Silk Road into the Modern Era Evidence of the shorter-lived but similar relationship between the Soviet Union and
the People’s Republic of China in the 1950s can still be seen today (Millward 2013, 115). i 61 | Teaching Central Eurasia in Undergraduate Survey Courses Bringing the Silk Road into the Modern Era Yet, Mao and Chinese Communist Party members
who were disillusioned with Khrushchev’s Soviet present remained fascinated by the Soviet
past. They continued to study the work of Lenin and Stalin and adapt the Soviet past to
the Chinese present throughout the People’s Republic under Mao. While historians of the
Soviet Union may stress the Soviet leadership’s focus on the third world rather than China
as a destination for the export of socialism after the 1950s, a Chinese perspective shows the
continuing significance of Soviet institutions across the border. i
Millward suggests the usefulness of the term “silk road” in characterizing the Sino-
Soviet relationship as a whole. The long relationship between India and China, revolving
around Buddhism, entailed China’s study of another country’s religious system, the transla
tion of its textual corpus, the exchange of envoys, and imitation of technology, music and
art. Evidence of the shorter-lived but similar relationship between the Soviet Union and
the People’s Republic of China in the 1950s can still be seen today (Millward 2013, 115). Marxism-Leninism fulfilled the role of “religion” and involved personnel exchanges and the
popularization of Russian film, literature, and music in China. As Millward argues, “China
adopted Soviet-style apartment buildings in the twentieth century under circumstances
similar to those when it adopted the chair (and much else) from India in the tenth and
eleventh centuries. If we look at the silk-road phenomena broadly, we can see quantitative
difference but little qualitative difference between the exchange of goods and ideas across
Eurasia from prehistoric to early modern times and what we now speak of as ‘globalization’”
(Millward 2013, 117). Though this is an excellent example of continuity, many historical
issues may not be the same for premodern nomadic/sedentary relations or cross-regional
networks of exchange and those that existed in the twentieth century, but a comparison of
the two periods could be used to launch a discussion among students of how things have
changed over time. i
Millward suggests the usefulness of the term “silk road” in characterizing the Sino-
Soviet relationship as a whole. The long relationship between India and China, revolving
around Buddhism, entailed China’s study of another country’s religious system, the transla
tion of its textual corpus, the exchange of envoys, and imitation of technology, music and
art. 62 | Teaching Central Eurasia in Undergraduate Survey Courses Conclusion Recent scholarship by Central Eurasianists offers insights into world history that are not
captured by standard texts. This essay has attempted to touch on some of the ways in which
these ideas can be used to supplement lectures and assignments in world history and Asian
history survey courses for both the premodern and modern periods. Nonspecialists can 61 | Teaching Central Eurasia in Undergraduate Survey Courses ASIANetwork Exchange | Spring 2014 | volume 21 | 2 discuss the history of Central Eurasia in a way that corrects misconceptions, links Central
Eurasia to other parts of the world in new and engaging ways, and offers a narrative that
fulfills Christian’s call to view modernity as an indirect product of a long history of cross-
regional exchanges along silk roads that are still traveled today. rEFERENCES Beckwith, Christopher I. 2009. Empires of the Silk Road: A History of Central Eurasia from the Bronze Age to the
Present. Princeton: Princeton University Press.h Christian, David. 2000. “Silk Roads or Steppe Roads? The Silk Roads in World History.” Journal of World History
11, 1: 1-26.h Cosmo, Nicola. 2002. Ancient China and its Enemies: The Rise of Nomadic Power in East Asian History. Cam
bridge: Cambridge University Press. Di Cosmo, Nicola. 2002. Ancient China and its Enemies: The Rise of Nomadic Power in East Asian History. Cam
bridge: Cambridge University Press. Ebrey, Patricia Buckley, ed. 1993. Chinese Civilization: A Sourcebook. New York: Free Press. Ebrey, Patricia Buckley, ed. 1993. Chinese Civilization: A Sourcebook. New York: Free Press. Golden, Peter B. 2011. Central Asia in World History. Oxford: Oxford University Press.hh Harris, Rachel. 2008. The Making of a Musical Canon in Chinese Central Asia: The Uyghur Twelve Muqam. London:
Ashgate Publishing. Hansen, Valerie. 2012. Silk Road: A New History. Oxford: Oxford University Press. Light Nathan 2008 Intimate Heritage: Creating Uyghur Muqam Song in Xinjiang B g
g
g
yg
q
g
j
g
Liu, Xinru. 2010. The Silk Road in World History. Oxford: Oxford University Press. ---. 2012. The Silk Roads: A Brief History with Documents. Boston: Bedford/St. Martin’s. Liu, Xinru. 2010. The Silk Road in World History. Oxford: Oxford University Press.h h
---. 2012. The Silk Roads: A Brief History with Documents. Boston: Bedford/St. Martin’s. h
Millward, James. 2007. Eurasian Crossroads: A History of Xinjiang. New York: Columbia University Press.h ---. 2013. The Silk Road: A Very Short Introduction. Oxford: Oxford University Press.i Danke Li is a Professor in the
Department of History at Fair
field University Notes 1. Research Institute for Inner Asian Studies at the University of Indiana, Bloomington (http://www.indiana. edu/~rifias/RIFIAS_and_Inner_Asian_Studies.htm) 1. Research Institute for Inner Asian Studies at the University of Indiana, Bloomington (http://www.indiana.i 1. Research Institute for Inner Asian Studies at the University
edu/~rifias/RIFIAS_and_Inner_Asian_Studies.htm)h 1. Research Institute for Inner Asian Studies at the University of Indiana, Bloomington (http://www.indi
edu/~rifias/RIFIAS_and_Inner_Asian_Studies.htm)h i
2. The website also provides links to web resources, such as the Central Eurasian Studies Society, which
publishes the Central Eurasian Studies Review, edits the journal Central Asian Survey, and holds annual
conferences through the Sinor Research Institute for Inner Asian Studies at Indiana University (http://cen
traleurasia.org/). 62 | Teaching Central Eurasia in Undergraduate Survey Courses ASIANetwork Exchange | Spring 2014 | volume 21 | 2 63 | Teaching the History of Women in China and Japan Danke Li Abstract: Studying the history of Chinese and Japanese women provides American
students with a thematic approach to Asian Studies. This paper reflects on the chal
lenges I face in teaching women’s histories in China and Japan. It also discusses the
pedagogy and sources I use in teaching the course. The paper argues that teaching
the history of women in China and Japan will allow us to move beyond the con
ventional regional or nationally focused approach to Asian Studies and enable us to
reimagine old narratives and introduce students to new methods of understanding
both the universality and diversity within Asian history. Keywords China; Japan; Women Keywords China; Japan; Women Studying histories of China and Japan can provide American Students with a thematic
approach to Asian Studies instead of a conventionally geographical approach. Although
China, as an emerging economy, and Japan, as an existing economic powerhouse, are no
strangers to most American undergraduates, many do not know much about women in
China and Japan. Most textbooks on modern Chinese and Japanese histories cover very lit
tle about women. If there is coverage, it tends to be abstract and remote to many American
students of the twenty-first century. As recently as last year, I asked my American students
what first came to mind when thinking about Chinese and Japanese women during the
first class of my “Women in China and Japan” course. The predominant answers given still
cast Chinese and Japanese women as subservient victims of patriarchal society. Examples
often include foot binding, one-child family policy in China, or geishas in Japan. The overall
images are often ones of passive and agencyless women. While these images do reflect some
truth, it is only a partial if not a misleading one. The prevalence of such one-dimensional
impressions about Asian women underscores the fact that what and how to teach American
students about Chinese and Japanese women still remains a challenge in the twenty-first
century. These challenges are discussed and exemplary sources presented in the following. Challenge number one: what to teach—the contentshi The first challenge involved in teaching the history of women in China and Japan per
tains to content—what to include in the course materials. Most of the generalized and to
some degree stereotypical images of Chinese and Japanese women as viewed by American
students perhaps partially arise from a relative lack of representation of Chinese and Japa
nese women’s own voices in Western language scholarship.h There is an uneven representation of Western and non-Western scholarly voices in femi
nist academic work. For example, Japanese feminist scholars have noted an asymmetry in
dissemination of feminist scholarship. Since the 1970s, a considerable body of scholarship
and literature on women in Japan has been published by Western scholars and translated
and made available to Japanese readers. However, very few Japanese scholarly works on
the same subject become available in Western languages for Western audiences (Fujimura- 63 | Teaching the History of Women in China and Japan ASIANetwork Exchange | Spring 2014 | volume 21 | 2 Fanselow and Kameda 1995, xi). The situation in China is about the same. Additionally, in
most of the existing Western works published in the 1970s, 1980s, and even the early 1990s,
Chinese and Japanese women are treated mainly as the subjects of study and theorizing. And since feminist theories and the very field of women’s studies themselves first arose in
the West, Japanese and Chinese scholars also heavily relied on these Western theories in
their own work when they first began to engage in research on women and feminism dur
ing the early 1970s and throughout the 1980s. It was not until the late 1980s and the1990s
that some of the Japanese and Chinese scholars’ research became known in the United
States, though they remained in the academic periphery. Women’s studies is a relatively new academic field in Japan and China. In Japan, the
Women’s Studies Association was established in 1979, and in China it was established
in the mid to late 1980s. In both countries, the development of women’s studies into an
interdisciplinary discourse has been influenced by Western feminist and gender theories
and scholarly works. However, since the 1990s, especially after the Fourth World Women’s
Conference in Beijing in 1995, women’s studies has not only blossomed but also continually
matured in both countries. Challenge number one: what to teach—the contentshi Moving beyond the initial stage of absorbing Western theories
as models for women’s studies in their countries, Japanese and Chinese scholars are now
developing their own perspectives and theories on women in their respective countries and
believe that their studies can enrich the field both in and outside of Japan and China. For
example, in China, more and more scholars have explicitly begun to challenge the univer
sality of Western-born gender and feminist theories. One of the leading Chinese scholars of
women’s studies even states that Chinese scholars “respect Western-based feminist theory,
and yet they still believe that Chinese women’s studies has its own background and circum
stances unique to Chinese history and social reality. Western feminist theory is certainly
valuable as a rich source of reference, but Western feminist tradition can hardly provide a
standardized answer to all Chinese women’s questions” (Li and Zhang 1994, 148). Chinese
scholars are challenging the notion that gender representation is one size fits all. Western scholars—for example, Christina Gilmartin, Gail Hershatter, Lisa Rofel, and
Tyrene White—had advocated for the creation of “a dialogue that would challenge the
dichotomy between Europeans and Americans as theory makers and Chinese women as
objects of theory” (1994, 7). Many efforts, including joint conferences, publications, and
foundation-sponsored projects have been developed to promote such a dialogue. However,
while Western scholarship on women’s studies has continued to inspire the field in Japan
and China, Japanese and Chinese scholarship has still not attained the same status in the
West. In general, Western scholars and Japanese and Chinese scholars predominantly work
and publish in separate circles and have not had enough active intellectual cross-fertiliza
tion between them. In addition to the fact that Japan and China do not hold the same level
of global political and cultural clout as the U.S. does, the countries’ political, cultural, and
linguistic differences make it more difficult for Japanese and Chinese scholarship in the
field to gain the same prestige enjoyed by Western scholarship. Most of the scholarly works
published by Japanese and Chinese scholars in their respective languages remain read
mostly in Japan and China and have had little impact on the field in the U.S. and elsewhere,
except among scholars who can read Chinese or Japanese. This uneven representation of
scholarship was first observed by scholars in the United States. 64 | Teaching the History of Women in China and Japan Challenge number one: what to teach—the contentshi In its 2010 Call for Papers,
The National Women’s Studies Association (NWSA) pointed out: “Although the problem
of omissions, silences, and distortions in women’s studies has been analyzed for decades,
too often feminist scholarship continues to theorize on the basis of hegemonic frameworks,
false universals, and a narrow range of lived experiences. The legitimate terrain of feminist 64 | Teaching the History of Women in China and Japan ASIANetwork Exchange | Spring 2014 | volume 21 | 2 theory, inquiry, and politics remains contested.”1 The voices, lived experiences, and perspec
tives of Chinese and Japanese women in particular, and of Asian women in general, are part
of a still-underrepresented area of feminist scholarship. In China, for example, Chinese
scholars of women’s history have recognized the importance and have “shown a tendency to
approach women’s problems from women’s points of view and demonstrate a strong sense
of needing to reveal women’s actual life experiences” (Wang 2006, 318). But most of the
Japanese and Chinese scholarly works are not available in English. In light of this situation,
the question of what to include and how to recognize Japanese and Chinese scholars’ and
women’s voices into a gender and women course for American undergraduates is a signifi
cant challenge. In my “Women in China and Japan” course, I try to strike a balance between West
ern feminist and gender theories, interpretations, and the voices and lived experiences of
Japanese and Chinese women as well as their scholarly works. In addition to introducing
existing gender and feminist theories and framework by Western scholars, I try to include
materials written by Chinese and Japanese women to show that Japanese and Chinese
scholarship can enrich the field both in and outside of Japan and China. Many Japanese
and Chinese scholars in women’s studies are not only interested in writing about women in
their countries, but also in pushing for changes in women’s status in government policies
and society. Their insight on women’s studies and women’s movements in their respective
countries will enhance intellectual understanding for people outside of Japan and China. It is also important for American students to see the usefulness of Japanese and Chinese
scholarship as an integrated part of the field. To reach this goal, American students need to
hear the voices of Japanese and Chinese women. 65 | Teaching the History of Women in China and Japan Challenge number one: what to teach—the contentshi Since most students today are netizens, I include in the syllabus some credible websites
that provide useful information, for example, the Bibliography of Asian Studies, Stanford
University’s online source center on Japanese studies, the website of the Universities Service
Centre for China Studies, Chinese University of Hong Kong, and the German Heidelberg
University’s online information center on Chinese women.2 Clips of documentary and fiction films are also used in this course. For the history of
Chinese women, in addition to the usual suspects, such as Raise of the Red Lantern, Judou,
The Story of Qiu Ju, Road to Home, and Small Happiness, I also use the documentary films
China Blue, Not One Less, The Last Train Home, Marrying from the Heart of the Dragon
series, Chinese Foot Binding, Women in China: Up Against the Wall, Nushu, and A World
Without Fathers or Husbands to provide an array of visual images of Chinese women to
American students to show how diverse women in China are. For the history of Japanese
women, I use clips from Sandakan Brothel No 8, Black Rain, Street of Shame, No Regret for
the Youth, The Woman in the Dunes, Twenty-four Eyes, Kabe: Our Mother, and Granny Gabai
as well as documentary films such as Japanese Women, Faces of Japan, and Mother’s Way
Daughter’s Choice, to break the stereotyped presentation of the images of Japanese women. I also find that YouTube provides many useful clips on gender and women for courses on
gender and feminism. For example, I use a four-minute video presentation called “Socializa
tion and Gender Roles within the Family”3 on YouTube to stimulate conversation on what
gender is and how it is constructed in China and Japan. In choosing the assigned readings and audiovisual materials for the course, it is impor
tant to include materials that inform American undergraduate students about Japanese and
Chinese women beyond the conventional categories of family, marriage, motherhood, and
sexuality, in other words, women’s lives in domesticity. The course materials also encourage
students to study women and war and the LGBT movement, as well as women’s lives in edu
cation, work, religion, and politics. Challenge number one: what to teach—the contentshi To add Japanese and Chinese women’s perspectives and lived experiences, I rotate the
following books on Japanese women: Office Ladies and Salaried Men (Ogasawara 1998), on
Japanese women in the workplace; Sandakan Brothel #8: An Episode in the History of Lower-
Class Japanese Women (Yamazaki and Colligan-Taylor 1998), on Japanese women who
were sold into prostitution; Haruko’s World: A Japanese Farm Woman and Her Community 65 | Teaching the History of Women in China and Japan ASIANetwork Exchange | Spring 2014 | volume 21 | 2 (Bernstein 1983), on Japanese farm women and their community; The Secrets of Mariko:
A Year in the Life of a Japanese Woman and Her family (Bumiller 1995), on middle-class
urban women and their lives; In the Beginning, Woman Was the Sun: the Autobiography of
a Japanese Feminist (Raicho and Carig 2010), on one of the early Japanese feminists; and
the Prison Memoirs of a Japanese Woman (Fumiko and Inglis 1991), on a radical Japanese
woman who was executed for plotting to assassinate the emperor. I rotate the following
books on Chinese women: A Daughter of Han: the Autobiography of a Chinese Working
Woman (Pruitt 1945); A Woman Soldier’s Own Story: The Autobiography of Xie Bingying
(Brissman 2001); Echoes of Chongqing: Women in Wartime China (Li 2010), an oral history
of twenty Chongqing women who experienced the Second Sino-Japanese War; Personal
Voices: Chinese Women in the 1980s (Honig and Hershatter 1988), a study of Chinese
women during the early reform era; The Gender of Memory: Rural Women and China’s Col
lective Past (Hershatter 2012), a book on rural women during the early People’s Republic
of China; Some of Us: Chinese Women Growing Up in the Mao Era (Zhong, Wang, and Bai
2001), a collection of personal essays by China-born female scholars who live and teach in
the United States about being female under Mao’s China; and To the Storm: The Odyssey
of a Revolutionary Chinese Women (Yue 1985), an autobiography of a Chinese intellectual
woman who lived through the Maoist era and the Cultural Revolution. Challenge number one: what to teach—the contentshi The textbook I use to present Chinese scholarship on women is Holding Up Half the Sky:
Chinese Women, the Past, Present, and Future (Mow, Tao, and Zheng 2004). This book is the
first collection of essays on Chinese women written by Chinese women scholars. The book
covers the history of Chinese women from the Song dynasty to the early 2000s, as well as a
wide range of issues and themes, from Chinese women’s own efforts in promoting women’s
education in the nineteenth century, to women in the Peking opera, to women’s education
and women in politics in the twenty-first century. The textbook for Japanese women is
Transforming Japan: How Feminism and Diversity Are Making a Difference (Fujimura-Fan
selow 2011). This book is also a collection of essays on Japanese women written by Japanese
women scholars. The book is an updated version of Japanese Women: New Feminist Perspec
tives on the Past, Present, and Future (Fujimura-Fanselow and Kameda 1995). One reviewer
commented that the 1995 book “in offering Western readers Japanese research and points
of view, is path-breaking and full of opportunities for breaking down Western ethnocen
trism, even feminist ethnocentrism…” (Fujimura-Fanselow and Kameda 1995, back cover). The 2011 book retains five historical essays from the 1995 volume while developing new
topics ranging from single mothers’ struggles, lesbian partnerships, the formation and
growth of the men’s movement to activism for the rights of minorities and the politicization
of housewives in Japan. As the editor of the book points out, the text intends to show
case a comprehensive picture of diverse Japanese women living in a multicultural society
(Fujimura-Fanselow 2011, x). 66 | Teaching the History of Women in China and Japan Challenge number two: what to teach—the theoretical
perspectives The second challenge of teaching history of women in Japan and China is to introduce
and engage American undergraduate students with existing gender, feminist, and women’s
studies theories, and draw their attention to competing theories and ongoing debates on the
question of the universality of gender, feminist, and women’s studies theories. In her 2006
study of the rise of Chinese women’s history as part of the development of social history in
China, Shou Wang noticed that although gender, feminist, and women’s studies in China
were inspired and continued to be inspired by theories developed by scholars in the West,
since the 1990s more and more Chinese scholars have recognized the limitations of these
theories and their applicability to the Chinese situation. For instance, Wang points out “the
term ‘gender,’ which is widely used in Western works in women’s studies, does not have a
fixed Chinese equivalent.” Some of the Chinese scholars even worry about the danger of
“theoretical colonization” of the study of Chinese women’s history by Western scholars
(Wang 2006, 320). Thus, developing reading materials that help American undergraduate
students understand important concepts regarding gender, feminism, and women’s stud
ies while recognizing their usefulness and limitations for studying women in non-Western
societies is a challenge. To help students understand the importance of gender and feminist theories and stud
ies, I find several books particularly useful. The first is Women: Images and Realities (Kelly,
Parameswaran, and Schniedewind 2011). This book not only introduces the concepts of
women’s studies, gender, gender studies, and the question of the LGBT, but also showcases
how gender intersects with class, race, and ethnicity by providing examples of lived experi
ences of multicultural Americans. It also includes men’s voices in supporting women’s rights
movements and masculine studies. Another book is Gender in Modernism (Kime 2007). This is a collection of essays grouped into twenty-one thematic sections. I find the essays
on the theme of gender and global location particularly useful for the study of the history
of Japanese and Chinese women. The Evolution of American Women’s Studies: Reflections on
Triumphs, Controversies and Change (Ginsberg 2008) is useful as well, especially the intro
duction chapter. This chapter highlights the three waves of feminist movement and related
scholarly works in the United States and points out their weaknesses and challenges. Challenge number one: what to teach—the contentshi In the case of China, I include readings and visual mate
rials about non-Han Chinese women, for example the documentary films of Nushu, a film
about a unique women’s language developed and used exclusively by women in Yongjiang,
Hunan Province, and A World Without Fathers or Husbands, a film on the Mosuo women
who still live in a matrilineal society in Yunnan Province, to show that gender and feminist 66 | Teaching the History of Women in China and Japan ASIANetwork Exchange | Spring 2014 | volume 21 | 2 studies intersect not only with social class but also with ethnicity in China. China has fifty-
six recognized nationality groups, and conventional scholarship on Chinese women covers
mainly women from the Han majority, not minority groups. Readings encourage students
to consider the issue of gender and ethnicity in Japan as well. For example, they are given
readings on the experiences of Korean and “Other” Asian women who live in Japan (Faier
2008). 67 | Teaching the History of Women in China and Japan Challenge number two: what to teach—the theoretical
perspectives The
three books together can introduce American students to the basic theories and knowledge
on gender, feminism, and women’s studies and provide them with some frameworks to
probe what the prevailing theories are and whether they are useful in studying the history of
women in Japan and China. Another aspect of the theoretical challenge in teaching the history of women in Japan
and China is to make sure students understand that gender and feminist studies are not just
academic fields but also opportunities for everyday activism for promoting social justice
and equality in the world. I find Grassroots (Baumgardner and Richards 2005) inspiring and
a fun and absorbing text for undergraduates. It is aimed at pointing out to young people
that everyone, regardless of social and economic status and political standing, can be an 67 | Teaching the History of Women in China and Japan ASIANetwork Exchange | Spring 2014 | volume 21 | 2 activist and make a difference in the world. In teaching the history of women in Japan and
China, the above-mentioned books on gender, feminist, and women’s studies theories and
activism may be connected to the geolocation and realities of Japan and China presented in
the introduction chapters of Chinese Femininities and Chinese Masculinities (Wasserstrom
and Brownell 2002) and Transforming Japan: How Feminism and Diversity Are Making A
Difference (Fujimura-Fanselow and Kameda 2011). f
By connecting American undergraduate students to theories of gender, feminism, and
women’s studies in general and the Japanese and Chinese realities regarding women’s his
tory in particular, a course on the history of Japanese and Chinese women could engage
the students to think critically. For example, last year in the discussion of the articles
in Amy Kesselman’s Women: Images and Realities, students in my class noticed that the
authors argued that “feminism is continually developing a more multicultural and inclusive
perspective, mirroring the lives of women in all races, ethnic groups, and classes. Femi
nists of varied races and ethnicities are generating theory and practice that address their
particular experiences and consciousness, broadening and deepening the scope of femi
nist analysis” (Kesselman, McNair, and Schniedewind 2006, 12). Then, questions emerge:
if feminism allows different races and ethnicities to practice and address their particular
experiences and consciousness, do non-Western women groups, for example Chinese and
Japanese women, experience gender the same ways as white middle-class Western women
do? 68 | Teaching the History of Women in China and Japan Challenge number two: what to teach—the theoretical
perspectives According to Ginsberg, in the 1960s and 70s there was a clear goal for scholars and
activists about women’s studies and feminist movements in the United States. Do Chinese
and Japanese scholars and activists have a similarly clear goal about women’s studies and
movement in their respective countries? What is it? Is the goal of Chinese women the same
as that of Japanese women? Is the goal of Chinese and Japanese women the same as the goal
of women in other parts of the world? What unites and defines the category of women? Is womanhood defined universally in all countries? How do we strike a balance regarding
universalism and cultural relativism in gender and women’s studies? How do we create an
inclusive feminism that does not have the West-non-Western divide? Many of these ques
tions are probably relevant to all history courses, not only those on gender and women’s
studies, but also to Asian studies in general. Although we never reach consensual answers to these questions, the study of Japanese
and Chinese women’s history helps students engage critically with the renegotiation of gen
der theories. It makes them see the challenge of the common usage of Western theories as a
unitary standard for women’s histories elsewhere in the world despite their development in
the historical particulars of Western experience. In teaching the course on history of women
in China and Japan I realize that we need to encourage students to consider the neces
sity and possibilities for intellectual cross-fertilization to create a dialogue challenging the
dichotomy between Euro-Americans as theory makers and non-Western women as objects
of theory and see that gender representation is not one size fits all. Students need to learn
not only pertinent knowledge of gender and women in general, but also historiography of
gender and women’s studies in China and Japan. Studying history of women in Japan and China provides American students with
the opportunity to consider the commonly-shared concerns of all women as well as the
unique issues facing women in these countries. For example, students are asked to con
sider whether socialist revolution and industrial modernization liberate women in China
and whether industrialization and the Pacific War mobilization improved women’s status
in Japan. Challenge number three: how to teach—pedagogyh The third challenge in teaching women in Japan and China is what pedagogy to use and
how to make the course interesting. I employ a student-centered and interactive pedagogy
to motivate students to become active and engaged learners and critical thinkers.h The format of the course includes lecture, discussion based on assigned readings, debate,
and audiovisual material presentations. In addition, last year I also arranged an e-mail
penpal exchange between Chinese college students and my American students. A former
student of mine taught English at the Central China Normal University and I asked her to
mobilize her Chinese students to exchange emails with my students. This e-mail penpal
project enables American students in my class to actually interact with young Chinese peers
to exchange views on topics in the course. In my course, all students are expected to participate in class activities by contributing
thoughts, perspectives, anecdotes, and so forth on the readings, classroom experience, and
lectures. All students are required to lead one week of discussion, questioning and inte
grating the theories and arguments of the assigned readings and forging their own critical
opinions on the subject matter. To ensure the quality of each week’s discussion, a weekly
assignment is given. Before coming to each week’s class, students are required to finish a
two-page reflective essay on the week’s readings. They are asked to show that they under
stand the main ideas, themes, and assertions contained in each reading and are able to iden
tify the information supporting the author’s statement, as well as evaluate the weakness and
strength of the author’s ideas and presentation. In addition to the short essay, students write
out a few conceptual questions that will lead to a meaningful discussion. In order to help
students make intellectual connections to the various readings they are assigned each week,
they are asked to take one or more ideas and facts for the week and think about how they
can contrast, compare, clarify, call into question, or relate to knowledge and concepts they
have gained in previous weeks or from other sources, like outside readings, other courses,
and life experience. This course also requires students to produce a substantial final research
paper on their chosen subject pertaining to women in Japan and China at the end of the
semester. Challenge number two: what to teach—the theoretical
perspectives Teaching history of women in Japan and China helps students understand that
gender construction is complex and that a multicultural and historical approach to women’s 68 | Teaching the History of Women in China and Japan ASIANetwork Exchange | Spring 2014 | volume 21 | 2 histories is necessary. As women’s contributions and participation are included in Chinese
and Japanese history, subjects of inquiry are recast and causes and impact of major events
like China’s revolutions and Japan’s industrial modernization are also revised. By under
standing women’s position in gender relations, students gain a more realistic and compre
hensive knowledge of the values of Chinese and Japanese cultures, the functioning of their
societies, and the nature of historical continuities and changes. 69 | Teaching the History of Women in China and Japan Challenge number three: how to teach—pedagogyh As I see, at least in the realm of politics, Japanese women con
tinue to make strides, bit by bit, as seen through the profiles of Aokage Takako and Mitsui
Mariko, or the growing influx of ‘housewives’ into the political sphere, as described by Yoko
Sato in ‘From the Home to the Political Arena’.”4 A female student in the same course also commented on the readings on the subject of
the growth and movement of Japanese feminism, pointing out that the feminist movement
in Japan is “in some ways like the American feminist movement, a grassroots movement
that has undergone different waves and has had different leaders throughout; it is important
to realize that while there are similarities/universalities to the feminist movement through
out the world, each country’s movement has slightly different cultural issues it is dealing
with and there are different timelines that events occurred for each country. The readings
for this week focused on the growth of the Japanese feminist movement since the late 1890s
and traced its progress throughout today; the feminist movement is not a static organiza
tion; it has changed and its goals have progressed with the time period.”5 With proper guid
ance and intensive intellectual discussion and debate, undergraduate American students
can see that it is important to think like a historian and to strike a balance in the debate of
universalism and cultural relativism. Another common challenge encountered in my “Women in Japan and China” class
is that it is easy for American students to assume that there is no difference between the
experiences of Chinese and Japanese women. They often are surprised to see that although
women in the two countries share some common experiences and aspirations, they face
many different challenges and have different development stages pertinent to their own
history, society, and cultural traditions. For example, during the Pacific War, while Japanese
women’s organizations were banned or forced to serve the military state, the war pro
vided opportunities for Chinese women to develop their organizations and movement for
liberating their country and themselves. Another example is from the middle of the 1960s
to the late 1970s; when Chinese women dealt with the challenges of the Cultural Revolu
tion and were cut off from the feminist movement in the West, the Japanese women were
aware of the movement and started to launch a feminist movement of their own. Challenge number three: how to teach—pedagogyh Both the weekly reading reflective papers and the final research paper are useful
materials for assessing the learning outcomes of the course. In teaching the course I realize that it is easy for many students to unconsciously fall
into the trap of false gender universalism by using their own twenty-first century Western-
gendered glasses to view women in Chinese and Japanese history. By measuring women in
Japan and China with a yardstick determined by life in the United States in the twenty-first
century, it is easy for students to jump into a prematured and asymmetric comparison and
conclusion that women in Japan and China in the nineteenth and twentieth centuries are
much worse off than women in the twenty-first century United States. One of the male students from my course stated the following in his weekly short essay:
“In a way, I feel as if my original views on the status of Japanese women in their nation
and society are gradually being worn away, bit by bit. When I first began reading about the
stigmas, biases, means of discrimination, strict cultural expectations, and other such woes 69 | Teaching the History of Women in China and Japan ASIANetwork Exchange | Spring 2014 | volume 21 | 2 that Japanese women have had to and continue to endure, I more or less had a knee-jerk
reaction in which I wanted to more or less brand the whole society as a living trap of sexism
from my personal point view. However, according to the week’s readings, it would appear
that yet again, I was extraordinarily hasty to dole out my over-generalizations borne out my
strong emotional reaction. 70 | Teaching the History of Women in China and Japan Challenge number three: how to teach—pedagogyh Thus it is
important for students to know that in studying women in Chinese and Japanese history,
a multicultural and historical approach to women’s studies is necessary. It is important to
locate women’s history within a society’s national history as well as to describe its role in an
integrated history of East Asia. When we teach about East Asia, we need to strike a balance
between recognizing the region at large as an organizing unit in our teaching and drawing
attention to the individual East Asian countries’ unique identities and cultural traditions. Gender theories and feminism are not just scholarly engagements; they are also aimed
at promoting activism. One of the objectives of teaching a gender/women course is to help
students link classroom learning with activism for diversity. Richard Arum and Josipa
Roksa in their 2011 study point out that in the twenty-first century extracurricular activi
ties are an integral part of American undergraduate student life and play an important role
in shaping campus culture in many colleges (Arum and Roksa 2011, 3). Taking advantage
of this reality, last year when I taught the course on women in Japan and China, I chal
lenged students to connect our classroom learning with the real world of our everyday life. The students took the challenge and made the connection through a class project. After a 70 | Teaching the History of Women in China and Japan ASIANetwork Exchange | Spring 2014 | volume 21 | 2 brainstorming session, they decided to stage three mock Chinese weddings on campus to
promote diversity and cultural awareness of China. In the course, marriage is introduced as an institution that is vital in understanding a
society; wedding ceremonies symbolize and showcase a society’s values and rituals. The
mock weddings project provides a great opportunity for the students and the onlookers to
learn about and understand the importance of marriage and wedding in the construction
of womanhood, family, and gender relations, and in reproducing class structures in China
from the late imperial times to the current economic reforms. The students acted out three Chinese weddings: a traditional wedding, a revolution
ary wedding presumably during the Maoist era, and a post-Mao wedding of the economic
reform era. Challenge number three: how to teach—pedagogyh They staged a prewedding negotiation between the groom’s and bride’s families
to show the audience that marriage in traditional Chinese society was not just the union of
the young people who were getting married, but the union of the two families. They also
paraded the wedding dowries to show off the economic and social standings of the groom’s
and the bride’s families in the local community. During the ceremonies, a narrator told
the audience the historical background of the weddings, the meaning of each act, and the
change and continuities of the ceremonies from the traditional to the Maoist revolutionary
and the economic reform era as a result of the change and lack of change in China’s political
and social structures. During the course of preparing for the event, we not only learned a great deal about Chi
nese marriage and wedding culture, but also established our class as a learning community
which went beyond the classroom. Since this project was included as part of the Multicul
tural Monday events, we worked closely with the University Activities and Student Diversity
Programs Offices. The students wrote a grant proposal and got funding from a foundation
for the event. Throughout the preparation process the students not only learned organiza
tional skills and grant proposal writing, but also contributed to the promotion of diversity
and cultural awareness on campus. This project made the students realize that learning
about East Asia can be fun and meaningful to their everyday life. It provided them with an
opportunity to connect academic learning with the promotion of activism and multicultur
alism on campus and encouraged them to consider that as global citizens, they could think
globally but act locally. 71 | Teaching the History of Women in China and Japan rEFERENCES Arum, Richard, and Josipa Roksa. 2011. Academically Adrift: Limited Learning on College Campuses, Chicago: The
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y
Mow, Shirley, Tao Jie, and Zheng Bijun, eds. 2004. Holding up Half the Sky: Chinese Women, the Past, Present, and
Future. New York: the Feminist Press. Ogasawara, Yuko. 1998. Office Ladies and Salaried Men. Berkeley: University of California Press. Pruitt, Ida. 1945. A Daughter of Han: the Autobiography of a Chinese Working Woman. New Haven: Yale University
Press. Raicho Hiratsuka and Teruko Craig. 2010. In the Beginning, Woman Was the Sun: the Autobiography of a Japanese
Feminist. New York: Columbia University Press.hhh Wang Shou. 2006. “The ‘New Social History’ in China: The Development of Women’s History.” The History Teacher
39, no. 3: 315-323.f Wasserstrom, Jeffrey N., and Susan Brownell, ed. 2002. Chinese Femininities, Chinese Masculinities: A Reader. Wasserstrom, Jeffrey N., and Susan Brownell, ed. 2002. Chinese Femininities, Chinese Masculinities: A Reader. Berkeley: University of California Press. Wasserstrom, Jeffrey N., and Susan Brownell, ed. 2002. Chinese Femininities, Chinese Masculinities: A Reader. Berkeley: University of California Press. Yamazaki, Tomoko, and Karen Colligan-Taylor. 1998. Sandakan Brothel #8: An Episode in the History of Lower-class
Japanese Women Armonk: M E Sharpe Japanese Women. Armonk: M.E. Sharpe.h Yue Daiyun. 1985. To the Storm: The Odyssey of A Revolutionary Chinese Women. Berkeley: University of California
Press. Zhong, Xueping, Wang Zheng, Bai Di, eds. 2001. Some of Us: Chinese Women Growing Up in the Mao Era. New
Brunswick: Rutgers University Press. Conclusion Anyone teaching the history of women in China and Japan in America faces chal
lenges of how to select balanced course materials and introduce women-related theoretical
perspectives that include the voices of Chinese and Japanese women. I believe that with
carefully selected course materials, we can help American students hear Chinese and Japa
nese women’s voices, which will not only enable students to better understand the history
of these two countries, but also enrich the existing feminist and women’s studies theories
and practices in the U.S. A challenge of teaching Asian histories in the United States today is
that it is difficult to make a connection between the subject matter and American students’
everyday life. An East Asian women’s history course can employ feminist activism and an
active pedagogy to make such a connection. The study of Chinese and Japanese women’s
history may engage students critically with the renegotiation of gender theories and realities
in the East Asian countries under study, in particular acknowledging the common usage of
Western theories as a unitary standard for women’s histories elsewhere in the world despite
their development in the historical particulars of Western experience. Teaching the history
of women in China and Japan will allow us to move beyond the conventional “area studies” 71 | Teaching the History of Women in China and Japan ASIANetwork Exchange | Spring 2014 | volume 21 | 2 and the nation-state boundaries, to reimagine old narratives and introduce students to new
methods of understanding themes of both universalism and diversity within East Asian
history. 72 | Teaching the History of Women in China and Japan 73 | Teaching the History of Women in China and Japan Films Cited Black Rain. Directed by Shohei Imamura et al. 1989; Wilmington, NC: AnimEigo, 2009. DVD. Rain. Directed by Shohei Imamura et al. 1989; Wilmington, N China Blue. Directed by Micha Peled. 2005; Oley, PA: Bullfrog Films, 2005. DVD.h Chinese Foot Binding: The Vanishing Lotus. Directed by Fung Wing Chuen Tely. 2000; New York: Filmakers Library,
2004. DVD.i Faces of Japan: Woman Soldier. Directed by Dick Cavett, et al. 1988; New York: Pacific Mountain Network, 1988. VHS Faces of Japan: Woman Soldier. Directed by Dick Cavett, et al. 1988; New York: Pacific Mountain Network, 1988. VHS.̄ Granny Gabai. Directed by Hitoshi Kurauchi. 2006; Tōei Bideo, 2006. DVD. Heart of the Dragon: Marrying. Directed by Mischa Scorer et al. 1985; MediaOutlet.com, 2012. DVD. 72 | Teaching the History of Women in China and Japan ASIANetwork Exchange | Spring 2014 | volume 21 | 2 Japanese Woman. Directed by Kalle Lasn. 1984; Irwindale, CA: Barr Films, 1984. VHS. u. Directed by Zhang Yimou. 1990; Studio City, CA: Razor Dig Ju Dou. Directed by Zhang Yimou. 1990; Studio City, CA: Razor Digital Entertainment, 2005. DVD. Kabei: Our Mother. Directed by Yoji Yamada. 2008; Culver City, CA: Strand Home Video, 2009. DVD. The Last Train Home. Directed by Lixin Fan. 2009; Zeitgeist Films, 2011. DVD. h
Mother’s Way, Daughter’s Choice. Directed by Kyoko Gasha. 2009; New York: Filmakers Library, 2011. DVD. Not One Less. Directed by Zhang Yimou. 1999; Culver City, CA: Columbia TriStar Home Entertainment, 2000. DVD. Nu Shu: A Hidden Language of Women in China. Directed by Yue-Qing Yang. 1997; New York: Women Make Mov
ies, 1999. DVD. Raise the Red Lantern. Directed by Zhang Yimou. 1991; Beverly Hills, CA: Twentieth Century Fox Home Enter
tainment, 2007. DVD. The Road Home. Directed by Zhang Yimou. 1999; Culver City, CA: Columbia TriStar Home Entertainment, 2001. DVD. Sandakan No. 8. Directed by Kei Kumai. 1974; Video Action, 1986. VHS. Small Happiness. Directed by Carma Hinton and Richard Gordon. 1984; New York: New Day Films, 1990. VHS.h Happiness. Directed by Carma Hinton and Richard Gordon. 19 The Story of Qiu Ju. Directed by Zhang Yimou. 1992; Culver City, CA: Sony Pictures Home Entertainment, 2006. h
Street of Shame. Directed by Kenji Mizoguchi. 1956; Irvington, NY: Criterion Collection, 2008. DVD. Twenty-four Eyes. Directed by Keisuke Kinoshita. 1954; Irvington, NY: Criterion Collection, 2008. DVD.h The Woman in the Dunes. Directed by Hiroshi Teshigahara. 1964; Irvington, NY: Criterion Collection, 2007. Films Cited DVD Women in China: Up Against the Wall. Directed by Laurie Layton Schapira. 1998; Derry, NH: Chip Taylor Com
munications, 1998. DVD.h A World Without Fathers or Husbands. Directed by Eric Blavier and Thomas Lavachery. 2000; New York: Filmakers
Library, 2001. DVD. Book Review: A Guide to Asian Philosophy Classics Puqun Li (with Arthur K. Ling), A Guide to Asian Philosophy Classics (Broadview
Publishers, Peterborough, Ontario and Buffalo, New York, 2012)
ISBN: 978-1-55481-034-5 Puqun Li has provided a helpful entryway for newcomers into Asian philosophy. He has
done so not by resorting to what are always doomed to be indefensible generalities about
this vast and philosophically complex group of cultures, but rather by prudently and help
fully providing cogent readings of ten Indian, Chinese, and Japanese classics. h The book begins with a consideration of the Indian heritage. Hinduism is introduced
by an analysis of the Upanishads. Unfortunately, Hinduism only gets a single chapter and
the Bhagavad-Gita, for example, does not receive substantial commentary. Indian Bud
dhism fares better with a consideration of both its early expression and its refashioning as
the Mahayana (or Great Vehicle). Early Theravada Buddhism is discussed by taking up the
Dhammapada, and Mahayana is introduced through Nagarjuna’s Madyamaka or Middle
Way as Li takes on the famously vexing Mulamadyamakakarika (The Fundamental Verses on
the Middle Way). Li examines select arguments from Garfield’s edition of this text, including
a helpful discussion of Nagarjuna’s critique of substantial accounts of time. Li then turns to China, which forms the majority of the book. Confucian philosophy is
presented through an explication of the Analects, the Mengzi, and finally the Xunzi, which
defends Confucian philosophy while adapting elements of rival schools. Li also discusses
early philosophical Daoism by analyzing this tradition’s two most famous and celebrated
works, the Daodejing and the Zhuangzi. Li finishes his consideration of China by turning to
Chan Buddhism. Using the Dun-huang version of the manuscript made famous in the six
ties by Philip Yampolsky, Li takes up the Platform Sutra, historically attributed to Hui Neng,
but really the work of multiple authors. China receives three-fifths of the attention here, and
Li’s discussions of Chinese texts are the book’s best chapters, his readings enhanced by clear
and accessible analyses of some of the original Chinese terms and phrases. Li concludes by turning to Dogen Zenji’s Kamakura period Soto Zen masterpiece, the
Shobogenzo (Treasury of the True Dharma Eye). The entirety of this complex and interlock
ing collection of fascicles is longer then all of the other works that Li considers in this book
combined, so he wisely only concentrates on a few emblematic fascicles that he takes from
Waddell and Abe’s The Heart of Dogen’s Shobogenzo. Notes 1. http://lists.csustan.edu/pipermail/facnet-l/2010-January/002138.html (accessed May 3, 2013) p://jguide.stanford.edu, http://www.usc.cuhk.edu.hk, http://ww 2. http://jguide.stanford.edu, http://www.usc.cuhk.edu.hk, http://www.sino.uni-heidelberg.de/wo p
y
y
4. Geoffrey Staysniak’s weekly reflective essay for HI 366, Women in China and Japan, 2010. 5
Eli
b h M ll ’
kl
fl
i
f
HI 366 W
i Chi
d J
2010 4. Geoffrey Staysniak’s weekly reflective essay for HI 366, Women in China and Japan, ey Staysniak’s weekly reflective essay for HI 366, Women in Chi 5. Elisabeth Muller’s weekly reflective essay for HI 366, Women in China and Japan, 2010. ASIANetwork Exchange | Spring 2014 | volume 21 | 2 74 | Book Review: A Guide to Asian Philosophy Classics Book Review: A Guide to Asian Philosophy Classics Li considers Genjokoan (“Manifesting
Suchness”) as well as the famous discussion of the nonseparation of being and time (Uji),
Shoji, Dogen’s consideration of the great matter of living and dying, and Bussho, Dogen’s
meditation on the problem of Buddha nature. fi Li’s analyses, written as accessibly as such difficult material allows, may be of interest to
general readers who are trying to find their way into these traditions, but his target audience
seems to be undergraduate philosophy students working at a largely introductory level. Li
keeps discussions of the scholarly debates around these texts to a minimum (although he is
always sure to mention the most salient issues). Moreover, Li has inserted many features that one normally expects to find in the more 74 | Book Review: A Guide to Asian Philosophy Classics ASIANetwork Exchange | Spring 2014 | volume 21 | 2 traditional textbook format. Each chapter is linked to a suggested primary source, namely
the translation and passages of the work that Li will be discussing. Li then provides “learn
ing objectives.” For example, if you make it through Li’s chapter on the Platform Sutra,
you will be able to 1) “describe the general features of Chinese Chan Buddhism” as well
as, among other objectives, be able to “compare and evaluate Shen Xiu’s ‘gradual awaken
ing’ and Hui Neng’s ‘sudden awakening’” (259). I am not sure what Hui Neng would have
thought about the transformation of the great question of awakening into a learning out
come. I personally lament the incursion of such bureaucratic compromises into the practice
of philosophy, but I also recognize that this battle may already be lost. For teachers hard
pressed to produce such rhetoric, Li’s objectives will be highly welcome. Li then provides a vocabulary list (“key words”) of the important critical terms for each
work, as well as questions that his own analysis will try to answer. For example, one of the
questions that guides Li’s analysis of the Platform Sutra is: “How did Indian Buddhism fuse
with the indigenous Chinese Confucianism and Daoism?” (260) Li also interpolates boxed
areas that either include terse and often helpful microreflections or provide sample writing
assignments and prompts. Li wisely avoids speaking in wild and indefensible generalities about Asian philosophy. Jason M. Wirth, Professor of Philoosphy, Seattle University. 75 | Book Review: A Guide to Asian Philosophy Classics Mission Statement The mission of the ASIANetwork Exchange: A Journal for Asian Studies in the Liberal Arts is
to highlight the central role of reflective research to teaching about Asian societies and cul
tures. The Exchange shares information useful to educators in liberal arts settings through
the publication of original research and media reviews in order to provide materials to assist
teachers in their own professional development and to deepen the understanding of Asia
among campus communities. Book Review: A Guide to Asian Philosophy Classics When he makes comparisons, they are between specific ideas and texts, and Li does so not
to catalogue coincidences and differences, but rather to “prompt dialogue, engagement, and
fresh learning” (xv). His textual analyses recreate the original philosophical context of the
work, demonstrate their inner coherence, and judiciously bring select ideas into dialogue
with select Western philosophical ideas (xiii). Li also incorporates some very helpful appendices, including some handy tips on pinyin
pronunciation, a glossary of key terms, some useful websites and journals, and even some
suggestions of programs for graduate study in Chinese philosophy. While Li’s analysis helps students appreciate some of the vast philosophical range of
Asian philosophy, he also draws three attractive conclusions about how these works can
help us live a “human life.” Although the accounts of the self vary, none of these texts argue
that an “ego-centered” life will help us flourish (312). Rather, to live well, one must become
present to every moment and, finally, although teachers may help, every serious negotiator
of the Way must learn to stand on her or his own two feet (313). Li leans heavily toward China, but he also gives each of the ten included texts an appre
ciative and lucid reading. As such, this is a helpful guide for introductory students to enter
the extraordinary world of these philosophical treasures. ASIANetwork Exchange | Spring 2014 | volume 21 | 2 Requirements for Manuscript Submission When preparing a manuscript, please note that the ASIANetwork Exchange: A Journal for
Asian Studies in the Liberal Arts is committed to publishing current research, as well as
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Use parenthetical in-text citation, and author-date formatting for references per Chi
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ASIANetwork Exchange | Spring 2014 | volume 21 | 2
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The Role of the Community in Efforts to Reduce the Prevalence of Stunting in Kembang Village, Pacitan Regency, East Java
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Dinamisia
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Abstrak Angka prevalensi stunting di Pacitan masih menyentuh angka 22,77 persen. Tentunya, angka ini
masih terbilang tinggi, jauh dari target dari WHO, yakni di bawah 20 persen. Kenyataan ini menjadi
dorongan masyarakat Desa Kembang Kabupaten Pacitan untuk melakukan kegiatan atau upaya menekan
angka prevalensi stunting. Bentuk kegiatan yang dilakukan ialah pertama, mengadakan seminar
pencegahan stunting, di mana menghadirkan narasumber yang kompeten seperti Ir. Kuriyah, M.M selaku
perwakilan dari PPKB dan P3A, Kemudian Khuzaini dari Dinas Kesehatan dan juga Yuli Rahmawati Mutiah,
M.A dari University Of Warith Al Anbiya. Kedua, mengadakan penyuluhan mengenai stunting pada ibu hamil
dan menyusui dengan melibatkan Bidan Desa, Kader Posyandu, dan juga Dokter dari Puskesmas Pacitan. Ketiga, memberikan makanan tambahan yang seimbang dan bergizi yang kemudian kemudian hasil dari
olahan makanan sehat tersebut dibagikan kepada anak-anak dari masyarakat Desa Kembang. Upaya-upaya
yang dilakukan berjalan dengan baik dan lancar. Hal ini disebabkan oleh dukungan dari berbagai pihak
yang turut serta mensukseskan kegiatan yang dilakukan terutama masyarakat Desa Kembang. Kata Kunci: Peran, Prevelensi, Stunting, Desa Kembang Abstract
T The stunting prevalence rate in Pacitan still stands at 22.77 per cent. Of course, this figure is still
relatively high, far from the target from WHO, below 20 per cent. This fact has encouraged the Kembang
Village, Pacitan Regency community to carry out activities or efforts to reduce the prevalence of stunting. First, the form of activities is holding a stunting prevention seminar, where competent speakers such as Ir. Kuriyah, M.M as a representative of PPKB and P3A, then Khuzaini from the Health Office and Yuli Rahmawati
Mutiah, M.A from the University Of Warith Al Anbiya. It was second, conducting counselling on stunting in
pregnant and lactating women by involving Village Midwives, Posyandu Cadres, and Doctors from Pacitan
Health Center. Third, providing balanced and nutritious supplementary food and then distributing the results
of processed healthy food to children from the Kembang Village community. The efforts made went well and
smoothly. It's due to the support of various parties who participated in the activities' success, especially the
people of Kembang Village. Vol. 7, No. 4 Agustus 2023, Hal. 919-929
DOI: https://doi.org/10.31849/dinamisia.v7i4.15197 Vol. 7, No. 4 Agustus 2023, Hal. 919-929
DOI: https://doi.org/10.31849/dinamisia.v7i4.15197 DINAMISIA: Jurnal Pengabdian Kepada Masyarakat Peran Masyarakat dalam Upaya Menekan Angka Prevalensi Stunting di
Desa Kembang Kabupaten Pacitan Jawa Timur Rizal Al Hamid*1, Muhammad Fathun Niam2
1,2 Universitas Islam Negeri Sunan Kalijaga Yogyakarta
E-mail co author: rizal.alhamid@uin-suka.ac.id 1. PENDAHULUAN Stunting telah menjadi masalah nasional sejak lama dan menjadi tantangan tersendiri
untuk mencetak generasi berkualitas.(Rahmadhita, 2020) Bukan hanya itu, permasalahan gizi
ini telah menjadi masalah global.(Handayani, Putri, & Dewi, 2022) Sebagaimana yang tercatat
oleh Global Nutrition Report pada tahun 2018, terdapat lebih dari 150 juta balita di dunia yang
teridentifikasi stunted.(Juwita, Andayani, Bakhtiar, Sofia, & Anidar, 2019) Berbagai upaya
pemerintah dilakukan, di mana permasalahan gizi akan sangat berdampak pada kualitas aset
manusia atau sumber daya manusia di masa depan nanti.(Rahmawati et al., 2020) Tentunya,
pemerintah dengan segala upaya menjadikan masalah gizi ini sebagai prioritas(Boli, Baliwati, &
Sukandar, 2018), bahkan dengan target lima tahun bebas stunting, guna meraih bonus
demografi pada tahun 2045 nanti.(Oktavianty, Affrian, Kusbandrijo, & Rochim, 2023) Bukan P-ISSN 2614-7424 | E-ISSN 2614-8927 919 DINAMISIA: Jurnal Pengabdian Kepada Masyarakat Vol. 7, No. 4 Agustus 2023, Hal. 919-929
DOI: https://doi.org/10.31849/dinamisia.v7i4.15197 hanya itu, tentu juga untuk meningkatkan kualitas SDM Indonesia sebagai upaya peningkatan
taraf ekonomi.(N. Hidayah & Marwan, 2020) Jika generasi saat ini lahir dalam keadaan stunting,
maka bonus demografi akan hanya menjadi wacana saja.(Ngasuko, 2019) Di mana seharusnya
Indonesia akan didominasi oleh generasi produktif, tetapi karena stunting, generasi-generasi
kita akan tidak berkualitas. Yang mana, hal ini disebabkan oleh masalah gizi: salah satunya
adalah stunting.(Malik, Utsman, Mulyono, Arbarini, & Desmawati, 2021) hanya itu, tentu juga untuk meningkatkan kualitas SDM Indonesia sebagai upaya peningkatan
taraf ekonomi.(N. Hidayah & Marwan, 2020) Jika generasi saat ini lahir dalam keadaan stunting,
maka bonus demografi akan hanya menjadi wacana saja.(Ngasuko, 2019) Di mana seharusnya
Indonesia akan didominasi oleh generasi produktif, tetapi karena stunting, generasi-generasi
kita akan tidak berkualitas. Yang mana, hal ini disebabkan oleh masalah gizi: salah satunya
adalah stunting.(Malik, Utsman, Mulyono, Arbarini, & Desmawati, 2021) Dari hasil Survei Status Gizi Indonesia, kasus stunting di Indonesia saat ini 2021,
prevalensi stunting menyentuh angka 24,4%,(Gita, Surya, & Setyaningsih, 2023) Angka tersebut
telah mengalami penurunan yang cukup signifikan dari 37,2% di tahun 2013. 1. PENDAHULUAN Kemudian pada
tahun 2016 presentase tersebut turun menjadi 27,5%,(Aryastami & Tarigan, 2017) tetapi angka
itu belum sesuai dengan batas wajar yang diberikan oleh WHO (World Healt Organization),
yakni di bawah 20%.(Zulyanies & Palupi, 2021)Upaya pemerintah dalam menangani stunting ini
cukup perlu diperhatikan, bukan hanya frekuensi, intensi dari pengadaan sosialisasi stunting,
pengadaan fasilitas, dan pemerataan pembangunan, tetapi juga perlu memperhatikan
keakurasian pemerintah sendiri dalam pengadaan, baik penyuluhan maupun bantuan.(Saputri,
2019) Di tengah masyarakat kita sendiri, telah kita temui banyak kasus ketidakpahaman orang
tua maupun generasi tua dalam memperhatikan buah hati mereka(Sugitanata & Ilyas, 2023). Sebagai contoh sederhana, yakni: orang tua yang memberikan anaknya (umur 6 bulan) air gula
sebagai pengganti ASI.(D. I. Hidayah, 2022) Adapun kasus kematian bayi sebab keteledoran
orang tua dalam pemberian makanan berupa nasi, di mana usia 6 bulan bukan usia yang baik
untuk diberi makan selain ASI ekslusif.(Maghfiroh & Laksono, 2020) Dalam dua kasus tersebut,
bisa diperhatikan bagaimana para orang tua tidak memahami pentingnya memperhatikan gizi
buah hati. Terlebih masa yang paling perlu diperhatikan, atau masa emas anak, yakni masa yang
mampu menentukan kualitas seorang anak adalah pada 1000 HPK (1000 Hari Pertama
Kehidupan).(Marni & Ratnasari, 2021) Di mana, pada masa-masa ini, seorang anak dapat
ditentukan menjadi anak yang berkualitas dengan pemberian gizi yang baik, atau menumbuhkan
anak yang tidak berkualitas sebab dari kekurangam gizi, atau kurangnya perhatian atas
psikososial, nutrisi dan gizi, kebersihan. Jika hal tersebut tidak diperhatikan, baik nutrisi ibu
yang mengandung maupun ketercukupan nutrisi dan gisi anak yang lahir maka, akan terlahir
bayi yang stunted, atau balita pendek, atau keterlambatan.(Ruaida, 2018) Masalah stunting
menjadi problem serius yang harus dihadapi oleh pemerintahan kabupaten Pacitan, Jawa
Timur.(Sutopo, 2021) Tertanggal 23 Agustus 2022, Kabupaten Pacitan memperoleh 2744 kasus
bayi stunted.(Wibowo, 2022) Dikutiip dari Radar Madiun, pada 17 Juni 2022, angka prevalensi
stunting di Pacitan masih menyentuh angka 22,77 persen.(Mediun, 2022) Tentunya, angka ini
masih terbilang tinggi, jauh dari target dari WHO, yakni di bawah 20 persen. Beberapa penelitian telah melakukan kajian mengenai stunting seperti Khoirun Ni’mah
dan Siti Rahayu Nadhiroh(Ni’mah & Nadhiroh, 2015) mengungkapkan bahwa salah satu faktor
yang mampu mempengaruhi terjadinya balita stunting adalah pendidikan sang ibu, yang
berimplikasi pada pengetahuan yang dimilikinya. Sementara tidak dengan pendidikan seorang
ayah, yang rupanya tidak memiliki pengaruh langsung atas kejadian balita stunting. Dalam
penelitian yang dilakukan oleh Khusnul Khotimah(Khotimah, 2022) stunting mampu
mempengaruhi pendapatan negara secara signifikan. 1. PENDAHULUAN Misal dari penelitian di Albania yang
menunjukkan kerugian negara yang diterima karena kasus stunting adalah sebesar 50%
(Khotimah, 2022). Sebuah penelitian yang dilaksanakan oleh Irmaida(Irmaida, Briawan, &
Martianto, 2021) menunjukkan bahwa dampak dari stunting ini akan sangat berdampak pada
penghasilan seumur hidup sebesar 10%, dan menyebabkan kemiskinan antargenerasi. Begitu P-ISSN 2614-7424 | E-ISSN 2614-8927 920 DINAMISIA: Jurnal Pengabdian Kepada Masyarakat Vol. 7, No. 4 Agustus 2023, Hal. 919-929
DOI: https://doi.org/10.31849/dinamisia.v7i4.15197 juga dengan berkurangnya SDM (Sumber Daya Manusia) berkualitas, dan produktif, maka akan
berdampak pada Produk Domestik Bruto (Gross Domestic Product). Di mana sebab dari turunnya
kualitas SDM kita, maka akan menurunkan nilai dari produk dan jasa.(Adam & Mas’ud, 2022)
Begitu juga dengan penelitian yang dilakukan oleh Brigitte Sarah Renyoet(Renyoet, Martianto, &
Sukandar, 2016) yang menunjukkan bahwa kerugian negara yang ditanggung dari akibat
masalah penurunan produktivitas masyarakatnya adalah sekitar 3.057 miliar rupiah hingga
13.758 miliar rupiah dari total PDB (Produk Domestik Bruto) Indonesia. Penelitian yang dilakukan oleh Rini Archda Saputri(Saputri, 2019)yang berfokus pada
kejadian dan upaya penanggulangan kasus bayi stunted di provinsi Bangka Belitung, di mana,
pemerintah sendiri telah memberi fasilitas air bersih, sanitasi, jaminan kesehatan, dan
pendidikan untuk orang tua serta pendidikan di usia dini. Namun, bukan hanya itu, pemerintah
daerah secara inisiatif memberikan layaan dan program-progam guna menekan angka
prevalensi bayi stunted, misal: pemberian makanan tambahan untuk balita dan ibu hamil,
pemberian tablet tambah darah untuk remaja putri dan ibu hamil sebagai langkah dalam pra-
konsepsi, peningkatan cakupan imunisasi dasar lengkap pada bayi dan balita, pemberian
vitamin A, pemberian zinc pada pengidap diare ibu hamil dan balita. Sebagaimana fakta besarnya kasus stunting di Pacitan, rupanya rasa awareness, peduli dari
masyarakat terhadap masalah stunting masih terbilang kurang.(Mediun, 2022) Terlebih bangsa
Indonesia akan mendapatkan bonus demografi di tahun 2045, yang tentunya akan memberi
peluang Indonesia menjadi negara yang produktif.(Gunardi, 2021) Namun, bagaimana jika
pemuda-pemuda yang nanti akan memimpin negeri memiliki kualitas yang buruk sebab
stunting? Oleh karena itu, kegiatan pengabdian ini mengajak masyarakat untuk berperan dalam
mencegah stunting dengan beberapa indikator yang dicapai yakni menjelaskan dampak yang
diakibatkan oleh masalah gizi berupa stunting? Selanjutnya mendiskripsikan kasus stunting di
Desa Kembang, Kabupaten Pacitan, provinsi Jawa Timur? Kemudian memaparkan peran
masyarakat dalam upaya menekan angka prevalensi stunting di Desa Kembang Kabupaten
Pacitan Jawa Timur. A. Potret Umum Tentang Stunting Stunting suatu kondisi gagal tumbuh yang dialimi bayi dari akibat kekurangan gizi
kronis.(Hamalding, Said, & Nurmiati, 2020) Di mana stunting atau juga dikenal sebagai
kejadian kerdil atau pendek adalah ketika balita memiliki tinggi badan yang lebih kecil
atau kurang jika dibandingkan dengan umur.(Mantasia & Sumarmi, 2022) Oleh karenanya,
balita yang terindikasi stunted akan memiliki tinggi yang abnormal dari ukuran bayi
normal.(Nasution & Susilawati, 2023) Hal ini dapat diukur dengan menggunaka standar
deviasi (SD) dari WHO (World Health Organization), jika balita memiliki ukuran -2
SD(Anzar, 2019), maka diindikasikan stunted, demikian menurut Peraturan Menteri
Kesehatan Nomor 20 tahun 2020. Adapun pemahaman di tengah masyarakat, bahwa
tinggi badan dipengaruhi oleh gen, sementara menurut hasil sebuah studi menunjukkan
bahwa pengaruh gen terhadap tinggi badan tidak melebihi angka 15%. Faktor terbesar
dalam menentukan baik tidaknya tumbuh kembang anak adalah asupan zat gizi, hormon
pertumbuhan, beserta penyakit infeksi berulang.(Rahayu, Yulidasari, Putri, & Rahman,
2015) Secara umum terdapat beberapa faktor yang dapat menyebabkan bayi stunted,
diantaranya adalah: panjang badan bayi saat lahir yang pendek, asupa gizi, kurangnya
pengetahuan ibu tentang gizi, pendidikan ibu yang rendah yang akan berpengaruh bahka
pada masa pra-kosepsi, dan pendapatan keluarga yang cenderung rendah.(Adam &
Mas’ud, 2022) Faktor-faktor tersebut sedikit banyak mempunyai hubungan yang saling
berkaitan dengan kejadian stunting pada balita.(Putra, Anggraini, & Putri, 2023) 2. METODE Berdasakan penjabaran masalah yang dijabarkan. Upaya-upaya yang dilakukan masyarakat
Desa Kembang dalam menekan angka prevalensi stunting menggunakan beberapa tahapan
kegiatan yakni: a. Mengadakan Seminar Pencegahan Stunting
Seminar dilakukan sebagai bentuk untuk memberikan suatu pemahaman terhadap
masyarakat mengenai stunting. Dalam pelaksanaan seminar, turut dihadirkan narasumber
dari Dinas PPKB dan P3A, Dinas Kesehatan dan akademisi dari University Of Warith Al
Anbiya. Masyarakat yang hadir dalam penyuluhan ini ialah masyarakat dari Desa Kembang,
Kabupaten Pacitan Jawa Timur. b. Mengadakan Penyuluhan Mengenai Stunting Pada Ibu Hamil dan Menyusui
Guna mengoptimalkan penyampaian dan penyebaran informasi mengenai stunting. Penyuluhan mengenai stunting terhadap ibu hamil dan menyusui dilakukan di Posyandu
yang ada di Dusun Bubakan Desa Kembang dengan melibatkan Bidan Desa, Kader
Posyandu, dan juga Dokter dari Puskesmas Pacitan. c. Melakukan Pengolahan Makanan Yang Bergizi Dan Seimbang
Kegiatan pemberian dan pengolahan makanan yang seimbang dan bergizi dilakukan
menggunkan olahan pangan lokal yang terbuat dari ikan. Kegiatan pemberian dan pengolahan makanan yang seimbang dan bergizi dilakukan
menggunkan olahan pangan lokal yang terbuat dari ikan. P-ISSN 2614-7424 | E-ISSN 2614-8927 921 Vol. 7, No. 4 Agustus 2023, Hal. 919-929
DOI: https://doi.org/10.31849/dinamisia.v7i4.15197 Vol. 7, No. 4 Agustus 2023, Hal. 919-929 DINAMISIA: Jurnal Pengabdian Kepada Masyarakat B. Pemetaan Temuan Kasus dan Faktor Yang Melatarbelakangi Stunting di Desa
Kembang Kecamatan Pacitan Desa Kembang merupakan bagian wilayah dari Kabupaten Pacitan di Jawa
Timur,(“Https://Kembang.Kabpacitan.Id,” 2023) di mana wilayahnya terdiri dari sebagian
besar berupa daratan rendah yang ada di Teluk Pacitan. Kemudian juga dikelilingi dengan
perbukitan dan pegunungan. Desa Kembang terdiri dari 5 Dusun yang di mana terdapat
Dusun Krajan yang menjadi pusat dari pemerintahan Desa, selanjutnya Dusun Sedayu,
Dusun Karang, Dusun Bubakan dan Dusun Kiteran yang merupakan wilayah terluas dari
Desa Kembang.(“Arsip Profil Desa Kembang,” 2023) Tingkat pendidikan dari penduduk Desa Kembang masih didominasi oleh tamatan
SD sederajat dengan jumlah sebanyak 710 orang. Disusul dengan tingkat pendidikan yang
paling minim atau sedikit yakni tamatan S1 dan S2 yang peneliti dapatkan datanya pada
tahun 2019 sekitar 5 orang. (“Arsip Profil Desa Kembang,” 2023) Wilayah Desa Kembang yang berada di pesisir menjadikan masyarakat atau
penduduk Desa Kembang banyak bertumpu atau menjadikan mata pencahariannya
sebagai nelayan. Kemudian sebagian lainnya menjadi petani sebanyak 445 orang, buruh
tani sebanyak 364 orang, pekerja migran sebanyak 2 orang, PNS sebanyak 29 orang,
nelayan sebanyak 512 orang, TNI sebanyak 4 orang, POLRI sebanyak 2 orang, Dosen
swasta sebanyak 1 orang, pedagang sebanyak 3 orang, Asisten Rumah Tangga sebanyak 3
orang, karyawan swasta 426 orang dan karyawan pemerintah sebanyak 14 orang
kemudian terdapat juga pensiunan sebanyak 11 orang.(“Arsip Profil Desa Kembang,”
2023) P-ISSN 2614-7424 | E-ISSN 2614-8927 922 DINAMISIA: Jurnal Pengabdian Kepada Masyarakat Vol. 7, No. 4 Agustus 2023, Hal. 919-929 Vol. 7, No. 4 Agustus 2023, Hal. 919-929
DOI: https://doi.org/10.31849/dinamisia.v7i4.15197 Dari penjelasan singkat mengenai Desa Kembang di atas. Desa Kembang ternyata
masih menyisakan permasalahan yakni kasus stunting. Hal ini berdasarkan data temuan
yang peneliti lakukan dan peneliti gambarkan dalam bentuk tabel sebagai berikut: Dari penjelasan singkat mengenai Desa Kembang di atas. Desa Kembang ternyata
masih menyisakan permasalahan yakni kasus stunting. Hal ini berdasarkan data temuan
yang peneliti lakukan dan peneliti gambarkan dalam bentuk tabel sebagai berikut: Gambar 1. Temuan kasus stunting di Desam Kembang Gambar 1. Temuan kasus stunting di Desam Kembang Gambar 1. Temuan kasus stunting di Desam Kembang Sebagaimana dijelaskan sebelumnya bahwa hingga saat ini, masalah stunting
menjadi problem serius yang harus dihadapi oleh pemerintahan kabupaten Pacitan, Jawa
Timur. Tertanggal 23 Agustus 2022, kabupaten Pacitan memperoleh 2744 kasus bayi
stunted.(Wibowo, 2022) Dikutiip dari Radar Madiun, pada 17 Juni 2022, angka prevalensi
stunting di Pacitan masih menyentuh angka 22,77 persen.(Mediun, 2022) Tentunya, angka
ini masih terbilang tinggi, jauh dari target dari WHO, yakni di bawah 20 persen. C. Peran Masyarakat Desa Kembang Menekan Angka Prevalensi Stunting Di Desa
Kembang Kabupaten Pacitan Jawa Timur Upaya yang dilakukan masyarakat Desa Kembang dalam menekan angka prevalensi
stunting di Desa Kembang Kabupaten Pacitan Jawa Timur dapat peneliti jabarkan sebagai
berikut: B. Pemetaan Temuan Kasus dan Faktor Yang Melatarbelakangi Stunting di Desa
Kembang Kecamatan Pacitan Hal ini rupanya terjadi karena kurangnya kesadaran masyarakat terhadap
pentingnya memperhatikan kecukupan gizi, nutrisi, baik untuk anak maupun untuk ibu
hamil. Jayuk Susilaningtyas mengungkapkan, selaku kepala Dinas Pengendalian Penduduk
Keluarga Berencana Pemberdayaan Perempuan dan Perlindungan Anak, Pacitan, Di mana
kurangnya kesadaran masyarakat menjadi biang tingginya agka prevalensi stunting di
Pacitan.(“Wawancara Dengan Kepala Dinas Pengendalian Penduduk Keluarga Berencana
Pemberdayaan Perempuan Dan Perlindungan Anak, Pacitan,” 2023) Demikian itu juga disebabkan oleh permasalahan ekonomi, yang tentunya
merembet pada kurangnya nutrisi yang bisa diberikan kepada anak ataupun ibu
hamil.(Mediun, 2022) Hal ini juga dipertegas oleh dr Hendra Purwaka, kepala Dinas
Kesehatan kabupaten Pacitan, bahwa penyebab kejadian stunting ini adalah kurangnya
akses masyarakat terhadap asupan nutrisi, dan makanan bergizi yang hal ini juga
berkaitan dengan kondisi ekonomi masyarakat; asupan protein dan mineral, serta vitamin
yang dirasa masih kurang.(Wibowo, 2022) Bahkan, Dinas Kesehatan Pacitan memprediksi,
terdapat lebih dari sepuluh ribu bayi sekitar 14,76 persen yang terancam mengidap
stunting.(Mediun, 2022) P-ISSN 2614-7424 | E-ISSN 2614-8927 923 DINAMISIA: Jurnal Pengabdian Kepada Masyarakat Vol. 7, No. 4 Agustus 2023, Hal. 919-929 Vol. 7, No. 4 Agustus 2023, Hal. 919-929
DOI: https://doi.org/10.31849/dinamisia.v7i4.15197 1) Mengadakan Seminar Pencegahan Stunting 1) Mengadakan Seminar Pencegahan Stunting Semangat masyarakat Desa Kembang dalam meminimalisir kasus stunting
menjadi peran penting keberhasilan terhadap bebasnya kasus stunting. Hal ini
dibuktikan dengan mengadakan seminar pencegahan stunting dengan tema “Peran
Masyarakat Desa Kembang Menekan Angka Prevalensi Stunting Di Desa Kembang,
Kecamatan Pacitan, Kabupaten Pacitan, Provinsi Jawa Timur” yang dilakukan pada hari
senin tanggal 7 agustus 2023 di Balai Desa Kembang. Kegiatan ini turut menghadirkan
narasumber seperti Ir. Kuriyah, M.M selaku perwakilan dari PPKB dan P3A, Kemudian
Khuzaini dari Dinas Kesehatan dan juga Yuli Rahmawati Mutiah, M.A dari University Of
Warith Al Anbiya. Gambar 2. Dokumentasi dengan Narasumber Seminar Pencegahan Stunting
Gambar 3. Pemaparan Materi Seminar Oleh Narasumber Gambar 2. Dokumentasi dengan Narasumber Seminar Pencegahan Stunting Gambar 2. Dokumentasi dengan Narasumber Seminar Pencegahan Stunting Gambar 3. Pemaparan Materi Seminar Oleh Narasumber Gambar 3. Pemaparan Materi Seminar Oleh Narasumber Gambar 3. Pemaparan Materi Seminar Oleh Narasumber P-ISSN 2614-7424 | E-ISSN 2614-8927 924 DINAMISIA: Jurnal Pengabdian Kepada Masyarakat Vol. 7, No. 4 Agustus 2023, Hal. 919-929 Vol. 7, No. 4 Agustus 2023, Hal. 919-929
DOI: https://doi.org/10.31849/dinamisia.v7i4.15197 Gambar 5 dan 6. Peserta Seminar Pencegahan Stunting Gambar 5 dan 6. Peserta Seminar Pencegahan Stunting Indikator dari diadakannya seminar yang diadakan di Masyarakat Desa Kembang
dengan menghadirkan narasumber yang berkompeten dari bidangnya ialah memberikan
manfaat bagi masyarakat itu sendiri sehingga goldnya seperti yang disampaikan diawal
yakni bebas dari kasus stunting. 2) Mengadakan Penyuluhan Mengenai Stunting Pada Ibu Hamil dan Menyusui Foto Dokumentasi Pengolahan Makanan Yang Bergizi dan Seimbang
Gambar 10 dan 11. Penyerahan Hasil Olahan Makanan Yang Bergizi Dan Seimbang Vol. 7, No. 4 Agustus 2023, Hal. 919-929
DOI: https://doi.org/10.31849/dinamisia.v7i4.15197 Vol. 7, No. 4 Agustus 2023, Hal. 919-929
DOI: https://doi.org/10.31849/dinamisia.v7i4.15197 Vol. 7, No. 4 Agustus 2023, Hal. 919-929 DINAMISIA: Jurnal Pengabdian Kepada Masyarakat Gambar 9. Foto Dokumentasi Pengolahan Makanan Yang Bergizi dan Seimbang Gambar 9. Foto Dokumentasi Pengolahan Makanan Yang Bergizi dan Seimbang
Gambar 10 dan 11. Penyerahan Hasil Olahan Makanan Yang Bergizi Dan Seimbang
Gambar 12. Dokumentasi Dengan Salah Satu Kordinator Pengolahan Makanan Yang
Bergizi Dan Seimbang Gambar 9. Foto Dokumentasi Pengolahan Makanan Yang Bergizi dan Seimbang Gambar 10 dan 11. Penyerahan Hasil Olahan Makanan Yang Bergizi Dan Seimbang Gambar 10 dan 11. Penyerahan Hasil Olahan Makanan Yang Bergizi Dan Seimbang Gambar 12. Dokumentasi Dengan Salah Satu Kordinator Pengolahan Makanan Yang
Bergizi Dan Seimbang Gambar 12. Dokumentasi Dengan Salah Satu Kordinator Pengolahan Makanan Yang
Bergizi Dan Seimbang Gambar 12. Dokumentasi Dengan Salah Satu Kordinator Pengolahan Makanan Yang
Bergizi Dan Seimbang 2) Mengadakan Penyuluhan Mengenai Stunting Pada Ibu Hamil dan Menyusui Penyuluhan mengenai stunting terhadap ibu hamil dan menyusui dilakukan di
Posyandu yang ada di Dusun Bubakan Desa Kembang dengan melibatkan Bidan Desa,
Kader Posyandu, dan juga Dokter dari Puskesmas Pacitan. Kegiatan penyuluhan ini
dilakukan pada tanggal 5 Agustus 2023. Materi dari kegiatan penyuluhan ini ialah
menjelaskan apa itu stunting, kemudian bagimana ciri-ciri dari stunting, apa saja
dampak dari stunting, dan bagaimana upaya dalam mencegah terjadinya stunting. Gambar 7. Dokumentasi dengan Penyuluh dari Bidan Desa, Kader Posyandu dan Dokter
Puskesmas Pacitan. Gambar 7. Dokumentasi dengan Penyuluh dari Bidan Desa, Kader Posyandu dan Dokter
Puskesmas Pacitan. P-ISSN 2614-7424 | E-ISSN 2614-8927 925 DINAMISIA: Jurnal Pengabdian Kepada Masyarakat Vol. 7, No. 4 Agustus 2023, Hal. 919-929
DOI: https://doi.org/10.31849/dinamisia.v7i4.15197 Gambar 8. Kegiatan Penyuluhan dari Bidan Desa, Kader Posyandu dan Dokter Puskesmas
Pacitan. Gambar 8. Kegiatan Penyuluhan dari Bidan Desa, Kader Posyandu dan Dokter Puskesmas
Pacitan. Kegiatan penyuluhan mengenai stunting pada ibu hamil dan menyusui memiliki
tujuan utama yakni mencegah stunting. Sebagaimana tujuan dilakukan penyuluhan yang
dijelaskan diawal ialah menjelaskan apa itu stunting, kemudian bagimana ciri-ciri dari
stunting, apa saja dampak dari stunting, dan bagaimana upaya dalam mencegah
terjadinya stunting. Kemudian juga para narasumber yang berkompeten pada bidangnya
melakukan pengarahan dan penyerahan makanan-makanan yang memiliki kandungan
untuk mengatasi protein kronis dalam hal ini dengan menyasar ibu-ibu hamil dan juga
memberi makanan tambahan guna mengentaskan kurangnya energi ibu-ibu hamil
tersebut. Kemudian pihak penyuluh juga melakukan edukasi tentang bagaimana
memberikan makan pada bayi dan anak yang disarankan baik pada ibu hamil dan
khususnya pada ibu menyusui. Berikutnya memberikan edukasi dalam proses menyusui
dengan benar, dilanjutkan dengan edukasi dalam hal memberikan pendamping ASI pada
bayi yang berumur 6 hingga 24 Bulan. 3) Melakukan Pengolahan Makanan Yang Bergizi Dan Seimbang
Dalam memenuhi dan meningkatkan protein, melalui kesadaran masyarakat
Desa Kembang, maka diadakan kegiatan tata cara mengolah makanan yang sehat,
seimbang dan tentunya bergizi. Kegiatan ini dilaksanakan pada tanggal 5 agustus 2023. Kegiatan ini ditujukan untuk meningkatkan kesadaran masyarakat Desa Kembang
mengenai kebutuhan nutrisi dari anak-anak mereka. Di mana dalam pengolahan
makanan yang bergizi dan seimbang memanfaatkan keunggulan dari Desa yakni ikan
laut. Hal ini dikarenakan masyarakat Desa Kembang mayoritas mata pencahariannya
ialah sebagai nelayan. Hasil dari olahan yang dilakukan selama kegiatan berlangsung
kemudian dibagikan kepada anak-anak dari masyarakat Desa Kembang. P-ISSN 2614-7424 | E-ISSN 2614-8927 926 NAMISIA: Jurnal Pengabdian Kepada Masyarakat
Vol. 7, No. 4 Agustus 2023, Hal. 919-929
DOI: https://doi.org/10.31849/dinamisia.v7i4.15197
Gambar 9. DAFTAR PUSTAKA Adam, A., & Mas’ud, H. (2022). Komunikasi Perubahan Perilaku"Cegah Stunting Itu Penting"
Melalui Pemanfaatan E-Modul. Jurnal Pengabdian Masyarakat Sasambo, 3(2), 28–36. Anzar, J. (2019). Nutrisi Untuk Stunting. Conferences of Medical Sciences Dies Natalis Faculty of
Medicine Universitas Sriwijaya, 1(1). Arsip Profil Desa Kembang. (2023). Aryastami, N. K., & Tarigan, I. (2017). Kajian Kebijakan dan Penanggulangan Masalah Gizi
Stunting di Indonesia. Buletin Penelitian Kesehatan, 45(4), 233–240. Boli, E. B., Baliwati, Y. F., & Sukandar, D. (2018). Komitmen Politik dan Peluang Pengembangan
Kebijakan Gizi Pemerintah Daerah Provinsi Nusa Tenggara Timur. J MKMI, 14(4), 351–359. Gi
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Hamalding, H., Said, I., & Nurmiati, S. (2020). Analisis Determinan Kejadian Stunting Di Desa
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Percepatan Penurunan Stunting: Studi Kualitatif Di Kabupaten Bogor. Media Gizi Indonesia. Juwita, S., Andayani, H., Bakhtiar, B., Sofia, S., & Anidar, A. (2019). Hubungan Jumlah Pendapatan
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Sektor Publik), 2(1), 113–132. Maghfiroh, M. S., & Laksono, A. D. (2020). “Diberi air gula... awalnya nangis menjadi diam, Maghfiroh, M. S., & Laksono, A. D. (2020). “Diberi air gula... 4. KESIMPULAN Beberapa upaya yang dilakukan masyarakat Desa Kembang dalam menekan angka
prevalensi stunting di Desa Kembang Kabupaten Pacitan Jawa Timur diantaranya pertama,
mengadakan seminar pencegahan stunting, kedua, mengadakan penyuluhan mengenai P-ISSN 2614-7424 | E-ISSN 2614-8927 927 DINAMISIA: Jurnal Pengabdian Kepada Masyarakat Vol. 7, No. 4 Agustus 2023, Hal. 919-929
DOI: https://doi.org/10.31849/dinamisia.v7i4.15197 stunting pada ibu hamil dan menyusui, ketiga, memberikan makanan tambahan yang
seimbang dan bergizi. Upaya-upaya yang dilakukan berjalan dengan baik dan lancar. Hal ini
disebabkan oleh dukungan dari berbagai pihak yang turut serta mensukseskan kegiatan yang
dilakukan yakni diantaranya pemerintah Desa Kembang, narasumber seminar seperti Ir. Kuriyah, M.M selaku perwakilan dari PPKB dan P3A, Kemudian Khuzaini dari Dinas
Kesehatan dan juga Yuli Rahmawati Mutiah, M.A dari University Of Warith Al Anbiya, Bidan
Desa, Kader Posyandu, dan juga Dokter dari Puskesmas Pacitan, ibu-ibu hamil dan menyusui,
dan tentunya seluruh masyarakat Desa Kembang. Melalui kegiatan atau upaya-upaya yang dilakukan dalam menekan angka prevalensi
stunting di Desa Kembang secara umum menghasilkan pemahaman-pemahaman dan
pengetahuan dari masyarakat Desa Kembang mengenai stunting dan tata cara
pencegahannya secara umum. Indikator setelah masyarakat memahami dan mengetahui
mengenai stunting dan tata cara pencegahannya mampu menekan angka prevalensi stunting
di Desa Kembang. DAFTAR PUSTAKA awalnya nangis menjadi diam, karena P-ISSN 2614-7424 | E-ISSN 2614-8927 928 DINAMISIA: Jurnal Pengabdian Kepada Masyarakat Vol. 7, No. 4 Agustus 2023, Hal. 919-929
DOI: https://doi.org/10.31849/dinamisia.v7i4.15197 kenyang, gak lemas, daya tahan tubuhnya meningkat”; Studi Pola Asupan Pada Bayi. Amerta
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Ngasuko, T. A. (2019). Invest in Human Resource, Invest in Economy. Warta Fiskal, 2(1). Ni’mah, K., & Nadhiroh, S. R. (2015). Faktor Yang Berhubungan Dengan Kejadian Stunting Pada
Balita. Media Gizi Indonesia, 10(1), 13–19. Oktavianty, P. A., Affrian, R., Kusbandrijo, B., & Rochim, A. I. (2023). Evaluasi Program Perbaikan
Gizi Masyarakat Kategori Belita Berstatus Stunting Di Kecamatan Sungai Pandan
Kabupaten Hulu Sungai Utara (Studi Program Gerakan Atasi Stunting Dengan ASI “GUSI”). Jurnal Niara, 15(3), 388–399. Putra, A., Anggraini, A. S., & Putri, R. F. D. (2023). Systematic Literature Review: Literasi
Penurunan Angka Stunting. Jurnal Pendidikan Luar Sekolah, 17(1), 25–35. Rahayu, A., Yulidasari, F., Putri, A. O., & Rahman, F. (2015). Riwayat Berat Badan Lahir Dengan
Kejadian Stunting Pada Anak Usia Bawah Dua Tahun. KESMAS: Jurnal Kesehatan
Masyarakat Nasional (National Public Health Journal), 10(2), 67–73. y
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Sandi Husada, 9(1), 225–229. Rahmawati, R., Bagata, D. T. R., Raodah, R., Almah, U., Aziz, M. I., Zadi, B. S., … Putri, M. S. K. (2020). Sosialisasi Pencegahan Stunting Untuk Meningkatkan Sumber Daya Manusia
Unggul. DAFTAR PUSTAKA Jurnal Pembelajaran Pemberdayaan Masyarakat (JP2M), 1(2), 79–84. Renyoet, B. S., Martianto, D., & Sukandar, D. (2016). Potensi Kerugian Ekonomi Akibat Biaya
Rawat Inap Dan Rawat Jalan Pada Belita Yang Mengalami Obesitas Sampai Dewasa Di
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Pendek) di Indonesia. Global Health Science, 3(2), 139–151. Saputri, R. A. (2019). Upaya Pemerintah Daerah Dalam Penanggulangan Stunting di Provinsi
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Perempuan dan Perlindungan Anak, Pacitan. (2023). Wibowo, A. (2022). 2744 Anak Balita di Pacitan Menderita Stunting. Retrieved from
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Pranggang Kecamatan Plosoklaten Kabupaten Kediri. Prosiding Seminar Penelitian
Kesehatan, 3(1). P-ISSN 2614-7424 | E-ISSN 2614-8927 929
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Electrochemical Vicinal Difluorination of Alkenes: Scalable and Amenable to Electron‐Rich Substrates
|
Angewandte Chemie
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Doobary, S., Sedikides, A. T., Caldora, H. P., Poole, D. L., & Lennox,
A. J. J. (2020). Electrochemical Vicinal Difluorination of Alkenes:
Scalable and Amenable to Electron-Rich Substrates. Angewandte
Chemie - International Edition, 59(3), 1155-1160. https://doi.org/10.1002/anie.201912119 Publisher's PDF, also known as Version of record
License (if available):
CC BY
Link to published version (if available):
10.1002/anie.201912119 Link to publication record on the Bristol Research Portal
PDF-document This is the final published version of the article (version of record). It first appeared online via Wiley at
https://onlinelibrary.wiley.com/doi/full/10.1002/anie.201912119 . Please refer to any applicable terms of use of
the publisher. Doobary, S., Sedikides, A. T., Caldora, H. P., Poole, D. L., & Lennox,
A. J. J. (2020). Electrochemical Vicinal Difluorination of Alkenes:
Scalable and Amenable to Electron-Rich Substrates. Angewandte
Chemie - International Edition, 59(3), 1155-1160.
https://doi.org/10.1002/anie.201912119 Doobary, S., Sedikides, A. T., Caldora, H. P., Poole, D. L., & Lennox,
A. J. J. (2020). Electrochemical Vicinal Difluorination of Alkenes:
Scalable and Amenable to Electron-Rich Substrates. Angewandte
Chemie - International Edition, 59(3), 1155-1160. https://doi.org/10.1002/anie.201912119 Electrochemical Vicinal Difluorination of Alkenes: Scalable and
Amenable to Electron-Rich Substrates** However, in both cases the product
selectivity is poor and the alkene-types amenable to the
reaction are limited, with success demonstrated on only very
simple substrates. Yoneda employed p-tolyl difluoro l3-
iodane (1a) as the oxidant,[13,14] which improved product
selectivity, however 1a is light- and temperature-sensitive,
highly hygroscopic and expensive.[15] Thus, the in situ for-
mation of 1a using aryl-iodide, HF-salts and Selectfluor or
mCPBA, was the subject of elegant work by both Gilmour[16]
and Jacobsen.[17] The use of these stoichiometric oxidants then
permits the use of sub-stoichiometric quantities of the aryl-
iodide catalyst. render their use impractical. While the required oxidizing
equivalents are self-contained in these reagents, subsequent
methods have relied on the combination of HF-salts and
oxidants (Figure 1B). Oxidation with the use of electro-
chemistry[11] or Selectfluor[12] provided the earliest confirma-
tions of this strategy. However, in both cases the product
selectivity is poor and the alkene-types amenable to the
reaction are limited, with success demonstrated on only very
simple substrates. Yoneda employed p-tolyl difluoro l3-
iodane (1a) as the oxidant,[13,14] which improved product
selectivity, however 1a is light- and temperature-sensitive,
highly hygroscopic and expensive.[15] Thus, the in situ for-
mation of 1a using aryl-iodide, HF-salts and Selectfluor or
mCPBA, was the subject of elegant work by both Gilmour[16]
and Jacobsen.[17] The use of these stoichiometric oxidants then
permits the use of sub-stoichiometric quantities of the aryl-
iodide catalyst. [*] S. Doobary, A. T. Sedikides, H. P. Caldora, Dr. A. J. J. Lennox
School of Chemistry, University of Bristol
Cantock’s Close, Bristol, BS8 1TS (UK)
E-mail: a.lennox@bristol.ac.uk
Dr. D. L. Poole
Medicines Design, GSK Medicines Research Centre
Gunnels Wood Rd, Stevenage, SG1 2NY (UK)
[**] A previous version of this manuscript has been deposited on
a preprint server (https://doi.org/10.26434/chemrxiv.9974687.v1). Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under https://doi.org/10. 1002/anie.201912119. T 2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. 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. [**] A previous version of this manuscript has been deposited on
a preprint server (https://doi.org/10.26434/chemrxiv.9974687.v1). Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under https://doi.org/10. 1002/anie.201912119. T 2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. T 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Electrochemical Vicinal Difluorination of Alkenes: Scalable and
Amenable to Electron-Rich Substrates** Electrochemical Vicinal Difluorination of Alkenes: Scalable and
Amenable to Electron-Rich Substrates**
Sayad Doobary, Alexi T. Sedikides, Henry P. Caldora, Darren L. Poole, and
Alastair J. J. Lennox* Electrochemical Vicinal Difluorination of Alkenes: Scalable and
Amenable to Electron-Rich Substrates**
S
d D
b
Al
i T S dikid
H
P C ld
D
L P
l
d Figure 1. A) Significance and application of vicinal difluorides. B) Methods to prepare vicinal difluorides. Abstract: Fluorinated alkyl groups are important motifs in
bioactive compounds, positively influencing pharmacokinetics,
potency and conformation. The oxidative difluorination of
alkenes represents an important strategy for their preparation,
yet current methods are limited in their alkene-types and
tolerance of electron-rich, readily oxidized functionalities, as
well as in their safety and scalability. Herein, we report
a method for the difluorination of a number of unactivated
alkene-types that is tolerant of electron-rich functionality,
giving products that are otherwise unattainable. Key to success
is the electrochemical generation of a hypervalent iodine
mediator using an “ex-cell” approach, which avoids oxidative
substrate decomposition. The more sustainable conditions give
good to excellent yields in up to decagram scales. Th The inclusion of fluorine in bioactive compounds is becom-
ing more important:[1] since 2006, prevalence has increased
from 6% to 31% in the top 100 best-selling small-molecule
drugs.[2] Fluorinating alkyl groups can increase potency,
lipophilicity and metabolic stability,[3] while reducing the
basicity of neighbouring amines,[4] all of which can improve
bioactivity and pharmacokinetics. The vicinal difluoroethane
unit has attracted recent attention, particularly as a bioisostere
of ethyl or trifluoromethyl groups[5] (Figure 1A) and for its
unique propensity to adopt a gauche conformation in
solution.[6] Exploiting this stereo-electronic effect is an
emerging strategy for molecular design,[7] and has found
application in, for example, organocatalysis[8] and peptide
mimics.[9] Figure 1. A) Significance and application of vicinal difluorides. B) Methods to prepare vicinal difluorides. Simple vicinal difluorides have been prepared from
alkenes with the use of ambiphilic fluoride reagents, F2 and
XeF2.[10] However, their high reactivity, toxicity and high cost render their use impractical. While the required oxidizing
equivalents are self-contained in these reagents, subsequent
methods have relied on the combination of HF-salts and
oxidants (Figure 1B). Oxidation with the use of electro-
chemistry[11] or Selectfluor[12] provided the earliest confirma-
tions of this strategy. 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/ Angewandte
Chemie Angewandte
Chemie Angewandte
Chemi Communications German Edition:
DOI: 10.1002/ange.201912119
International Edition: DOI: 10.1002/anie.201912119 German Edition:
DOI: 10.1002/ange.201912119
International Edition: DOI: 10.1002/anie.201912119 German Edition:
DOI: 10.1002/ange.201912119
International Edition: DOI: 10.1002/anie.201912119 Angew. Chem. Int. Ed. 2020, 59, 1155 –1160
T 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim [*] S. Doobary, A. T. Sedikides, H. P. Caldora, Dr. A. J. J. Lennox
School of Chemistry, University of Bristol
Cantock’s Close, Bristol, BS8 1TS (UK)
E-mail: a.lennox@bristol.ac.uk
Dr. D. L. Poole
Medicines Design, GSK Medicines Research Centre
Gunnels Wood Rd, Stevenage, SG1 2NY (UK)
[**] A previous version of this manuscript has been deposited on
a preprint server (https://doi.org/10.26434/chemrxiv.9974687.v1).
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under https://doi.org/10.
1002/anie.201912119.
T 2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co.
KGaA. 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. [*] S. Doobary, A. T. Sedikides, H. P. Caldora, Dr. A. J. J. Lennox
School of Chemistry, University of Bristol
Cantock’s Close, Bristol, BS8 1TS (UK)
E-mail: a.lennox@bristol.ac.uk
Dr. D. L. Poole
Medicines Design, GSK Medicines Research Centre
Gunnels Wood Rd, Stevenage, SG1 2NY (UK) Communications Communications Figure 2. Reaction optimisation: electrolyses in undivided cell,
19F NMR yields. A) Yields of 3a from 2a (25 mm) in the presence of
different aryl-iodides (1) 4 Fmol@1. E1/2 vs. Fc/Fc+. B) Yields of 4 (blue)
and 3a (green) from 1a and 2a (both 25 mm) in different HF:amine
ratios after 4 Fmol@1. “Amine”=NEt3 or py. C) Yields of 1a after
4 Fmol@1, 12 mA (PtkPt) in different amounts of HFIP in CH2Cl2. D) Fluorination of 2as with and without Tol-I. 4 Fmol@1 passed at
12 mA (PtkPt). Chemie
nications rich moieties; due to competitive substrate oxidation, result-
ing in either decomposition or unselective fluorination. Moreover, owing to the potential of fluorinated alkyl groups
in high-value bioactive compounds, a method that is readily
scaled, and therefore is safe, inexpensive and does not
produce much waste, is still required. To address these shortfalls, we sought to access the l3-
iodane 1 mediators[18] using electrochemical[19] oxidation. The
unique spatial control of redox events, along with the control
of potential and rate, should facilitate the expansion of
substrate classes. As well as the inherent safety and scalability
of electrochemistry,[20] the addition of a chemical oxidant is
not required, as protons can ultimately accept electrons at the
cathode to form H2 as the sole by-product, thereby rendering
the process more atom-economical.[21] The electrochemical generation of many hypervalent l3-
iodane species from aryl-iodides is known.[22] Difluoro l3-
iodanes (1) has been comparatively less explored,[23] with
Waldvogel recently reporting the only example in the
presence of alkenes,[23e] which are liable to preferentially
oxidise. A number of additional problems are reported to
occur when 1 is generated at an electrode,[24] which include
dimerization, benzylic fluorination and the formation of
“many other complicated products”.[23d] The application of
high potentials with HF-salts also causes anode passivation,
where a non-conducting polymer coating forms on the
electrode surface that suppresses faradaic current and can
attenuate reaction.[25] Thus, our primary objective was to
address these issues in our optimisations. We started by examining different aryl-iodide mediators
for the electrochemical difluorination of allylbenzene (2a)
(Figure 2A). No conversion to difluorinated alkane 3a was
observed with the use of 4-iodo-anisole (R = 4-OMe), the
most readily oxidized derivative we tested. However, we
observed the formation of 3a using aryl-iodides with higher
oxidation potentials. Communications Further increases in potential beyond
R = Me (tolyl) led to a subsequent decline in yield, as direct
substrate oxidation out-competes aryl-iodide oxidation (ca. Eox = 1.9 V). Although iodotoluene gave the highest yield of
3a, substantial quantities of benzylic fluorination (to 4) were
observed (< 18%) (Figure 2B). We confirmed that 4 itself is
a very poor mediator, as only 7% of 3a was returned when
using 4 in place of 1a.[26] As this side reaction requires
deprotonation, we reasoned that it should be attenuated by
reducing the availability of basic fluoride by increasing the
proportion of HF to amine (in mixes of commercially
available 3HF·NEt3 and 9HF·py (amine = py or NEt3). Indeed, by increasing this ratio from 3 to 5.6, benzylic
fluorination decreased with an accompanying increase in
product 3a (Figure 2B). This trend also mirrors the enhanced
activation of 1a with acid expected from the presence of more
HF. Further increases beyond 5.6 maintained the lack of 4
formation, but led to a decline in product 3a, possibly
reflecting a decrease in fluoride activity. This sweet-spot
demonstrates the delicate balance between activation of 1a
and deactivation of fluoride. Figure 2. Reaction optimisation: electrolyses in undivided cell,
19F NMR yields. A) Yields of 3a from 2a (25 mm) in the presence of
different aryl-iodides (1) 4 Fmol@1. E1/2 vs. Fc/Fc+. B) Yields of 4 (blue)
and 3a (green) from 1a and 2a (both 25 mm) in different HF:amine
ratios after 4 Fmol@1. “Amine”=NEt3 or py. C) Yields of 1a after
4 Fmol@1, 12 mA (PtkPt) in different amounts of HFIP in CH2Cl2. D) Fluorination of 2as with and without Tol-I. 4 Fmol@1 passed at
12 mA (PtkPt). under these conditions.[25] This insulating effect was reflected
by a large rise in cell potential during reaction compared to
other solvents tested,[26] none of which improved the yield
beyond that of CH2Cl2. However, when hexafluoro-isopro-
panol (HFIP) was added as a co-solvent, higher yields of 3a
were observed, as also noted in other halogenation[27] and
electrochemical reactions.[28] 30% HFIP in CH2Cl2 led to the
greatest enhancement.[26] Control reactions in the absence of
alkene revealed that the concentration of HFIP determined
the amount of 1a formation (Figure 2C) after the same
amount of charge was passed. T 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2020, 59, 1155 –1160 www.angewandte.org
T 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. C Electrochemical Vicinal Difluorination of Alkenes: Scalable and
Amenable to Electron-Rich Substrates** 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. T 2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. 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. While these examples represent great advances in access-
ing vicinal difluorides, there is no general method for
accessing these motifs in compounds containing electron- 1155 Angew. Chem. Int. Ed. 2020, 59, 1155 –1160 Angewandte
Chemie
nications Angewandte
Chem Communications Communications Allylic ethers and amines (2i–t) were
both tolerated if containing electron poor (hetero)aromatics. Ester (2h), alcohol (2 f), sulfonate (2g) and halide (Br, Cl,
e.g., 2l and 2q) functional groups were all untouched,
providing useful products for further derivatization. While
acid sensitive functionality, such as boc groups, was not
tolerated,[26] a cyclopropyl ring (2t) avoided competing
oxidative ring opening.[30] Figure 3. [a] Isolated yields (NMR yields in parentheses). [b] 2
(1.2 mmol (0.3m) or 0.8 mmol (0.2m)) and iodotoluene (1 equiv.) in 3
parts 5.6HF:amine (where amine=py or NEt3) and 1 part
CH2Cl2:HFIP (7:3); electrolysis: 8 mA (0.2m) or 12 mA (0.3m),
3.5 Fmol@1, PtkPt, undivided cell, then stirring (12 h). [c] Reaction
performed with Electrasyn 2.0 in a PTFE vial. The scalability of the method was demonstrated by
yielding gram-scale quantities of products 3e, 3i and 3s and
a deca-gram-scale quantity of 3s. In each of these cases, 70 +
% of pure iodotoluene was recovered. The commercially
available ElectraSyn 2.0 set-up was also tested-in combina-
tion with a PTFE vial-and was found to give product 3d in
a comparable yield to our set-up, validating the robustness of
the conditions. formation of 1a in a divided cell, followed by the subsequent
addition of substrate 2u.[26] With this approach the yield of 3u
was raised from 45% to 73% (Figure 4). The scope of electron-rich or easily oxidised substrates
was now tested with this “ex-cell” method (Figure 4). Electron-rich allyl arenes (2u–x) were now tolerated, return-
ing moderate to very good yields of product. Success was
achieved with the very electron-rich dimethylaniline 2w,
however, the anisole derivative 2y was less well tolerated,
which may be due to the known C@H activation pathway of
these arenes-types to generate diaryliodonium species.[31] A
pharmaceutically relevant morpholine amide (2z) was also
tolerated. These “in-cell” conditions performed worse with the more
electron-rich allyltoluene (2u) (Figure 4), returning only
a moderate yield of difluorinated product 3u. This reactivity
is consistent with previous methods that also struggle with
readily
oxidized
substrate
classes.[16]
Therefore,
a
new
approach was sought to specifically gain access to products
containing electron-rich moieties. Analysis of a range of
electron-rich substrates by cyclic voltammetry revealed their
preferential oxidation to iodotoluene,[26] thereby eluding the
vital formation of l3-iodane 1a. Communications The addition of allylpenta-
fluorobenzene (2e) to these mixtures confirmed its more
efficient transformation to 3e in the presence of more 1a.[26]
To rationalise the downward trend of 1a (Figure 2C), we
observed a reduced solubility of iodotoluene with increased
HFIP. CV studies revealed that the oxidation potential of The influence of solvent was then examined. MeCN
performed worse than CH2Cl2, which was unsurprising
considering anode passivation is particularly predominant 1156 Angewandte
Chem Communications Figure 3. [a] Isolated yields (NMR yields in parentheses). [b] 2
(1.2 mmol (0.3m) or 0.8 mmol (0.2m)) and iodotoluene (1 equiv.) in 3
parts 5.6HF:amine (where amine=py or NEt3) and 1 part
CH2Cl2:HFIP (7:3); electrolysis: 8 mA (0.2m) or 12 mA (0.3m),
3.5 Fmol@1, PtkPt, undivided cell, then stirring (12 h). [c] Reaction
performed with Electrasyn 2.0 in a PTFE vial. Angewan
nications g
Chemie iodotoluene under the reaction conditions decreases with
more HFIP present,[26,29] resulting in a milder oxidizing
environment, which should contribute to improved functional
group tolerance. Finally,
19F NMR analysis of a genuine
sample of 1a, supported our assumption that 1a was formed
under the optimized conditions.[26] The importance of the iodotoluene mediator to product
selectivity was confirmed by control reactions in its absence
(Figure 2D). Without iodotoluene, direct oxidation of 2as led
to benzylic, rather than alkene, fluorination with low mass
balance. The use of sub-stoichiometric quantities of the
mediator led to a decline in yield of 3a,[26] reflecting
a mismatch of reaction-rates in the electrochemical and
chemical steps (EC mechanism). We could drop the loading
of iodotoluene to 20 mol% by applying 5 cycles of 0.7 Fmol@1
with 6 h stirring in between each cycle.[26] However, the vastly
increased reaction time was deemed to be an inferior
adjustment to the conditions than using an equivalent of
iodotoluene and running the reaction in one go. Moreover, we
are able to recover pure iodotoluene in greater than 70%
yields, and thus recycle it for use in subsequent reactions. With this “in-cell” optimised method now in hand, we
proceeded to explore the substrate scope (Figure 3). Previ-
ously published protocols do not report substrate classes that
contain electron-rich moieties, therefore, we initially avoided
these substrates. Indeed, electron-poor allyl arenes were well
tolerated (2a–e) and gave difluorinated products in good to
excellent yields. Long-chain alkenes, 2 f–h, returned excellent
yields, demonstrating that a proximal aromatic ring is not
necessary for reactivity. Angew. Chem. Int. Ed. 2020, 59, 1155 –1160
T 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Communications To avoid this problem, an “ex-
cell” method was devised that spatially and temporally
separates the electrochemical oxidation and fluorination
steps, thereby avoiding competitive direct oxidation of the
substrates. Thus, conditions were re-optimized for the initial Anilines are a substrate class that have also not previously
been demonstrated, as they are very readily oxidized. Aniline
2aa posed problems using the “in-cell” method, cf. only 25%
3aa was isolated. However, by adopting the “ex-cell” method,
this was increased to 70%. The greater electron withdrawing
effect of nosyl (3ab) vs. tosyl (3aa) translated into greater 1157 www.angewandte.org Figure 4. [a] Isolated yields with NMR yields in parentheses, black=“in-cell”, green=“ex-cell”. [b] Electrolysis of iodotoluene (1.2 mmol, 0.2m) in
3 parts 5.6HF:amine and 1 part CH2Cl2:HFIP (7:3): 12 mA, 3.0 Fmol@1, PtkPt, divided cell, then addition of 2 (1.2 mmol), stir (12 h). [c]
Selectfluor + Tol-I conditions from Ref. [16a]; mCPBA + Ar-I conditions from Ref. [17a]; Selectfluor conditions from Ref. [12]. [d] CHCl3 used
nstead of CH2Cl2 with 4.5HF:amine. [e] Styrene difluorination conditions from Ref. [16b]. Selectfluor conditions from Ref. [16a] generated only
(17%) 3ap. Angewandte
Chemie
Communications Angewandte
Chem Angewandte
Chemie Communications Communications Figure 4. [a] Isolated yields with NMR yields in parentheses, black=“in-cell”, green=“ex-cell”. [b] Electrolysis of iodotoluene (1.2 mmol, 0.2m) in
3 parts 5.6HF:amine and 1 part CH2Cl2:HFIP (7:3): 12 mA, 3.0 Fmol@1, PtkPt, divided cell, then addition of 2 (1.2 mmol), stir (12 h). [c]
Selectfluor + Tol-I conditions from Ref. [16a]; mCPBA + Ar-I conditions from Ref. [17a]; Selectfluor conditions from Ref. [12]. [d] CHCl3 used
instead of CH2Cl2 with 4.5HF:amine. [e] Styrene difluorination conditions from Ref. [16b]. Selectfluor conditions from Ref. [16a] generated only
(17%) 3ap. yields. Good to excellent yields of other difluorinated anilines
containing electron rich and poor rings were generated (3ac–
ag), including mesityl. Other terminal alkenes containing
electron-rich moieties were tolerated, including an allyl amine
(2am) and ether (2as). Styrenes were tolerated if they were
electron poor, such as 2ai, otherwise geminal fluorination
occurred (2ah), which is a process that has previously been
observed with this substrate class.[32] Substituted styrenes
(2aj–al) were also well tolerated. alkenes (2ag, 2an–ar, 2at–au). Good to excellent yields were
produced in each case. Both cis and trans isomers of internal
alkenes were viable substrates, and their stereochemical
information was translated to their products with a diastereo-
selectivity between 5–9:1. Readily oxidised trialkylamines
(2aq–ar) were again tolerated functionality. T 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2020, 59, 1155 –1160 www.angewandte.org
T 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int Communications To ascertain the
effect of steric bulk or hydrophobic shielding on internal
alkenes, 4 regio-isomers of trans-octene were tested in the
reaction (2av–ay). It was found that yields improved as the
bulk either side of the alkene decreased. Substituted alkenes that are unactivated are problematic
substrates for other methods, and so we were pleased to
discover that our “ex-cell” conditions readily translate
electron-rich di- and tri-substituted terminal and internal The difluorination of several electron-rich substrates
using our electrochemical method was compared to methods
in the literature, including those that employ Selectfluor and 1158 Angewandte
Chemie Angewandte
Chem Communications Gilmour, ACS Med. Chem. Lett. 2019, https://doi.org/10.1021/
acsmedchemlett.9b00287. mCPBAwith aryl-iodide, and Selectfluor alone. In each of the
examples tested, the electrochemical method gave superior
yields (Figure 4), thereby validating the importance of the
“ex-cell” approach. The sustainability of the electrochemical
method is reflected in the low E-factor[33] (ratio of total waste
to product) calculated for the reaction,[34] which was consis-
tently lower than the Selectfluor or mCPBA methods. The
main improvement to waste reduction originates from the
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Angew. Chem. Int. Ed. 2020, 59, 1155 –1160 www.angewandte.org
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Version of record online: November 28, 2019 1160 www.angewandte.org
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https://openalex.org/W2559340251
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https://www.frontiersin.org/articles/10.3389/fmicb.2016.01930/pdf
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English
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Identification and Characterization of Novel Compounds Blocking Shiga Toxin Expression in Escherichia coli O157:H7
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Frontiers in microbiology
| 2,016
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cc-by
| 8,767
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ORIGINAL RESEARCH
published: 30 November 2016
doi: 10.3389/fmicb.2016.01930 Keywords: Shiga toxin, E. coli, RecA, phage, expression Identification and Characterization of
Novel Compounds Blocking Shiga
Toxin Expression in Escherichia coli
O157:H7 Alejandro Huerta-Uribe1, Zoe R. Marjenberg1, Nao Yamaguchi2, Stephen Fitzgerald2,
James P. R. Connolly1, Nuria Carpena1, Hanna Uvell3, Gillian Douce1, Michael Elofsson3,
Olwyn Byron4, Rudi Marquez5, David L. Gally2 and Andrew J. Roe1* Alejandro Huerta-Uribe1, Zoe R. Marjenberg1, Nao Yamaguchi2, Stephen Fitzgerald2,
James P. R. Connolly1, Nuria Carpena1, Hanna Uvell3, Gillian Douce1, Michael Elofsson3,
Olwyn Byron4, Rudi Marquez5, David L. Gally2 and Andrew J. Roe1* 1 Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow,
Glasgow, UK, 2 Division of Immunity and Infection, The Roslin Institute and R(D)SVS, The University of Edinburgh, Edinburgh,
UK, 3 Laboratories for Chemical Biology Umeå, Department of Chemistry, Umeå University, Umeå, Sweden, 4 School of Life
Sciences, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, UK, 5 Department of Chemistry,
Xi’an Jiaotong-Liverpool University, Suzhou, China Edited by: Edited by:
Axel Cloeckaert,
French National Institute
for Agricultural Research, France Reviewed by:
Paul Alan Hoskisson,
University of Strathclyde, UK
Victor Krylov,
I.I. Mechnikov Research Institute
of Vaccines and Sera, Russia *Correspondence:
Andrew J. Roe
andrew.roe@glasgow.ac.uk Specialty section:
This article was submitted to
Antimicrobials, Resistance
and Chemotherapy,
a section of the journal
Frontiers in Microbiology Infections caused by Shiga toxin (Stx)-producing E. coli strains constitute a health
problem, as they are problematic to treat. Stx production is a key virulence factor
associated with the pathogenicity of enterohaemorrhagic E. coli (EHEC) and can result
in the development of haemolytic uremic syndrome in infected patients. The genes
encoding Stx are located on temperate lysogenic phages integrated into the bacterial
chromosome and expression of the toxin is generally coupled to phage induction
through the SOS response. We aimed to find new compounds capable of blocking
expression of Stx type 2 (Stx2) as this subtype of Stx is more strongly associated
with human disease. High-throughput screening of a small-molecule library identified
a lead compound that reduced Stx2 expression in a dose-dependent manner. We show
that the optimized compound interferes with the SOS response by directly affecting the
activity and oligomerization of RecA, thus limiting phage activation and Stx2 expression. Our work suggests that RecA is highly susceptible to inhibition and that targeting
this protein is a viable approach to limiting production of Stx2 by EHEC. This type of
approach has the potential to limit production and transfer of other phage induced and
transduced determinants. Infections caused by Shiga toxin (Stx)-producing E. coli strains constitute a health
problem, as they are problematic to treat. Stx production is a key virulence factor
associated with the pathogenicity of enterohaemorrhagic E. coli (EHEC) and can result
in the development of haemolytic uremic syndrome in infected patients. The genes
encoding Stx are located on temperate lysogenic phages integrated into the bacterial
chromosome and expression of the toxin is generally coupled to phage induction
through the SOS response. We aimed to find new compounds capable of blocking
expression of Stx type 2 (Stx2) as this subtype of Stx is more strongly associated
with human disease. High-throughput screening of a small-molecule library identified
a lead compound that reduced Stx2 expression in a dose-dependent manner. We show
that the optimized compound interferes with the SOS response by directly affecting the
activity and oligomerization of RecA, thus limiting phage activation and Stx2 expression. Our work suggests that RecA is highly susceptible to inhibition and that targeting
this protein is a viable approach to limiting production of Stx2 by EHEC. This type of
approach has the potential to limit production and transfer of other phage induced and
transduced determinants. INTRODUCTION coli O157:H7 was November 2016 | Volume 7 | Article 1930 1 Frontiers in Microbiology | www.frontiersin.org Blocking Stx Expression and Transduction Huerta-Uribe et al. linked to the development of HUS (Karmali et al., 1983), a
disease predominantly affecting children and carrying a 5−10%
mortality rate. Since then, EHEC have been recognized as
responsible for hundreds of food and waterborne outbreaks, with
O157:H7 being the most prevalent serotype and the leading cause
of HUS in Europe and the United States (Tarr et al., 2005; Lim
et al., 2010). activity. Based on our phenotypic studies, we hypothesized that
the inhibition of Stx expression might be due to inhibition of
the SOS response protein RecA, which we tested by a series of
biochemical and biophysical analyses. Our work demonstrates
that RecA is highly susceptible to inhibition and that targeting
this protein is a viable approach to limiting production of Stx2 by
EHEC. The development of haemorrhagic colitis and HUS is
dependent on the production of Stx, a family of related toxins that
are essential for disease. Stx produced by EHEC strains are largely
differentiated into two types that share 55% sequence homology
(Fraser et al., 2004): Stx1, which differs from Shigella dysenteriae
Stx by a single amino acid, and Stx2, which is structurally similar
to Stx1 but antigenically distinct. Stx1 and Stx2 can be further
classified into several subtypes based on the sequence-based
relatedness of the proteins. These comprise three Stx1 subtypes
(1a, 1c, 1d) and seven Stx2 subtypes (2a, 2b, 2c, 2d, 2e, 2f, and 2g). INTRODUCTION Huerta-Uribe A, Marjenberg ZR,
Yamaguchi N, Fitzgerald S,
Connolly JPR, Carpena N, Uvell H,
Douce G, Elofsson M, Byron O,
Marquez R, Gally DL and Roe AJ
(2016) Identification
and Characterization of Novel
Compounds Blocking Shiga Toxin
Expression in Escherichia coli
O157:H7. Front. Microbiol. 7:1930. doi: 10.3389/fmicb.2016.01930 Huerta-Uribe A, Marjenberg ZR,
Yamaguchi N, Fitzgerald S,
Connolly JPR, Carpena N, Uvell H,
Douce G, Elofsson M, Byron O,
Marquez R, Gally DL and Roe AJ
(2016) Identification
and Characterization of Novel
Compounds Blocking Shiga Toxin
Expression in Escherichia coli
O157:H7. Front. Microbiol. 7:1930. doi: 10.3389/fmicb.2016.01930 Huerta-Uribe A, Marjenberg ZR,
Yamaguchi N, Fitzgerald S,
Connolly JPR, Carpena N, Uvell H,
Douce G, Elofsson M, Byron O,
Marquez R, Gally DL and Roe AJ
(2016) Identification
and Characterization of Novel
Compounds Blocking Shiga Toxin
Expression in Escherichia coli
O157:H7. Front. Microbiol. 7:1930. doi: 10.3389/fmicb.2016.01930 Enterohemorrhagic E. coli (EHEC) are a group of Shiga toxin (Stx) producing pathogenic E. coli
strains, which are associated with a broad spectrum of disease ranging from mild diarrhea to severe
haemorrhagic colitis and haemolytic uremic syndrome (HUS) (Paton and Paton, 1998). The first
recognition of an EHEC strain as a foodborne pathogen occurred in the US in 1982 during an
investigation of customers from a fast-food restaurant chain who had bloody diarrhea and severe
abdominal cramping with no fever (Riley et al., 1983). The E. coli serotype O157:H7, which had
not previously been associated with human disease, was isolated from infected individuals and
traced to contaminated hamburger meat (Riley et al., 1983). Shortly thereafter, E. coli O157:H7 was Enterohemorrhagic E. coli (EHEC) are a group of Shiga toxin (Stx) producing pathogenic E. coli
strains, which are associated with a broad spectrum of disease ranging from mild diarrhea to severe
haemorrhagic colitis and haemolytic uremic syndrome (HUS) (Paton and Paton, 1998). The first
recognition of an EHEC strain as a foodborne pathogen occurred in the US in 1982 during an
investigation of customers from a fast-food restaurant chain who had bloody diarrhea and severe
abdominal cramping with no fever (Riley et al., 1983). The E. coli serotype O157:H7, which had
not previously been associated with human disease, was isolated from infected individuals and
traced to contaminated hamburger meat (Riley et al., 1983). Shortly thereafter, E. Frontiers in Microbiology | www.frontiersin.org Chemical Synthesis Tetrahydrofuran and dichloromethane were purified through
a Pure Solv 400-5MD solvent purification system (Innovative
Technology, Inc). Solvents were evaporated under reduced
pressure at 40◦C. All reactions described were performed under
argon atmosphere unless otherwise stated and monitored by thin
layer chromatography (TLC) with pre-coated TLC plates (Merck
Silica Gel 60 F254). Plates were visualized by UV light (254 nm),
iodine vapors or stained with anisaldehyde. Shiga toxin are AB5 toxins, which bind via the B subunits
to the globotriaoslyceramide (Gb3) receptor, expressed on the
surface of vascular endothelial cells as well as Paneth cells in the
intestinal mucosa. B subunit binding leads to clathrin-dependent
endocytosis of the A-B subunits. Following endosomal processing
and trafficking to the golgi, the internalized A subunit cleaves
the 28S ribosomal RNA of the 60S ribosomal subunit, preventing
binding of elongation factor to the ribosome and thus inhibiting
protein synthesis, resulting in cell death by apoptosis (Bergan
et al., 2012). Details
of
the
chemical
synthesis
are
provided
in
Supplementary Methods and an overview in Supplementary
Figure
S1. Compounds
were
purified
via
flash
column
chromatography using silica gel (FlurochemSilica LC 60A)
as the stationary phase. 1H NMR and 13C NMR spectra were
recorded at 400 and 100 MHz or at 500 and 125 MHz using either
a Bruker DPX Avance400 instrument or a Bruker Avancell500
instrument, respectively. IR spectra were obtained using a
Shimadzu FTIR-8400 spectrometer. The genes for Stx in E. coli are exclusively located on temperate
lysogenic phages that integrate into the genome of their host
bacterium. In the lysogenic state, Stx genes are replicated as an
integral part of the bacterial genome. Expression of the phage
genes occurs when the phage lytic cycle is activated by induction
of the SOS response. This triggers both the packaging of the genes
encoding Stx to be into phage particles, which are assembled and
released through lysis of the cell, and simultaneous production of
the Stx protein. The released Stx-encoding bacteriophages have
the potential to transduce other E. coli leading to dissemination
of this virulence phenotype. High-resolution mass spectra were obtained using a JEOL
JMS-700 mass spectrometer by electrospray and chemical
ionization operating at resolution of 15,000 full width at half
height. High Throughput Screens In preparation for screening experiments, plasmids pNY16,
pNY14, and pAJR145 were transformed into ZAP0273 (E. coli
Sakai stx-). These plasmids comprise the promoter region of
the gene encoding stx2, sulA and rpsM, respectively fused to
green fluorescent protein (GFP). The ChemBridge library of
17,500 chemically diverse compounds was screened with the
pNY16, pNY14, and pAJR145 reporter plasmids at 50 µM and
1% DMSO. Gain settings for the fluorescence readings were
optimized according to the zero percent effect (ZPE) values from
each assay. The library is organized into 56, 384-well plates. The
library was screened once with each assay and the Z′-values were
analyzed for each plate. Fluorescence levels were measured at
both t = 0 and at the assay end-points. Hits were defined as
compounds that affected the fluorescence signal by greater than 3
SDs. The SOS response, a high-activity repair response to damage
of chromosomal DNA, is regulated by the interplay of the two
major proteins RecA, an ATP-dependent protein with DNA-
binding abilities, and LexA, the key repressor of SOS-induced
genes. Activation of RecA in response to DNA damage mediates
auto-cleavage of both LexA and prophage repressors, leading to
bacteriophage and Stx production. The importance of Stx for EHEC pathogenesis has driven
efforts to develop novel compounds that interfere with this potent
toxin. In this work we designed a high throughput screen (HTS)
to identify compounds that preferentially affect expression of
stx2. We focused on Stx2 because studies in primates have shown
that administration of Stx2 alone can produce the symptoms of
HUS, while administration of Stx1 at the same dose does not. In
addition, epidemiological and in vitro studies demonstrate that
Stx2a is more likely to be associated with more serious human
disease (Persson et al., 2007; Manning et al., 2008; Fuller et al.,
2011). For the most effective lead compound, we then modified
key functional groups to reveal the components required for Phage Transduction g
Prophage strains E. coli JP10819 and S. aureus JP5011 (Quiles-
Puchalt et al., 2014) were grown in LB (E. coli) or TSB (S. aureus)
media at 37◦C, and 50 µM AHU3 and 2 µg/ml MMC added
at OD600 = 0.25. Cultures were then incubated at 32◦C with
80 rpm shaking for 4 h, followed by 21◦C without shaking for
16 h. Cultures were filtered through a 0.2 µM filter, and serially
diluted in phage buffer (100 mM NaCl, 0.5 M tris pH 7.8, 1 mM
MgSO4, 4 mM CaCl2). Hundred microliter of phage lysate was
added to 1 ml aliquots of MG1655 (E. coli) or RN4220 (S. aureus)
at an OD600 of 1.4. Samples were incubated without shaking at
37◦C for 30 min before addition of 3 ml 0.75% agar and plating
onto LB or TSB agar plates containing 6 µg/ml tetracycline and
1.7 mM sodium citrate. A negative control lacking addition of
phage lysate was also included to confirm the absence of phage
contamination. Colonies were subsequently counted to define the
number of transduction events per sample. Colorimetric ATPase Assay Colorimetric ATPase assays were performed using an Innova
Biosciences ATPase assay kit (#601-0120), and reactions carried
out following the manufacturer’s protocol, using 50 µM RecA
(NEB), 500 µM poly dT ssDNA (Midland Certified Reagent
Company) and 0.1−100 µM AHU3. Generation of Reporter Plasmids to
Facilitate HTS Cultures of strain ZAP1620, a phage-type (PT) 21/28 EHEC
isolate, were cultured overnight in LB medium. After dilution
1/100, cultures were grown in the presence or absence of AHU3
(50 µM) to exponential phase (OD600 = 0.3–0.4) before phage
lysis was induced by the addition of mitomycin C (5 µg/ml). Lysis
was allowed to proceed for 1.5 h after which cultures were filtered
(0.22 µm filter). Supernatant samples containing the toxin were
applied directly to a RIDASCREEN
R⃝verotoxin (R-Biopharm)
96-well microtitre plate and assayed for toxin levels by ELISA
according to manufacturer guidelines. The main aim of our study was to identify compounds that
specifically reduced Stx2 production by ZAP0273. To address
this, we designed a HTS for compounds that preferentially
affected expression of stx2, we also included a screen for
sulA expression to examine generic effects on SOS induction
(Mizusawa et al., 1983). The promoter regions of stx2 and sulA
were amplified from E. coli O157:H7 Sakai by PCR and cloned
into the XbaI site upstream of the GFP gene in plasmid pKC26,
thus creating transcriptional GFP reporter plasmids pNY16 and
pNY14. A further control, pAJR145, was also used, comprising
the promoter for rpsM, encoding the 30S ribosomal protein S13,
fused to GFP. Expression of this plasmid is closely linked to
bacterial growth rate. The use of GFP reporters allowed us to
use intact bacteria in the screen, which ensures that any active
compounds are likely to penetrate cells and reach intracellular
targets. Reporter Assays Shiga toxin expression assays were performed using the
pstx2::GFP reporter fusion plasmid (pNY16). The plasmid was
transformed by electroporation into ZAP0273 and transformants
cultured overnight in LB medium containing 35 µg/mL
chloramphenicol. Overnight cultures were diluted into fresh November 2016 | Volume 7 | Article 1930 2 Blocking Stx Expression and Transduction Huerta-Uribe et al. LB at an OD600 of 0.08, and grown to an OD600 of 0.6 at
37◦C and 200 rpm. Compounds and mitomycin C (MMC,
1 µg/mL) were added at this point, and fluorescence and optical
density of cultures measured hourly. GFP fluorescence of 200 µL
aliquots was measured in a 96-well blank microtiter plate using a
FLUOstar Optima plate reader (BMG, Germany). Sedimentation equilibrium data were analyzed with SEDPHAT
(Vistica et al., 2004) using the single species analysis model in
order to gain a model-independent measure of the whole-cell
weight average molecular mass. LB at an OD600 of 0.08, and grown to an OD600 of 0.6 at
37◦C and 200 rpm. Compounds and mitomycin C (MMC,
1 µg/mL) were added at this point, and fluorescence and optical
density of cultures measured hourly. GFP fluorescence of 200 µL
aliquots was measured in a 96-well blank microtiter plate using a
FLUOstar Optima plate reader (BMG, Germany). HTS for Compounds that Reduce Stx
Expression p
To identify new compounds with the potential to reduce
expression of Stx we performed a HTS of the Chem-Bridge
library at the Laboratory for Chemical Biology, Umeå. The
library comprises 17,500 chemically diverse compounds that
have previously proven valuable for the identification of
novel therapeutics. Each compound was initially tested at a
concentration of 50 µM for the ability to suppress MMC-
induced expression of stx2::GFP in ZAP0273. Further details of
the HTS are provided in the Section “Materials and Methods”
and Figure 1. The primary selection criteria was for drugs
that inhibited pNY16 (stx2::GFP) fluorescence induction but
which had limited effects on pAJR145 (rpsm::GFP) and pBY14
(sulA::GFP) expression. However, compounds that showed
strong suppression of stx2::GFP expression and some suppression
of sulA::GFP were also taken forward for further investigation. In
total 76 hits, comprising 48 compounds that reduced fluorescence
in the pNY16 screen only and 28 compounds that reduced
fluorescence in pNY16 and pNY14 screens, were selected. November 2016 | Volume 7 | Article 1930 Sedimentation Equilibrium (SE)
Analytical Ultracentrifugation (AUC) Analytical ultracentrifugation (AUC) was performed using an
Optima XL-I analytical ultracentrifuge (Beckman Coulter), with
samples in 50 mM Na-citrate, 5% (w/v) glycerol, pH 6.0, buffer
conditions optimal for maximizing the oligomeric state of RecA
(Brenner et al., 1990). Rotor speeds of 6, 10, and 14 k rpm
at a temperature of 4 and 20◦C were used, and absorbance
scans (280 nm) were taken every 3 h until analysis of the
scans with WinMATCH (Jeffrey Lary, University of Connecticut,
Storrs, CT, USA) indicated that equilibrium had been reached. To validate the candidates and eliminate false positives,
compounds from the primary screens were studied in dose-
response (DR) experiments. Concentrations of compounds
ranging from 0.2 to 200 µM were prepared using 2-fold serial
dilutions. The DR experiments were performed with the pNY16,
pNY14, and pAJR145 reporters to evaluate inhibitor potency
and specificity. From the initial screen of 76 primary hits, 6
compounds showed a reproducible dose-dependent reduction
of MMC-induced stx::GFP expression (Supplementary Table S1). November 2016 | Volume 7 | Article 1930 Frontiers in Microbiology | www.frontiersin.org 3 Huerta-Uribe et al. Blocking Stx Expression and Transduction FIGURE 1 | Flow chart summarizing the screening process and DR experiments. A library of 17,500 compounds was screened using stx2::GFP, rpsm::GFP
and sulA::GFP reporter fusions. Seventy-six compounds that showed significant activity on stx2::GFP and suIA::GFP were identified from this initial screen, and
further dose-response assays narrowed this to six hits that reduced stx2::GFP expression in a dose-dependent manner. The single most potent compound was
taken forward for mechanistic studies. FIGURE 1 | Flow chart summarizing the screening process and DR experiments. A library of 17,500 compounds wa FIGURE 1 | Flow chart summarizing the screening process and DR experiments. A library of 17,500 compounds was screened using stx2::GFP, rpsm::GFP
and sulA::GFP reporter fusions. Seventy-six compounds that showed significant activity on stx2::GFP and suIA::GFP were identified from this initial screen, and
further dose-response assays narrowed this to six hits that reduced stx2::GFP expression in a dose-dependent manner. The single most potent compound was
taken forward for mechanistic studies. of MMC-induced stx2::GFP expression (Figure 3A) with
no effect on growth rate at concentrations up to 50 µM
(Figure 3B). Increasing the AHU1 concentration to 100 µM
further reduced stx2 expression but also inhibited the growth
rate by 10%. Sedimentation Equilibrium (SE)
Analytical Ultracentrifugation (AUC) As the genes encoding Stx are encoded on lytic
phages we also tested the ability of AHU1 to affect bacterial
lysis caused by phage mobilization. Addition of MMC to
ZAP0273 results in phage-mediated lysis that can be assayed
by monitoring the optical density of the culture. Lysis is
evident around 3 h after addition of MMC but is markedly
reduced by AHU1 at concentrations of 50 µm or greater
(Figure 3C). FIGURE 2 | Structures of the compounds used in this study. Chemical
structures of the hit compound AHU1 (A), and its derivatives AHU2 (B), AHU3
(C), and AHU4 (D). The maleimide moiety was determined to be essential for
the molecule to be biologically active since its loss in the derivative AHU4 led
to inactivity. Generation of a Focused Sub-Set of
Compounds to Study Structure-Activity
Relationship AHU1 is a planar molecule composed of a maleimide group and
a morpholine moiety, connected by a benzene ring (Figure 2A). In order to acquire information about the structure-activity
relationship of this compound, chemical derivatives of this
compound were synthesized. The morpholine group was chosen
for modification, as the presence of a heteroatom on the six-
membered ring readily facilitates chemical manipulation. Briefly,
the morpholine ring was replaced with its bioisostere piperazine
and the addition of a methyl group and a butyl chain on this
position gave the compounds AHU2 and AHU3, respectively
(Figures 2B,C). As we considered the maleimide part of the
compound was likely to be central to the activity of the molecule,
compound AHU4 was produced by the addition of a phenyl
group to the maleimide moiety (Figure 2D). FIGURE 2 | Structures of the compounds used in this study. Chemical
structures of the hit compound AHU1 (A), and its derivatives AHU2 (B), AHU3
(C), and AHU4 (D). The maleimide moiety was determined to be essential for
the molecule to be biologically active since its loss in the derivative AHU4 led
to inactivity. Effect of AHU1-4 on stx Expression Experiments were performed in triplicate, and
data plotted as the mean with standard deviation from the mean displayed by
error bars. FIGURE 4 | Inhibition of stx2::GFP expression and MMC-induced lysis
by AHU1-4. (A) Inhibition of stx2::GFP expression by 25 and 50 µM AHU1-4
at 6 h after addition of AHU1-4 and MMC. Data were calculated from triplicate
experiments and displayed as the mean inhibition with error bars showing the
standard deviation from the mean. Asterisks indicate a significant difference
(∗∗p < 0.001) from the original AHU1 concentration inhibition, determined by
Student’s unpaired t-test. (B) Inhibition of MMC-induced bacterial lysis after
addition of AHU1-4 and MMC. Experiments were performed in triplicate, and
data plotted as the mean with standard deviation from the mean displayed by
error bars. (Figure 4A). AHU3 produced a greater increase in inhibition
at 25 µM when compared with AHU1. AHU2 and AHU3
also resulted in inhibition of phage-mediated bacterial lysis
(Figure 4B). As AHU3 was the most effective inhibitor out
of the four compounds, it was selected for use in all further
experiments. FIGURE 3 | Effect of AHU1 on stx2::GFP expression, growth rate and FIGURE 3 | Effect of AHU1 on stx2::GFP expression, growth rate and
lysis of E. coli. ZAP0273 transformed with pNY16 was cultured in the
presence of 25−200 µM AHU1, and induced with 1 µg/ml MMC. Expression
of stx2::GFP expression was measured by fluorescence, and bacterial growth
by OD600. (A) Inhibition of MMC-induced stx2::GFP expression by AHU1. (B) Effect of AHU1 on bacterial growth in the absence of MMC. (C) Inhibition
of MMC-induced bacterial lysis by AHU1. Experiments were performed in
triplicate, and data plotted as the mean with standard deviation from the mean
displayed by error bars. FIGURE 3 | Effect of AHU1 on stx2::GFP expression, growth rate and
lysis of E. coli. ZAP0273 transformed with pNY16 was cultured in the
presence of 25−200 µM AHU1, and induced with 1 µg/ml MMC. Expression
of stx2::GFP expression was measured by fluorescence, and bacterial growth
by OD600. (A) Inhibition of MMC-induced stx2::GFP expression by AHU1. (B) Effect of AHU1 on bacterial growth in the absence of MMC. (C) Inhibition
of MMC-induced bacterial lysis by AHU1. Experiments were performed in
triplicate, and data plotted as the mean with standard deviation from the mean
displayed by error bars. Effect of AHU1-4 on stx Expression The most effective of these was Hit2Lead compound ID
5324836 (Figure 2A), a phenyl pyrroledione henceforth called
AHU1. This compound displays dose-dependent suppression p
To determine the activity of AHU1-4 we used the stx2::GFP
reporter and monitored expression following addition of MMC. Experiments were performed over a range of concentrations November 2016 | Volume 7 | Article 1930 Frontiers in Microbiology | www.frontiersin.org 4 Blocking Stx Expression and Transduction Huerta-Uribe et al. FIGURE 4 | Inhibition of stx2::GFP expression and MMC-induced lysis
by AHU1-4. (A) Inhibition of stx2::GFP expression by 25 and 50 µM AHU1-4
at 6 h after addition of AHU1-4 and MMC. Data were calculated from triplicate
experiments and displayed as the mean inhibition with error bars showing the
standard deviation from the mean. Asterisks indicate a significant difference
(∗∗p < 0.001) from the original AHU1 concentration inhibition, determined by
Student’s unpaired t-test. (B) Inhibition of MMC-induced bacterial lysis after
addition of AHU1-4 and MMC. Experiments were performed in triplicate, and
data plotted as the mean with standard deviation from the mean displayed by
error bars. FIGURE 3 | Effect of AHU1 on stx2::GFP expression, growth rate and
lysis of E. coli. ZAP0273 transformed with pNY16 was cultured in the
presence of 25−200 µM AHU1, and induced with 1 µg/ml MMC. Expression
of stx2::GFP expression was measured by fluorescence, and bacterial growth
by OD600. (A) Inhibition of MMC-induced stx2::GFP expression by AHU1. (B) Effect of AHU1 on bacterial growth in the absence of MMC. (C) Inhibition
of MMC-induced bacterial lysis by AHU1. Experiments were performed in
triplicate, and data plotted as the mean with standard deviation from the mean
displayed by error bars. FIGURE 4 | Inhibition of stx2::GFP expression and MMC-induced lysis
by AHU1-4. (A) Inhibition of stx2::GFP expression by 25 and 50 µM AHU1-4 FIGURE 4 | Inhibition of stx2::GFP expression and MMC-induced lysis
by AHU1-4. (A) Inhibition of stx2::GFP expression by 25 and 50 µM AHU1-4
at 6 h after addition of AHU1-4 and MMC. Data were calculated from triplicate
experiments and displayed as the mean inhibition with error bars showing the
standard deviation from the mean. Asterisks indicate a significant difference
(∗∗p < 0.001) from the original AHU1 concentration inhibition, determined by
Student’s unpaired t-test. (B) Inhibition of MMC-induced bacterial lysis after
addition of AHU1-4 and MMC. Effect of AHU3 on RecA Expression and
Activity Given that AHU3 inhibits expression and formation of functional
phage particles, we hypothesized that the compound may
function by affecting the activity of a protein involved in
the SOS response required for induction of phage expression. When the SOS response is initiated, RecA binds to single
stranded DNA, forming filaments with ATPase activity. These
activated RecA filaments mediate autocleavage of the key SOS
response repressor LexA and prophage repressors, allowing
gene expression and production of assembled phage particles. Therefore, one candidate target protein was RecA, the activity
of which can be measured using assays that quantify the amount
of free phosphate as a measure of ATP hydrolysis. This provides
an indirect measure of RecA nucleoprotein filament assembly
(Wigle and Singleton, 2007; Wigle et al., 2009; Sexton et al., 2010). FIGURE 5 | Reduction of MMC-induced phage production by 50 µM
AHU3. (A) Induction of JP10819 with 2 µg/ml MMC resulted in significantly
increased Stx phage production. Addition of 50 µM AHU3 produced a
significant decrease in phage production by both non-induced and
MMC-induced JP10819. The interaction between MMC and AHU3 was
determined to be significant (p < 0.001), implying that the reduction in phage
production by MMC-induced cells observed is not the result of the inhibitory
effect of AHU3 on bacterial growth. (B) Induction of JP5011 with 2 µg/ml
MMC resulted in significantly increased SLT phage production. Addition of
50 µM AHU3 produced a significant decrease in phage production by
MMC-induced JP5011. Asterisks indicate a significant difference (∗p < 0.05,
∗∗p < 0.001) in phage production between the groups indicated by capped
lines. The data shown are the average of triplicate individual experiments with
standard deviation from the mean displayed as error bars. Statistical
significance was determined by GLM analysis. Using an ATPase assay (Innova Biosciences) we optimized
RecA and ssDNA concentrations at 250 nM and 5 µM,
respectively. DMSO, required for solubility of AHU3, was kept
at a final concentration (2%), which did not interfere with the
enzymatic activity of RecA. The ability of AHU3 to inhibit RecA-
mediated ATP hydrolysis was investigated at concentrations
between 0.1 and 100 µM, revealing a concentration-dependent
decrease in enzymatic activity. The data were used to generate a
dose-response curve (Figure 6A) which was used to determine
a relative IC50 value of 7.72 µM using GraphPad Prism. Effect of AHU3 on RecA Expression and
Activity The
known RecA inhibitor curcumin (15) was used as a positive
control (Figure 6B) giving a relative IC50 of 6.69 µM. Since the
AHU4 compound showed no reduction in stx::GFP expression,
it was used as the negative control (Figure 6C). As expected,
AHU4 showed no dose-dependent effect on RecA-mediated ATP
hydrolysis 50 µM AHU3 reduced Stx2 expression to 47% activity (±3%)
compared with the positive control, demonstrating that AHU3
markedly inhibits Stx2 production in wild-type isolates. Effect of AHU3 on Stx2 Production by
EHEC 50 µM AHU3 reduced Stx2 expression to 47% activity (±3%)
compared with the positive control, demonstrating that AHU3
markedly inhibits Stx2 production in wild-type isolates. When no MMC was added to E. coli JP10819, basal levels
of Stx phage (104 phage/ml) were observed which increased
by four logs to 108 phage/ml following induction with MMC
(Figure 5A). The presence of 50 µM AHU3 resulted in a
significantly (P < 0.001) lower amount of phage produced with
a titre of 105 phage/ml; demonstrating that the production of
functional Stx phage particles is inhibited by AHU3. As the AHU compounds appeared to inhibit phage-mediated
bacterial lysis, we next explored whether these drugs could inhibit
expression of other prophages. To evaluate their activity in Gram-
positive bacteria, Staphylococcus aureus JP5011, which carries the
φSLT phage containing a tetracycline resistance cassette inserted
into the plv gene, was used. When induced by 2 µg/ml MMC,
approximately 106 transductants/ml of JP5011 culture were
produced. In contrast, only 102 transductants/ml were generated
from uninduced JP5011. Treatment of the MMC-induced culture
with 50 µM AHU3 showed greater than a log fold reduction in
the number of transductants generated (Figure 5B). Effect of AHU3 on Stx2 Production by
EHEC To assess if AHU3 could impact on Stx2 production in EHEC we
directly assayed toxin production in ZAP1620, a wild-type strain
that is lysogenized with both Stx2a- and Stx2c-encoding phages
and was isolated from a human patient. ZAP1620 was cultured
to mid-exponential phase and MMC added to induce toxin
expression. Culture supernatants were isolated and the levels
of toxin assessed using an ELISA-based assay (RIDASCREEN
R⃝
verotoxin, R-Biopharm). Data were calculated as a percentage of
expression compared with the positive control (100%), consisting
of inactivated Stx. Analysis of the supernatant of ZAP1620
showed strong MMC-dependent production of Stx2, at 85%
(±7%) activity compared with the positive control. Addition of (10−200 µM) for all four compounds. The effectiveness of
AHU1-4 was directly compared by calculating the percentage
inhibition of stx2::GFP expression 6 h post-MMC induction,
at two concentrations of the compounds, 25 and 50 µM,
respectively. Addition
of
AHU4,
containing
the
phenyl
substitution, resulted in no reduction in stx::GFP expression
thereby demonstrating the importance of the maleimide group
for activity. However, both AHU2 and AHU3 at 50 µM produced
near-complete inhibition of stx2::GFP, which was significantly
(p < 0.05) higher than AHU1, which when used at the same
concentration inhibited expression by approximately 60% November 2016 | Volume 7 | Article 1930 Frontiers in Microbiology | www.frontiersin.org 5 Huerta-Uribe et al. Blocking Stx Expression and Transduction FIGURE 5 | Reduction of MMC-induced phage production by 50 µM
AHU3. (A) Induction of JP10819 with 2 µg/ml MMC resulted in significantly
increased Stx phage production. Addition of 50 µM AHU3 produced a
significant decrease in phage production by both non-induced and
MMC-induced JP10819. The interaction between MMC and AHU3 was
determined to be significant (p < 0.001), implying that the reduction in phage
production by MMC-induced cells observed is not the result of the inhibitory
effect of AHU3 on bacterial growth. (B) Induction of JP5011 with 2 µg/ml
MMC resulted in significantly increased SLT phage production. Addition of
50 µM AHU3 produced a significant decrease in phage production by
MMC-induced JP5011. Asterisks indicate a significant difference (∗p < 0.05,
∗∗p < 0.001) in phage production between the groups indicated by capped
lines. The data shown are the average of triplicate individual experiments with
standard deviation from the mean displayed as error bars. Statistical
significance was determined by GLM analysis. Effect of AHU3 on Phage Transduction g
Having demonstrated that AHU3 was indeed a bona fide inhibitor
of Stx expression, we focused our attention on understanding the
mechanism of action underpinning its activity. As AHU3 was
able to reduce phage-induced lysis of EHEC, we hypothesized
that it might be able to reduce the generation of transducible
phage particles. Phage transduction assays were performed using
E. coli JP10819 that carries only the lysogenic Stx2 prophage
φP27. This prophage contains a tetracycline resistance cassette
inserted into the stx2 toxin gene, and lacks other lysogenic
prophages. Phage production was induced by addition of MMC
in the presence and absence of 50 µM AHU3 and the resultant
phages produced were isolated by filtration. The non-lysogenic
strain E. coli MG1655 was used as a recipient for the phage
isolated from E. coli JP10819. DISCUSSION In this study we employed a HTS to identify compounds with
an inhibitory effect on stx2 expression. We used bacterial GFP
reporter assays to ensure that any hits were active against intact
bacteria. Our selection criteria included a marked reduction
in stx2 expression, with minimal effects on growth rate or
off-target genes. When coupled with studies to determine the
dose-dependency of the response, only 6 compounds met our
criteria and we selected the best performing of these, AHU1, for
further study. Structure-activity studies resulted in the synthesis
of AHU3 that showed inhibition of stx2 gene expression and
phage production at a concentration that had minimal impact on
bacterial growth. Moreover, AHU3 was shown to be a genuine
inhibitor of Stx2 production and reduced toxin expression in a
wild-type EHEC O157 strain. However, the ability of AHU3 to
inhibit bacteriophage-mediated lysis, along with its activity in
both Gram-negative and Gram-positive strains indicated that the
activity of this compound was specific to an essential component
of the SOS response, rather than specifically on Stx production. FIGURE 6 | AHU3 inhibits RecA-mediated ATP hydrolysis. (A) Dose-response curve for AHU3 showing the inhibition of ATPase activity
with 0.1−100 µM AHU3. Data are plotted as a percentage of the average
absorbance of the assay wells containing 0 µM AHU3. The curve exhibits an
IC50 value of 7.72 µM and a Hill slope of 0.109. (B) Dose-response curve for
curcumin showing the inhibition of ATPase activity with 0.1−100 µM
curcumin. The curve exhibits an IC50 value of 6.69 µM and a Hill slope of
2.400. (C) Dose-response curve for AHU4 showing no inhibition of ATPase
activity. FIGURE 6 | AHU3 inhibits RecA-mediated ATP hydrolysis. FIGURE 6 | AHU3 inhibits RecA-mediated ATP hydrolysis. FIGURE 6 | AHU3 inhibits RecA-mediated ATP hydrolysis. (A) Dose-response curve for AHU3 showing the inhibition of ATPase activity
with 0.1−100 µM AHU3. Data are plotted as a percentage of the average
absorbance of the assay wells containing 0 µM AHU3. The curve exhibits an
IC50 value of 7.72 µM and a Hill slope of 0.109. (B) Dose-response curve for
curcumin showing the inhibition of ATPase activity with 0.1−100 µM
curcumin. The curve exhibits an IC50 value of 6.69 µM and a Hill slope of
2.400. (C) Dose-response curve for AHU4 showing no inhibition of ATPase
activity. oligomerization of E. coli RecA, revealing monomers in reversible
equilibrium with trimers, hexamers and dodecamers (12). Effect of AHU3 on RecA oligomerization M
inhibitor of Stx2 p
wild-type EHEC O
inhibit bacterioph
both Gram-negativ
activity of this com FIGURE 7 | AHU3 decreases RecA oligomerization. Whole cell weight
average molecular mass of RecA at AUC rotor speeds of 6, 10, and 14 k rpm. Ten micrometer RecA was studied alone, in the presence of 0.5% DMSO, and
in the presence of 0.5% DMSO and 100 µM AHU3. FIGURE 7 | AHU3 decreases RecA oligomerization. Whole cell weight
average molecular mass of RecA at AUC rotor speeds of 6, 10, and 14 k rpm. Ten micrometer RecA was studied alone, in the presence of 0.5% DMSO, and
in the presence of 0.5% DMSO and 100 µM AHU3. protein, we performed SE for RecA in the presence and absence of
AHU3. We also controlled for any influence of DMSO, included
as a solvent for AHU3, on protein behavior. The SE data were
analyzed with SEDPHAT using the species analysis model with
a single species, in order to gain a model-independent measure
of the whole-cell weight average molecular mass. The average
molecular mass of RecA oligomers in the absence of AHU3 and
DMSO was 556 kDa at 10,000 rpm and 404 kDa at 14,000 rpm
(Figure 7). When AHU3 was present in the sample, the average
molecular mass of RecA oligomers was greatly reduced at all rotor
speeds tested. The greatest changes were observed at 6,000 rpm,
with a 70% decrease in the average molecular mass compared
with untreated RecA (Figure 7). At 10,000 and 14,000 rpm,
the decreases were still marked, at 56 and 52%, respectively. As a control we also compared the average molecular mass of
RecA with that of the same protein in the presence of DMSO
and found only very minor effects on RecA oligomerization. These data demonstrate that AHU3 is affecting the formation of
larger oligomeric RecA species, resulting in an increase in lower
molecular mass oligomers. Effect of AHU3 on RecA oligomerization Effect of AHU3 on RecA oligomerization
Analytical ultracentrifugation allows the quantitative analysis of
macromolecules in solution. Previous studies have used AUC
sedimentation equilibrium experiments (SE) to analyze the November 2016 | Volume 7 | Article 1930 Frontiers in Microbiology | www.frontiersin.org 6 Huerta-Uribe et al. FIGURE 6 | AHU3 inhibits RecA-mediated ATP hydrolysis. (A) Dose-response curve for AHU3 showing the inhibition of ATPase activity
with 0.1−100 µM AHU3. Data are plotted as a percentage of the average
absorbance of the assay wells containing 0 µM AHU3. The curve exhibits an
IC50 value of 7.72 µM and a Hill slope of 0.109. (B) Dose-response curve for
curcumin showing the inhibition of ATPase activity with 0.1−100 µM
curcumin. The curve exhibits an IC50 value of 6.69 µM and a Hill slope of
2.400. (C) Dose-response curve for AHU4 showing no inhibition of ATPase
activity. Blocking Stx Expression and Transduction Huerta-Uribe et al. FIGURE 6 | AHU3 inhibits RecA-mediated ATP hydrolysis. (A) Dose-response curve for AHU3 showing the inhibition of ATPase activity
with 0.1−100 µM AHU3. Data are plotted as a percentage of the average
absorbance of the assay wells containing 0 µM AHU3. The curve exhibits an
IC50 value of 7.72 µM and a Hill slope of 0.109. (B) Dose-response curve for
curcumin showing the inhibition of ATPase activity with 0.1−100 µM
curcumin. The curve exhibits an IC50 value of 6.69 µM and a Hill slope of
2.400. (C) Dose-response curve for AHU4 showing no inhibition of ATPase
activity. oligomerization of E. coli RecA, revealing monomers in reversible
equilibrium with trimers hexamers and dodecamers (12) To
FIGURE 7 | AHU3 de
average molecular ma
Ten micrometer RecA
in the presence of 0.5
protein, we perform
AHU3. We also co
as a solvent for A
analyzed with SED
a single species, in
of the whole-cell
molecular mass of
DMSO was 556 kD
(Figure 7). When
molecular mass of
speeds tested. The
with a 70% decrea
with untreated Re
the decreases wer
As a control we a
RecA with that of
and found only v
These data demon
larger oligomeric R
molecular mass oli
DISCUSSION
In this study we e
an inhibitory effec
reporter assays to
bacteria. Our sele
in stx2 expression
off-target genes. W
dose-dependency
criteria and we sel
further study. Stru
of AHU3 that sho
phage production
bacterial growth. DISCUSSION To
assess whether AHU3 affects the oligomerization dynamics of the November 2016 | Volume 7 | Article 1930 Frontiers in Microbiology | www.frontiersin.org 7 Blocking Stx Expression and Transduction Huerta-Uribe et al. compounds (Wigle and Singleton, 2007; Wigle et al., 2009; Sexton
et al., 2010; Peterson et al., 2012). Bacterial strains with RecA
deletion or inactivation have displayed an increased susceptibility
to a number of antimicrobials and a delayed emergence of
resistance compared with wild-type strains (Singh et al., 2010; Thi
et al., 2011; Alam et al., 2016). Additionally, the production and
release of bacteriophages initiated by the SOS response can result
in horizontal transmission of genes located on mobile genetic
elements, and therefore inhibition of this process may limit
the dissemination of virulence factors and antibiotic resistance
(Beaber et al., 2004). compounds (Wigle and Singleton, 2007; Wigle et al., 2009; Sexton
et al., 2010; Peterson et al., 2012). Bacterial strains with RecA
deletion or inactivation have displayed an increased susceptibility
to a number of antimicrobials and a delayed emergence of
resistance compared with wild-type strains (Singh et al., 2010; Thi
et al., 2011; Alam et al., 2016). Additionally, the production and
release of bacteriophages initiated by the SOS response can result
in horizontal transmission of genes located on mobile genetic
elements, and therefore inhibition of this process may limit
the dissemination of virulence factors and antibiotic resistance
(Beaber et al., 2004). Initiation of the SOS response is controlled by RecA, the
activity of which requires formation of ssDNA-bound helical
homopolymeric filaments (Chen et al., 2008). We therefore
hypothesized that RecA was the biological target of AHU3, as
inhibition of either activation of the monomeric form of this
protein or function of assembled RecA filaments would prevent
autocleavage of the repressors of SOS and phage genes. The substitution of the AHU3 maleimide group with a phenyl
group, generating AHU4, caused the compound to become
inactive. Although natural maleimides are rare in nature, several
small compounds containing a modified maleimide moiety have
been identified in fungal and bacterial organisms. Pencolide,
produced by Penicillium strains, exhibits both bacteriostatic and
fungicidal activity (Lucas et al., 2007), while the fungal compound
farinomalein shows potent activity against plant pathogen
oomycetes (Putri et al., 2014). A Streptomycin showdoensis
maleimide compound, showdomycin, targets oxidoreductases,
and transferases involved in major cellular functions associated
with virulence, growth, and persistance in both Gram-negative
and Gram-positive strains (Böttcher and Sieber, 2010). FUNDING AH-U was supported by a Wellcome Trust studentship ref
089891/Z/09/Z. ZM was funded by the University of Glasgow
Industrial Postgraduate Programme. JC was supported by the
Yorkhill Hospital Trust, grant ref YRSS/PSG/2014/05. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found
online
at:
http://journal.frontiersin.org/article/10.3389/fmicb. 2016.01930/full#supplementary-material Several groups have suggested RecA as an attractive target
for inhibition and performed specific screens to identify suitable Bergan, J., Dyve Lingelem, A. B., Simm, R., Skotland, T., and Sandvig, K. (2012).
Shiga toxins. Toxicon 60, 1085–1107. doi: 10.1016/j.toxicon.2012.07.016
Böttcher, T., and Sieber, S. (2010). Showdomycin as a versatile chemical tool for
the detection of pathogenesis-associated enzymes in bacteria. J. Am. Chem. Soc.
132, 6964–6972. doi: 10.1021/ja909150y
Brenner, S. L., Zlotnick, A., and Stafford, W. F. (1990). RecA protein self-assembly.
II. Analytical equilibrium ultracentrifugation studies of the entropy-driven self-
association of RecA. J. Mol. Biol. 216, 949–964. doi: 10.1016/S0022-2836(99)
80013-8 DISCUSSION While RecA inhibitors might not show a high degree of
selectivity due to the high conservation of RecA across bacteria,
they may have potential as drugs administered alongside other
more specific treatments to increase bacterial susceptibility to
anti-bacterials or limit development of antibiotic resistance. Our work has demonstrated that RecA inhibition can also
significantly limit expression of prophage genes encoding
virulence factors, including Stx , emphasizing their additional
potential as therapies to limit bacterial production of key phage-
encoded virulence factors. Maleimide moieties are well known as Michael acceptors, and
react readily with sulfhydryl groups of cysteine residues. It is
therefore highly likely that AHU3 reacts with RecA through
this addition-elimination mechanism, covalently binding to one
or more of the three cysteine residues present in RecA. This
mechanism of inhibition has been reported for other small
maleimide-based molecules; a phenyl-substituted maleimide
with anti-angiogenic activity shows potential as a candidate for
treatment of proliferative retinal disorders (Nowak-Sliwinska
et al., 2012), while a series of maleimide-based compounds with
inhibitory activity on monoamine oxidase B have shown promise
as a potential therapy for Parkinson’s disease (Manley-King
et al., 2009). Interestingly, a chloromaleimide-based compound
bearing a striking structural resemblance to AHU3 has been
reported as an inhibitor of RAD51, the human homologue of
RecA. This compound, RI-1, was shown to covalently bind a
single cysteine residue on a RAD51 located in the interface
required for monomer-monomer interaction (Budke et al.,
2012). In support of this hypothesis, we showed that AHU3
directly affected RecA in solution by reducing the formation of
larger oligomeric species. The effects of these compounds on
eukaryotic cells will form part of ongoing studies into the possible
application of these molecules to treat infection-based pathology
in relation of phage-induced toxins. However, this is dependent
on lack of cross reactivity for RAD51. AUTHOR CONTRIBUTIONS AH-U and ZM contributed equally to this work and performed
the chemical synthesis and analysis of the mechanism of action. NY and HU performed the HTS. SF performed all work
associated with clinical isolates. NC and JC performed the phage
transduction assays. GD, ME, OB, RM, DG, and AR conceived
and supervised various aspects of the study. All authors have read,
contributed and approved the manuscript. REFERENCES Sexton, J. Z., Wigle, T. J., He, Q., Hughes, M., Smith, G. R., Singleton, S. F., et al. (2010). Novel inhibitors of E. coli RecA ATPase activity. Curr. Chem. Genomics
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©
2016
Huerta-Uribe,
Marjenberg,
Yamaguchi,
Fitzgerald,
Connolly,
Carpena,
Uvell,
Douce,
Elofsson,
Byron,
Marquez,
Gally
and
Roe. This
is
an
open-access
article
distributed
under
the
terms
of
the
Creative
Commons
Attribution
License
(CC
BY). The
use,
distribution
or
reproduction
in
other
forums
is
permitted,
provided
the
original
author(s) or licensor are credited and that the original publication in this
journal is cited, in accordance with accepted academic practice. No use,
distribution or reproduction is permitted which does not comply with these
terms. Peterson, E. J. R., Janzen, W. P., Kireev, D., and Singleton, S. F. (2012). High-
throughput screening for RecA inhibitors using a transcreener adenosine 5’-
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Sexton, J. Z., Wigle, T. J., He, Q., Hughes, M., Smith, G. R., Singleton, S. F., et al. REFERENCES I., Kinoshita, H., Kitani, S., Ihara, F., Sakihama, Y.,
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|
https://openalex.org/W4386849958
|
https://www.frontiersin.org/articles/10.3389/fbioe.2023.1261178/pdf?isPublishedV2=False
|
English
| null |
A novel “off-on” fluorescent probe for the detection of nickel ions and its clinical application
|
Frontiers in bioengineering and biotechnology
| 2,023
|
cc-by
| 6,839
|
TYPE Methods
PUBLISHED 18 September 2023
DOI 10.3389/fbioe.2023.1261178 TYPE Methods
PUBLISHED 18 September 2023
DOI 10.3389/fbioe.2023.1261178 OPEN ACCESS nickel ions, fluorescent probe, off-on fluorescent probe, clinical application, sequence-
specific cleavage OPEN ACCESS EDITED BY
Hamed Golmohammadi,
Chemistry and Chemical Engineering
Research Center, Iran Hua-Wei Yi1,2, Xian-Mo Wang1,2, Jia-Hao Wu1,2, Chang-Li Zhang3*
and Yi-Di Ding4* 1Laboratory Department, The First Affiliated Hospital of Yangtze University, Jingzhou, Hubei, China,
2Laboratory Department, The First People’s Hospital of Jingzhou, Jingzhou, Hubei, China, 3Department of
Pharmacy, The First Affiliated Hospital of Yangtze University, Jingzhou, Hubei, China, 4Department of
Infectious Diseases, Tangdu Hospital, Air Force Medical University, Xi’an, Shaanxi, China Nickel serves as an essential micronutrient for the human body, playing a vital role
in
various
enzymatic
processes. However,
excessive
nickel
entering
the
environment can cause pollution and pose serious risks to animals, plants, and
human health. High concentrations of nickel ions in the human body increase the
risk of various diseases, highlighting the need for accurate measurement of nickel
ions levels. In this study, we designed a sequence-specific cleavage probe for
nickel (II) ion called SSC-Ni. Similar to the TaqMan probe, SSC-Ni is an off-on
fluorescent probe with an exceptionally low background fluorescence signal. It
exhibits high detection specificity, making it highly selective for nickel ions, and the
detection limit of the probe towards Ni2+ is as low as 82 nM. The SSC-Ni probe can
be utilized for convenient and cost-effective high-throughput quantitative
detection of nickel ions in serum. Its user-friendly operation and affordability
make it a practical solution. By addressing the lack of simple and effective nickel
ion detection methods, this probe has the potential to contribute significantly to
environmental monitoring and the protection of human health. CITATION
Yi H-W, Wang X-M, Wu J-H, Zhang C-L
and Ding Y-D (2023), A novel “off-on”
fluorescent probe for the detection of
nickel ions and its clinical application. Front. Bioeng. Biotechnol. 11:1261178. doi: 10.3389/fbioe.2023.1261178 CITATION
Yi H-W, Wang X-M, Wu J-H, Zhang C-L
and Ding Y-D (2023), A novel “off-on”
fluorescent probe for the detection of
nickel ions and its clinical application. Front. Bioeng. Biotechnol. 11:1261178. doi: 10.3389/fbioe.2023.1261178 COPYRIGHT
© 2023 Yi, Wang, Wu, Zhang and Ding. This is an open-access article distributed
under the terms of the Creative
Commons Attribution License (CC BY). The use, distribution or reproduction in
other forums is permitted, provided the
original author(s) and the copyright
owner(s) are credited and that the original
publication in this journal is cited, in
accordance with accepted academic
practice. No use, distribution or
reproduction is permitted which does not
comply with these terms. Frontiers in Bioengineering and Biotechnology CITATION
Yi H-W, Wang X-M, Wu J-H, Zhang C-L
and Ding Y-D (2023), A novel “off-on”
fluorescent probe for the detection of
nickel ions and its clinical application.
Front. Bioeng. Biotechnol. 11:1261178.
doi: 10.3389/fbioe.2023.1261178 KEYWORDS
nickel ions, fluorescent probe, off-on fluorescent probe, clinical application, sequence-
specific cleavage 2.5 Calculation of detection limit The detection limit was calculated from the formula: DL = 3 × SD/
k, where SD is the standard deviation of the blank solution, k is the slope
of the calibration curve (Yu et al., 2016). In the probe solution without
nickel ions, there was no significant change in fluorescence intensity,
and low concentration of nickel ions can be used as a blank solution to
calculate SD value (Zare et al., 2017). Ten times of fluorescence intensity
curve of probe with 0.1 μM Ni2+ were test and SD could be calculated
from fluorescence enhancement rate (slope). The linear regression
curve of probe SSC-Ni was then fitted to fluorescence enhancement
rate of nickel ions at different concentrations to calculate the slope k. 2.7 Data analysis HPLC was used to detect the purity of probe SSC-Ni. The
C18 column with a volume of 10 μL was utilized, solvent A was 0.1%
trifluoroacetic
in
100%
acetonitrile,
solvent
B
was
0.1%
trifluoroacetic in 100% water, the flow rate was 1.0 mL/min, and
the detection wavelength was 220 nm. The molecular weight of the
probe was detected with the Agilent-6125B mass spectrometer, and
the theoretical molecular weight is 1,361.40 Da. The qPCR instrument was used to monitor the degradation of the
probe SSC-Ni by nickel ions in real-time. The raw data of fluorescence
intensity was exported, then the background fluorescence signal was
deducted,
and
finally
the
data
was
imported
into
the
Origin2018 software for linear fitting and mapping. The relevant
fitting parameters such as intercept, slope, and R2 could be obtained. 2.1 Synthesis of fluorescent probe SSC-Ni Four blood samples without anticoagulants were taken, and
serum was obtained by centrifugation at 4,000 rpm for 5 min after
the blood was fully coagulated. The probe SSC-Ni with a final
concentration of 1 μM was added to the serum, or additional 1,
10 μM of Ni2+ were added. Meanwhile, serum without probe was
used as a negative control, and the fluorescence signal of the FAM
channel was detected by qPCR instrument at 37°C. The amino acids sequence of the SSC-Ni probe is Gly-Ser-His-
His-Trp (-GSHHW-). During the polypeptide chains synthesis, the
Dabcyl group was introduced at the amino terminus and Lys (5-
FAM) was introduced at the carboxyl terminus. The synthesis of
probe SSC-Ni was completed by GL Biochem Ltd. (Shanghai,
China). 1 Introduction Inspired by the principles of the TaqMan
probe (Cao et al., 2012), the SSC-Ni probe utilizes an “off-on”
fluorescence mechanism by attaching a fluorescence quencher and
fluorophore at both ends of the polypeptide sequences (Gly-Ser-His-
His-Trp). To the authors’ knowledge, no fluorescent probes based
on sequence-specific cleavage by nickel ions have been reported. This probe demonstrates high specificity in detecting nickel (II) ion,
with a detection limit as low as 82 nM. Notably, the probe offers the
advantages of simple operation and low cost. Additionally, we have
conducted preliminary investigations into the application of the
SSC-Ni probe for detecting nickel ions in the blood. 2.4 Fluorescence signal detection of probe Different concentrations of nickel ions or other metal ions were
added to the probe SSC-Ni, and the final concentration of the probe was
1 μM. The buffer condition was 50 mM HEPES 100 mM NaCl, and
pH 8.2. Take 25 μL in a PCR tube and detect the real-time fluorescence
intensity of FAM by qPCR instrument. The program setting of the PCR
instrument included 60 cycles of hold at 37°C for 20 min, followed by
data acquisition at 37°C for 30 s on AGS 4800 real-time PCR system
(Bioanyu Technology Co., Ltd., Hangzhou, China). Building upon the work of Dang et al. (2019), who reported
polypeptide sequences specifically cleaved by nickel ions. We
designed a sequence-specific cleavage probe for nickel ions
known as SSC-Ni. Inspired by the principles of the TaqMan
probe (Cao et al., 2012), the SSC-Ni probe utilizes an “off-on”
fluorescence mechanism by attaching a fluorescence quencher and
fluorophore at both ends of the polypeptide sequences (Gly-Ser-His-
His-Trp). To the authors’ knowledge, no fluorescent probes based
on sequence-specific cleavage by nickel ions have been reported. This probe demonstrates high specificity in detecting nickel (II) ion,
with a detection limit as low as 82 nM. Notably, the probe offers the
advantages of simple operation and low cost. Additionally, we have
conducted preliminary investigations into the application of the
SSC-Ni probe for detecting nickel ions in the blood. Frontiers in Bioengineering and Biotechnology frontiersin.org 1 Introduction In recent years, nickel ion-selective
electrodes have been used for monitoring nickel ions (Abbaspour
and Izadyar, 2001; Tashkhourian, 2015; Sheikh et al., 2017). Nevertheless, many of these electrodes have high detection limits,
narrow working concentration ranges, and are susceptible to
interference from various ions, such as H+, Na+, K+, Ba2+, Ag+,
and Hg2+. Alternatively, fluorescent probes have gained the attention
of their high sensitivity, selectivity, and ease of use in nicked ion
detection (Song et al., 2021; Wang et al., 2022a; Wang et al., 2022b). Building upon the work of Dang et al. (2019), who reported
polypeptide sequences specifically cleaved by nickel ions. We
designed a sequence-specific cleavage probe for nickel ions
known as SSC-Ni. Inspired by the principles of the TaqMan
probe (Cao et al., 2012), the SSC-Ni probe utilizes an “off-on”
fluorescence mechanism by attaching a fluorescence quencher and
fluorophore at both ends of the polypeptide sequences (Gly-Ser-His-
His-Trp). To the authors’ knowledge, no fluorescent probes based
on sequence-specific cleavage by nickel ions have been reported. This probe demonstrates high specificity in detecting nickel (II) ion,
with a detection limit as low as 82 nM. Notably, the probe offers the
advantages of simple operation and low cost. Additionally, we have
conducted preliminary investigations into the application of the
SSC-Ni probe for detecting nickel ions in the blood. inductively coupled plasma mass spectrometry (Musil et al., 2021;
Xu et al., 2021), and flame photometry (de Oliveira et al., 2017;
Barahoei et al., 2021). However, these methods often require
expensive equipment and complex sample processing, limiting
their practical application. In recent years, nickel ion-selective
electrodes have been used for monitoring nickel ions (Abbaspour
and Izadyar, 2001; Tashkhourian, 2015; Sheikh et al., 2017). Nevertheless, many of these electrodes have high detection limits,
narrow working concentration ranges, and are susceptible to
interference from various ions, such as H+, Na+, K+, Ba2+, Ag+,
and Hg2+. Alternatively, fluorescent probes have gained the attention
of their high sensitivity, selectivity, and ease of use in nicked ion
detection (Song et al., 2021; Wang et al., 2022a; Wang et al., 2022b). Building upon the work of Dang et al. (2019), who reported
polypeptide sequences specifically cleaved by nickel ions. We
designed a sequence-specific cleavage probe for nickel ions
known as SSC-Ni. 1 Introduction Nickel, a widely used transition metal in industrial production, is also an essential
micronutrient distributed throughout the human body. It mainly exists as divalent ions and
plays a vital role in numerous enzymes (Genchi et al., 2020; Anchidin-Norocel et al., 2021). Nickel ions are found in various tissues such as the liver, kidney, spinal cord, brain, heart,
cartilage, lung connective tissue, and skin (Afzali and Mohammadi, 2011; Rajivgandhi et al.,
2021). However, excessive uptake of nickel ions can lead to various diseases, including
asthma, acute pneumonitis, dermatitis, lung cancer, and sinus cancer (Kang et al., 2017). Meta-analysis has indicated a correlation between high serum nickel levels and breast cancer,
suggesting nickel exposure as a risk factor (Yu and Zhang, 2017). Studies by Chiou et al. (2014) have also linked nickel accumulation in lung tissues to an increased risk of
p53
mutation
and
lung
carcinogenesis. Moreover,
excessive
nickel
entering
the
environment can cause pollution, making it critical to monitor nickel ion levels in the
environment, animals, and plants. Traditional methods for detecting trace amounts of nickel include flame atomic
absorption spectrometry (Lemes and Tarley, 2021; Rocha and Chemistry, 2021), Frontiers in Bioengineering and Biotechnology 01 frontiersin.org frontiersin.org Yi et al. Yi et al. 10.3389/fbioe.2023.1261178 the samples at room temperature (~25°C) for 12 h and observe the
color change of the solution. inductively coupled plasma mass spectrometry (Musil et al., 2021;
Xu et al., 2021), and flame photometry (de Oliveira et al., 2017;
Barahoei et al., 2021). However, these methods often require
expensive equipment and complex sample processing, limiting
their practical application. In recent years, nickel ion-selective
electrodes have been used for monitoring nickel ions (Abbaspour
and Izadyar, 2001; Tashkhourian, 2015; Sheikh et al., 2017). Nevertheless, many of these electrodes have high detection limits,
narrow working concentration ranges, and are susceptible to
interference from various ions, such as H+, Na+, K+, Ba2+, Ag+,
and Hg2+. Alternatively, fluorescent probes have gained the attention
of their high sensitivity, selectivity, and ease of use in nicked ion
detection (Song et al., 2021; Wang et al., 2022a; Wang et al., 2022b). inductively coupled plasma mass spectrometry (Musil et al., 2021;
Xu et al., 2021), and flame photometry (de Oliveira et al., 2017;
Barahoei et al., 2021). However, these methods often require
expensive equipment and complex sample processing, limiting
their practical application. FIGURE 1 FIGURE 1
Schematic diagram of the probe SSC-Ni. The Dabcyl quenching
group was attached at the amino end of the -GSHHW- sequence and
the FAM fluorophore was attached at the carboxyl terminus. The
fluorescence signal of FAM can be inhibited by Dabcyl. Nickel
ions specifically cleave the -GSHHW- sequence and FAM
fluorescence signals can be detected in the presence of appropriate
excitation light. FIGURE 1
Schematic diagram of the probe SSC-Ni. The Dabcyl quenching
group was attached at the amino end of the -GSHHW- sequence and
the FAM fluorophore was attached at the carboxyl terminus. The
fluorescence signal of FAM can be inhibited by Dabcyl. Nickel
ions specifically cleave the -GSHHW- sequence and FAM
fluorescence signals can be detected in the presence of appropriate
excitation light. We also used a qPCR instrument to detect the FAM fluorescence
signal of probe SSC-Ni in real-time, as shown in Figure 3B. Nickel
ions at final concentrations of 1 and 10 μM were added to the 1 μM
probe SSC-Ni, respectively. The fluorescence signal of FAM was
detected at 37°C. As shown in the figure the fluorescence intensity
gradually increases after the addition of nickel ions, and the
fluorescence intensity of 10 μM increases faster than that of 1 μM
nickel ions. Besides, there was no significant change in fluorescence
intensity when other metal ions such as Ca2+, Cu2+, Zn2+, Mg2+, Fe3+,
and K+ were added to the SSC-Ni probe. In addition, 10 μM Ni2+ and
other metal ions were simultaneously added to the probe to monitor
the fluorescence intensity, it was found that there was no significant
difference between its intensity and that of 10 μM Ni2+ alone. This
indicates that nickel ions can specifically degrade probe SSC-Ni, and
the probe was not sensitive to other metal ions. nickel ions, and the cleavage site of the -GSHHW- sequence is between
G and S residues (Dang et al., 2019). Using the peptide chains in vitro
synthesis method, we synthesized the -GSHHW- sequence and
connected Dabcyl fluorescent quencher and FAM dye at its amino
and carboxyl terminals, respectively. Thus, the probe SSC-Ni for
detecting nickel ions was designed as shown in Figure 1. Nickel ions
were able to degrade the -GSHHW- sequence and release the free FAM
fluorophore so that the fluorescence signal can be detected. Purity analysis of the SSC-Ni probe was performed using high-
performance liquid chromatography (HPLC), as shown in Figure 2A. 3.3 Preparation of standard curves To further investigate the correlation between nickel ion
concentration and degradation rate of probe SSC-Ni, nickel ions
with different concentrations (0.1–10 μM) were added to the 1 μM
probe to detect the fluorescence signal of FAM at 37°C, as shown in
Figure 4A. The higher the concentration of nickel ions, the faster the
fluorescence signal increases, and the fluorescence intensity data was
close to a straight line. Therefore, linear fitting was performed on the
fluorescence signal, and the intercept, slope, and R2 were shown in
Figure 4B, with all R2 values above 0.986. Further, the slope of each FIGURE 1 There was a high peak at the elution time of 9.107 min, and the
proportion of this peak was as high as 96.6% through area integration,
which indicates that the SSC-Ni probe has high purity. Meanwhile, we
also used a mass spectrometer to detect probe SSC-Ni, as shown in
Figure 2B, with an observed molecular weight of 1,360.39 Da, which is
very close to the theoretical molecular weight. 3.1 Design and detection of probe SSC-Ni Nickel ions with final concentrations of 1, 10, 100, and 1,000 μM
were added to the 5 μM SSC-Ni probe under the reported buffer
conditions (Dang et al., 2019), and the buffer for SSC-Ni probe in
this study were 50 mM HEPES, 100 mM NaCl, and pH 8.2. Leave Site-specific protein cleavage is essential for many protein
production protocols, and the polypeptide sequence Gly-Ser-His-
His-Trp (-GSHHW-) can be recognized and specifically cleaved by 02 frontiersin.org Yi et al. 10.3389/fbioe.2023.1261178 10.3389/fbioe.2023.1261178 Yi et al. FIGURE 1
Schematic diagram of the probe SSC-Ni. The Dabcyl quenching
group was attached at the amino end of the -GSHHW- sequence and
the FAM fluorophore was attached at the carboxyl terminus. The
fluorescence signal of FAM can be inhibited by Dabcyl. Nickel
ions specifically cleave the -GSHHW- sequence and FAM
fluorescence signals can be detected in the presence of appropriate
excitation light. were added to the 5 μM probe, respectively, and then placed at room
temperature (~25°C). A probe sample without nickel ions was used
as a negative control, as shown in Figure 3A. After 12 h of reaction at
room temperature, significant color changes in the solution can be
observed. The higher the concentration of nickel ions, the more
obvious the color change. There was no significant change in color
for the negative control group. In addition, we detected the
excitation and emission spectra of the probe (5 μM), as well as
emission spectra after incubation at 25°C for 12 h with equivalent
nickel ions (Supplementary Figure S1), and the fluorescence
intensity was enhanced after the addition of nickel ions. were added to the 5 μM probe, respectively, and then placed at room
temperature (~25°C). A probe sample without nickel ions was used
as a negative control, as shown in Figure 3A. After 12 h of reaction at
room temperature, significant color changes in the solution can be
observed. The higher the concentration of nickel ions, the more
obvious the color change. There was no significant change in color
for the negative control group. In addition, we detected the
excitation and emission spectra of the probe (5 μM), as well as
emission spectra after incubation at 25°C for 12 h with equivalent
nickel ions (Supplementary Figure S1), and the fluorescence
intensity was enhanced after the addition of nickel ions. 3.4 Application of probe SSC-Ni in the blood High concentrations of nickel ions in the human body will
increase the risk of breast cancer, lung cancer, and other tumors, and
this study attempts to use probe SSC-Ni to detect the concentration
of nickel ions in the blood. To avoid the interference of
anticoagulants on the detection, four blood samples without
anticoagulants
were
selected. The
serum
was
collected
by
centrifugation
and
nickel
ions
were
added
at
a
final
concentration of 1 μM. The fluorescence signal of FAM was
detected at 37°C as shown in Figure 5. 3.2 Degradation of probe SSC-Ni by nickel
ions To study the degradation of probe SSC-Ni by nickel ions, the
nickel ions with final concentrations of 1, 10, 100, and 1,000 μM FIGURE 2
HPLC and mass spectrometry detection of SSC-Ni probe. (A) The purity of probe SSC-Ni was detected by HPLC, using a C18 column with a volume
of 10 μL. The mobile phase flow rate was 1.0 mL/min and the detection wavelength was 220 nm. (B) The molecular weight of the probe SSC-Ni was
detected by the Agilent-6125B mass spectrometer, and the molecular weight was measured as 1,360.39 Da. FIGURE 2
HPLC and mass spectrometry detection of SSC-Ni probe. (A) The purity of probe SSC-Ni was detected by HPLC, using a C18 column with a volume
of 10 μL. The mobile phase flow rate was 1.0 mL/min and the detection wavelength was 220 nm. (B) The molecular weight of the probe SSC-Ni was
detected by the Agilent-6125B mass spectrometer, and the molecular weight was measured as 1,360.39 Da. Frontiers in Bioengineering and Biotechnology Frontiers in Bioengineering and Biotechnology 03 frontiersin.org Yi et al. 10.3389/fbioe.2023.1261178 FIGURE 3
Degradation of probe SSC-Ni by nickel ions. (A) Nickel ions with final concentrations of 1, 10, 100, and 1,000 μM were added to the 5 μM probe SSC-
Ni, respectively, and the color change of the solution was observed at room temperature. (B) qPCR instrument was used for real-time detection of SSC-Ni
probe degradation. 1 μM and 10 μM nickel ions or 10 μM other metal ions were added to the probe SSC-Ni at a final concentration of 1 μM. The
fluorescence signal of FAM was detected every 20 min at 37°C. (C) Study the interference effect of other metal ions on nickel ion detection. 10 μM
metal ions alone or 10 μM Ni2+ and other metal ions were added to the probe, and the fluorescence intensity was detected after holding at 37°C for 14 h. FIGURE 3
Degradation of probe SSC-Ni by nickel ions. (A) Nickel ions with final concentrations of 1, 10, 100, and 1,000 μM were added to the 5 μM probe SSC-
Ni, respectively, and the color change of the solution was observed at room temperature. (B) qPCR instrument was used for real-time detection of SSC-Ni
probe degradation. 1 μM and 10 μM nickel ions or 10 μM other metal ions were added to the probe SSC-Ni at a final concentration of 1 μM. Frontiers in Bioengineering and Biotechnology 3.2 Degradation of probe SSC-Ni by nickel
ions The
fluorescence signal of FAM was detected every 20 min at 37°C. (C) Study the interference effect of other metal ions on nickel ion detection. 10 μM
metal ions alone or 10 μM Ni2+ and other metal ions were added to the probe, and the fluorescence intensity was detected after holding at 37°C for 14 h. line was plotted against log10 nickel concentration to establish a
standard curve, as shown in Figure 4C. The R2 value of the standard
curve was 0.996. linearly fitted. The relevant parameters were shown in Figure 5,
all R2 values were above 0.984. The concentration of nickel ions
can be calculated based on the slope and standard curve, which
were 0.185 ± 0.006, 0.140 ± 0.004, 0.144 ± 0.004, 0.063 ±
0.003 μM, respectively (Table 1). Notably, the detection
results were extremely close to the reported serum nickel
concentration
(Yu
and
Zhang,
2017). Then
the
final
concentration of 1 μM and 10 μM Ni2+ were added to the
serum samples containing the probe. The experiments were
repeated for three time, and the concentration of nickel ions
were shown in Table 1. The recovery rates of Ni2+ in serum
samples were both greater than 90%. The detection limit (DL) of the probe can be calculated from the
formula: DL = 3 × SD/k (Yu et al., 2016). The detection limit of the
SSC-Ni probe was as low as 82 nM (Figure 4C), while the relative
standard deviation for 10 replicate detections of 0.1 μM Ni2+
was 4.8%. frontiersin.org 3.5 Comparison with other reported
fluorescent probes The probe SSC-Ni
with a final concentration of 1 μM were added to serum samples
one to four, and a serum sample without the probe was used as a
negative control. The fluorescence signal of the FAM was detected by
qPCR instrument at 37°C. Origin2018 software was used for linear
fitting of the fluorescence data, and the slope and R2 were labeled on
the figure. FIGURE 4
Preparation standard curve of nickel ions. (A) Nickel ions with final concentrations of 0, 0.1, 0.5, 1, 5, and 10 μM were added to the 1 μM probe SSC-
Ni, respectively. The fluorescence signal of the FAM channel of qPCR equipment was detected at 37°C. (B) Origin2018 software was used for linear fitting
of the fluorescence data, and related parameters such as intercept, slope, and R2 can be obtained. (C) The slope of each line was plotted against
log10 nickel concentration to establish a standard curve. FIGURE 4
Preparation standard curve of nickel ions. (A) Nickel ions with final concentrations of 0, 0.1, 0.5, 1, 5, and 10 μM were added to the 1 μM probe SSC-
Ni, respectively. The fluorescence signal of the FAM channel of qPCR equipment was detected at 37°C. (B) Origin2018 software was used for linear fitting
of the fluorescence data, and related parameters such as intercept, slope, and R2 can be obtained. (C) The slope of each line was plotted against
log10 nickel concentration to establish a standard curve. FIGURE 5
Concentration detection of nickel ions in blood samples. Four
blood samples without anticoagulant were taken and serum was
obtained by centrifugation at 4,000 rpm for 5 min. The probe SSC-Ni
with a final concentration of 1 μM were added to serum samples
one to four, and a serum sample without the probe was used as a
negative control. The fluorescence signal of the FAM was detected by
qPCR instrument at 37°C. Origin2018 software was used for linear
fitting of the fluorescence data, and the slope and R2 were labeled on
the figure. detection capability of SSC-Ni probe compared to optical chemical
sensor MMT (Aksuner et al., 2012). However, the probe designed
in this study is sufficient for nickel ions detection in serum
samples. In addition, the concentration of nickel ions in water
samples is also within the detection range of the SSC-Ni probe
(Alizadeh et al., 2016), which can theoretically detect nickel ions in
water samples. 3.5 Comparison with other reported
fluorescent probes To the authors’ knowledge, the SSC-Ni is the only nickel ion
detection probe that based on sequence-specific cleavage. The
limitation of this probe is that the detection time (greater than
20 h) is much longer than other probes, which may be related to
the low degradation efficiency of nickel ions on -GSHHW-
peptides. Further optimization of the peptide sequences may
shorten the detection time. Besides, the fluorescence intensity of
the probe can be automatically measured by qPCR instruments
and
achieve
high-throughput
detection
of
nickel
ions
concentration, which can partially compensate for the long
detection time. Concentration detection of nickel ions in blood samples. Four
blood samples without anticoagulant were taken and serum was
obtained by centrifugation at 4,000 rpm for 5 min. The probe SSC-Ni
with a final concentration of 1 μM were added to serum samples
one to four, and a serum sample without the probe was used as a
negative control. The fluorescence signal of the FAM was detected by
qPCR instrument at 37°C. Origin2018 software was used for linear
fitting of the fluorescence data, and the slope and R2 were labeled on
the figure. frontiersin.org 3.5 Comparison with other reported
fluorescent probes The SSC-Ni probe designed in this study for detecting nickel
ions was compared with other reported fluorescent probes, as
shown in Table 2. The detection limit and linear range of the probe
are not significantly different from other reported probes. Meanwhile, the probe has high selectivity and is not susceptible
to interference from other ions. There is still a significant gap in the The fluorescence signal of the serum samples gradually
increased with heat retention, and the fluorescence data were 04 frontiersin.org Yi et al. 10.3389/fbioe.2023.1261178 detection capability of SSC-Ni probe compared to optical chemical
sensor MMT (Aksuner et al., 2012). However, the probe designed
in this study is sufficient for nickel ions detection in serum
samples. In addition, the concentration of nickel ions in water
samples is also within the detection range of the SSC-Ni probe
(Alizadeh et al., 2016), which can theoretically detect nickel ions in
water samples. To the authors’ knowledge, the SSC-Ni is the only nickel ion
detection probe that based on sequence-specific cleavage. The
limitation of this probe is that the detection time (greater than
20 h) is much longer than other probes, which may be related to
the low degradation efficiency of nickel ions on -GSHHW-
peptides. Further optimization of the peptide sequences may
shorten the detection time. Besides, the fluorescence intensity of
the probe can be automatically measured by qPCR instruments
and
achieve
high-throughput
detection
of
nickel
ions
concentration, which can partially compensate for the long
detection time. 4 Discussion
In
conclusion,
this
paper
introduces
a
novel
off-on
fluorescent probe called SSC-Ni for the detection of nickel (II)
ion. Similar to the TaqMan probes, SSC-Ni operates on the
principle of off-on fluorescence, with a lower background
FIGURE 4
Preparation standard curve of nickel ions. (A) Nickel ions with final concentrations of 0, 0.1, 0.5, 1, 5, and 10 μM were added to the 1 μM probe SSC-
Ni, respectively. The fluorescence signal of the FAM channel of qPCR equipment was detected at 37°C. (B) Origin2018 software was used for linear fitting
of the fluorescence data, and related parameters such as intercept, slope, and R2 can be obtained. (C) The slope of each line was plotted against
log10 nickel concentration to establish a standard curve. FIGURE 5
Concentration detection of nickel ions in blood samples. Four
blood samples without anticoagulant were taken and serum was
obtained by centrifugation at 4,000 rpm for 5 min. 4 Discussion In
conclusion,
this
paper
introduces
a
novel
off-on
fluorescent probe called SSC-Ni for the detection of nickel (II)
ion. Similar to the TaqMan probes, SSC-Ni operates on the
principle of off-on fluorescence, with a lower background Frontiers in Bioengineering and Biotechnology 05 frontiersin.org Yi et al. Yi et al. 10.3389/fbioe.2023.1261178 TABLE 1 Detection results of nickel ions concentration in blood samples. Blood sample
Found Ni2+/μM
Added/μM
Found/μMa
Recovery %b
Sample 1
0.185 ± 0.006
1
1.130 ± 0.067
94.5
10
10.232 ± 0.120
100.5
Sample 2
0.140 ± 0.004
1
1.105 ± 0.019
96.5
10
10.084 ± 0.166
99.4
Sample 3
0.144 ± 0.004
1
1.095 ± 0.048
95.1
10
10.119 ± 0.031
99.8
Sample 4
0.063 ± 0.003
1
1.022 ± 0.007
95.9
10
10.064 ± 0.099
100.0
aNickel concentrations in the blood samples were measured after the addition of 1 and 10 μM nickel (II) ion. bRecovery % was calculated by the ratio of the difference in concentration obtained from two measurements to the content of added nickel ions. TABLE 2 Comparison with other fluorescent probes for nickel ion detection. Name
DL
Linear
ranges
Application
Main characteristics
References
Ra-Ni
26.2 nM
0–3 μM
water sample, living cells
a precise ratiometric fluorescent probe
Wang et al. (2022)
probe 6
0.1 μM
0–10 μM
water sample
a dual-responsive fluorescent turn-off probe
Song et al. (2020)
PAIC
2.82 μM
0–440 μM
water sample
a imidazole containing amide fluorescent turn-on probe
Annaraj et al. (2016)
compound- 3
—
0–10 μM
monitoring in EtOH solution
a fluorescent turn-off sensor comprised of pyrazoline and
benzimidazole
Han et al. (2017)
probe L
1.83 μM
0–20 μM
living cells
a reversible and highly selective fluorescence “on-off-on” probe
Yu et al. (2016)
CdTe QDs
7 nM
0.01–10 μM
water sample
a CdTe QDs fluorescent turn-off-on probe
Zare et al. (2017)
MMT
0.85 nM
1.0 nM-4.4 mM
tea leave and mushroom
sample
a optical chemical fluorescence quenching sensor
Aksuner et al. (2012)
SSC-Ni
82 nM
0.1–10 μM
serum and water sample
a sequence-specific cleavage fluorescent off-on probe
this study
DL: detection limit. TABLE 2 Comparison with other fluorescent probes for nickel ion detection. TABLE 2 Comparison with other fluorescent probes for nickel ion detection. fluorescence signal (Yao et al., 2017). 4 Discussion When exposed to nickel
ions, the probe undergoes degradation, leading to the release of
fluorescence signals, The rate of fluorescence signal enhancement
is directly proportional to the concentration of nickel ions. In
contrast to other probes, the SSC-Ni probe is a sequence-specific
cleavage fluorescent off-on probe that detects fluorescence
signals by specifically cutting the probe with nickel (II) ions. This probe exhibits high detection specificity and low detection
limit (82 nM). The SSC-Ni probe is a fluorescent probe that can
detect the concentration of nickel ions in the blood, which is of
great value in clinical disease diagnosis. was not shown), so the detection range of the SSC-Ni probe for
detecting nickel ions was between 82 nM–10 μM. This detection
range is coincidentally suitable for the detecting concentration of
nickel ions in human blood and the environment (Kazi et al.,
2008; Wang et al., 2022b). Nickel, as a heavy metal element, poses environmental pollution
risks and potential health hazards (Genchi et al., 2020). This study
successfully designed and synthesized the SSC-Ni probe, which can
be employed for large-scale detection of nickel ion concentration
using qPCR equipment. The probe demonstrated its capability for
detecting nickel ions in serum, yielding results that closely matched
the reported serum nickel concentration. This finding highlights the
significant clinical relevance of the SSC-Ni probe. Moreover, in
theory, the probe SSC-Ni can also be applied for the detection of
nickel ions in the environment. In the practical application of the probe SSC-Ni, we also have
found some defects such as the excessive detection time. This may
be related to the slow degradation rate of nickel ions on
polypeptide chains (Dang et al., 2019). In subsequent research,
more suitable peptide chains can be screened and further
optimized
for
the
SSC-Ni
probe. Moreover,
the
high
concentration
of
nickel
ions
has
a
quenching
effect
on
fluorophores (Yue et al., 2015). We have also tested for higher
concentrations of nickel ions (>10 μM), and found that the
fluorescence intensity of the probe could be significantly
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media. an experimental and theoretical investigation. Data availability statement The original contributions presented in the study are included in
the article/Supplementary Material, further inquiries can be directed
to the corresponding authors. 06 frontiersin.org frontiersin.org Yi et al. Yi et al. 10.3389/fbioe.2023.1261178 Publisher’s note H-WY: Conceptualization, Data curation, Funding acquisition,
Writing–original draft, Writing–review and editing. X-MW: Data
curation, Formal Analysis, Writing–original draft. J-HW: Data
curation, Formal Analysis, Writing and original draft. C-LZ:
Conceptualization,
Data
curation,
Formal
Analysis,
Writing–original draft. Y-DD: Conceptualization, Data curation,
Supervision, Writing–original draft, Writing–review and editing. All claims expressed in this article are solely those of the authors
and do not necessarily represent those of their affiliated organizations,
or those of the publisher, the editors and the reviewers. Any product
that may be evaluated in this article, or claim that may be made by its
manufacturer, is not guaranteed or endorsed by the publisher. Ethics statement Province, China [Grant Number 2021CFB261 (to H-WY)] and
Yangtze University Medical Innovation Fund of Jingzhou, China
[Grant Number 2022MIF08 (to H-WY)]. Province, China [Grant Number 2021CFB261 (to H-WY)] and
Yangtze University Medical Innovation Fund of Jingzhou, China
[Grant Number 2022MIF08 (to H-WY)]. The studies involving humans were approved by the Ethics
Committee of the First People’s Hospital of Jingzhou, China (no. KY202375). The studies were conducted in accordance with the local
legislation
and
institutional
requirements. Written
informed
consent for participation in this study was provided by the
participant’s legal guardians/next of kin. Funding The author(s) declare financial support was received for the
research, authorship, and/or publication of this article. This work
was supported by the Natural Science Foundation of Hubei The Supplementary Material for this article can be found online
at: https://www.frontiersin.org/articles/10.3389/fbioe.2023.1261178/
full#supplementary-material Conflict of interest The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be
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off-on fluorescence system based on carbon dots for trace pyrophosphate sensing. J. Fluoresc. 25, 585–594. doi:10.1007/s10895-015-1538-9 Xu, Y., Chen, B., He, M., and Hu, B. (2021). A homogeneous nucleic acid assay for
simultaneous detection of SARS-CoV-2 and influenza A (H3N2) by single-particle
inductively coupled plasma mass spectrometry. Anal. Chim. Acta 1186, 339134. doi:10. 1016/j.aca.2021.339134 Zare, H., Ghalkhani, M., Akhavan, O., Taghavinia, N., and Marandi, M. (2017). Highly sensitive selective sensing of nickel ions using repeatable fluorescence
quenching-emerging of the cdte quantum dots. Mater. Res. Bull. 95, 532–538. doi:10.1016/j.materresbull.2017.08.015 Yao, H., Cao, L., Zhao, W., Zhang, S., Zeng, M., and Du, B. (2017). An off-on
fluorescence probe targeting mitochondria based on oxidation-reduction response for 08 frontiersin.org Frontiers in Bioengineering and Biotechnology
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Supplementary Figure 5 from Induction of the Stem-like Cell Regulator CD44 by Rho Kinase Inhibition Contributes to the Maintenance of Colon Cancer–Initiating Cells
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Supplementary Figure S3. CD44high cells in the spheroids show characteristics of
cancer stem cells. A, flow cytometry analyses of spheroid cells (#20) double-stained with
anti-CD44 and anti-CD133 antibodies. B, the cell cycle profiles of CD44-/low, CD44med, and
CD44high cells. C, western blot analyses of FACS-sorted CD44high cells (#20) with the
indicated antibodies. CD44high cells were harvested immediately (day 0) or cultivated for a
week (day 7) under differentiation conditions before harvest. D, flow cytometry analyses
of sorted CD44high cells (#20) cultivated under spheroid conditions for indicated periods. E,
gene set enrichment analyses of gene expression profiles between FACS-sorted
CD44high and CD44-/low cells from four spheroids (#6, #17, #19, #20). The gene set
collection of version 3.7 of the Molecular Signatures Database (MSigDB) was used, and
two gene expression signatures related to the glycolytic pathway are shown.
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Effect of Selenium on the Responses Induced by Heat Stress in Plant Cell Cultures
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Received: 11 July 2018; Accepted: 10 August 2018; Published: 11 August 2018 Received: 11 July 2018; Accepted: 10 August 2018; Published: 11 August 2018 Abstract: High temperatures are a significant stress factor for plants. In fact, many biochemical
reactions involved in growth and development are sensitive to temperature. In particular, heat stress
(HS) represents a severe issue for plant productivity and strategies to obtain high yields under this
condition are important goals in agriculture. While selenium (Se) is a nutrient for humans and
animals, its role as a plant micronutrient is still questioned. Se can prevent several abiotic stresses
(drought, heat, UV, salinity, heavy metals), but the action mechanisms are poorly understood. Se
seems to regulate reactive oxygen species (ROS) and to inhibit heavy metals transport. In addition,
it has been demonstrated that Se is essential for a correct integrity of cell membranes and chloroplasts,
especially the photosynthetic apparatus. Previous results showed that in tobacco (Nicotiana tabacum cv. Bright-Yellow 2) cultures HS (5 min at 50 ◦C) induced cell death with apoptotic features, accompanied
by oxidative stress and changes in the levels of stress-related proteins. In this work we investigated
the effect of Se on the responses induced by HS. The obtained results show that Se markedly reduces
the effects of HS on cell vitality, cytoplasmic shrinkage, superoxide anion production, membrane
lipids peroxidation, activity of caspase-3-like proteases, and the levels of some stress-related proteins
(Hsp90, BiP, 14-3-3s, cytochrome c). Keywords: cell death; heat stress; plant cell cultures; selenium; tobacco BY-2 plants plants Article
Effect of Selenium on the Responses Induced by Heat
Stress in Plant Cell Cultures Massimo Malerba 1 and Raffaella Cerana 2,* ID Massimo Malerba 1 and Raffaella Cerana 2,* ID 1
Dipartimento di Biotecnologie e Bioscienze, Università degli Studi di Milano-Bicocca, 20126 Milan,
massimo.malerba@unimib.it 1
Dipartimento di Biotecnologie e Bioscienze, Università degli Studi di Milano-Bicocca, 20126 Milan, It
massimo.malerba@unimib.it
2
Dipartimento di Scienze dell’Ambiente e della Terra, Università degli Studi di Milano-Bicocca,
20126 Milan, Italy *
Correspondence: raffaella.cerana@unimib.it; Tel.: +39-02-64482932 *
Correspondence: raffaella.cerana@unimib.it; Tel.: +39-02-64482932 plants plants Keywords: cell death; heat stress; plant cell cultures; selenium; tobacco BY-2 1. Introduction Plants are heterothermic sessile organisms in thermal equilibrium with the environment. Strong
temperature variations exceeding lower or higher limits of the thermal optimum for the life of the
plant are sensed as thermal stress, cold stress or heat stress (HS), respectively. Thermal stress can
compromise the vital functions of the plant more or less severely, depending on the organ affected and
its developmental stage [1]. In fact, roots may tolerate lower temperatures than stems and expanded
leaves tolerate higher temperatures than the young ones. Thermal stress, in particular HS is one of the
main causes of the reduction in crops productivity because the light energy required for photosynthesis
results in a considerable increase of the temperature in the exposed tissues [2]. In addition, HS is
able to influence growth and biodiversity of forests [3]. This is very important considering the global
warming in progress in present years with increasing temperatures and decreasing precipitation with
a consequent reduction of wetlands and an increase in areas at risk of desertification [3]. In fact,
forests, thanks to their ability to fix carbon dioxide, absorb greenhouse gases and filter anthropogenic
pollutants, potentially play a crucial role in the moderation of these changes [3]. Thus, the study of the
effects of HS is of great interest for plant biologists. Plants 2018, 7, 64; doi:10.3390/plants7030064 www.mdpi.com/journal/plants www.mdpi.com/journal/plants Plants 2018, 7, 64 Plants 2018, 7, 64 2 of 10 Plants have evolved different responses to HS to minimize damage and ensure the conservation
of cell homeostasis. An intense HS causes a “Heat shock response” (Hsr), which involves the rapid
activation of “HS genes” due to the specific transcription factors, named “HS factors” (Hsf). The activity
of Hsf induces the synthesis of specific “Heat shock proteins” (Hsp), that act as molecular chaperones
involved in plant tolerance to a wide range of stresses [2]. HS can lead to protein denaturation and
alteration of the membrane fluidity. This effect may result in high production and accumulation of
reactive oxygen species (ROS) causing oxidative stress and, hence, cellular necrosis. On the other
hand, low ROS concentrations may act as a second messenger for signal transduction pathways
regulating a wide range of cellular functions including programmed cell death (PCD) [4,5]. PCD is an
ubiquitous genetically controlled process aimed at eliminating cells that are not necessary or harmful
for the proper development of the organism. 1. Introduction Among the others, the form of PCD that is the object of
major studies and is therefore better known, is the apoptosis of animal cells. Various forms of PCD
are also observed in plants, where they are induced by various biotic and abiotic stimuli, including
HS [6]. For a long time, selenium (Se) was considered toxic until it was recognized as a micronutrient
for humans and other animals [7]. Se is present as selenocysteine in the catalytic site of several
selenoproteins involved in important metabolic processes, such as thyroid hormone metabolism,
mechanisms of protection from oxidative stress and immune response [8]. In several countries the very
low soil concentration of Se causes deficiency in the diet of more than a billion people worldwide [9]. This implies important health problems [10]. Cultivated plants are an important source of Se for
humans and livestock. Being chemically analogous to sulphur, Se is absorbed by all plants by sulphate
transporters and is sequestered in the form of selenite and selenate [11]. The levels of Se accumulation
depend on the abundance of Se in the soil and the levels of the sulphur compounds that compete for
absorption [12]. Several attempts were made in order to increase Se content in plants. Changes in the
enzymes associated with sulphur metabolism have been widely used to vary Se levels in plants [13]. Recent researches use plant-microbome interactions to increase biofortification with Se and cultivate
accumulating plants on seleniferous soils, thus ameliorating soil characteristics for further cultivation. In addition, the biomass of these accumulating plants could be used to enrich the diet of people and
their livestock. Finally, given that different species of plants seem able to affect the accumulation of Se
from nearby plants and perhaps even their speciation, different co-cultivation techniques could be
tested to optimize biofortification with Se of the cultivated plants and their nutritional quality [14]. Despite these studies, until now, there has been no clear evidence of Se essentiality for plants growth. The metal seems to play a dual role: At high doses, it acts as a pro-oxidant agent, causing serious
damage to the plant, while low doses can counteract abiotic stress induced by high temperatures,
drought, intense light, UV rays, excess of water, salinity and heavy metals [15]. 1. Introduction The accumulation of
ROS in response to excess of Se may depend on an insufficient presence of antioxidant compounds
such as reduced glutathione, thiols, reduced ferredoxin and/or NADPH [16]. These compounds are
also involved in the assimilation of Se, thus their concentration can be insufficient to satisfy necessity
for Se uptake and at the same time to contrast accumulation of ROS [16]. In contrast, low levels of Se
can decrease accumulation of ROS, especially O2.−and/or H2O2, in plants subject to different stresses. Reduction of O2.−levels can depend on: spontaneous dismutation of O2.−to H2O2 (not catalyzed
by the enzyme superoxide dismutase SOD) [17], direct elimination of O2.−by Se compounds [18],
regulation of antioxidant enzymes [15]. However, the mechanisms associated with the protective effect
of Se against stresses appears complex and not yet fully understood. In addition to involvement in
the mechanism of ROS regulation, a role for Se has been proposed in the inhibition of absorption and
translocation of heavy metals. Furthermore, it seems to play a fundamental role in the reconstitution of
the cell structures and chloroplasts, and in the recovery of the photosynthetic apparatus after stress [15]. However, an excess of Se could exacerbate the damage to the photosynthetic apparatus and could
result in overproduction of starch [19]. Cultured cells are a good experimental material to investigate the responses elicited by HS due Cultured cells are a good experimental material to investigate the responses elicited by HS due
to their greater homogeneity compared to complex tissues. Furthermore, this system can be more 3 of 10 Plants 2018, 7, 64 controlled thus increasing the reproducibility of stress conditions. Previous results showed that
in tobacco (Nicotiana tabacum cv. Bright-Yellow 2) cultures HS (5 min at 50 ◦C) induced cell death
with apoptotic features, accompanied by oxidative stress and changes in the levels of stress-related
proteins [4,6]. In this work we investigated the effect of Se on the responses induced by HS. The
obtained results show that Se markedly reduces the effects of HS on cell vitality, cytoplasmic shrinkage,
superoxide anion production, membrane lipids peroxidation, activity of caspase-3-like proteases,
and the levels of some stress-related proteins (Hsp90, BiP, 14-3-3s, cytochrome c). tobacco (Nicotiana tabacum cv. Bright‐Yellow 2) cultures HS (5 min at 50 °C) induced cell death with
apoptotic features, accompanied by oxidative stress and changes in the levels of stress‐related
proteins [4,6]. 2.1. HS and Se Effects on Cell Viability and Cytoplasmic Shrinkage
2.1. HS and Se Effects on Cell Viability and Cytoplasmic Shrinkage To our knowledge, the effect of Se on plant cultured cells has never been investigated. Thus,
to identify the most appropriate Se concentration to use in subsequent experiments, in preliminary
experiments we evaluated the effects of different Se concentrations on the accumulation of dead cells
induced by HS. Figure 1 shows that in cell cultures not subjected to HS, the percentage of dead cells is
very low and does not vary during the experiment. HS determines a progressive accumulation of dead
cells, already evident after 3 h of treatment. The results show that there is a progressive protective
effect on the appearance of dead cells induced by HS by increasing the concentration of Se up to 1 mM. A further increase in Se concentration does not ameliorate the protective effect but rather seems to
reduce it. Therefore, the concentration of 1 mM Se was used in subsequent experiments. To our knowledge, the effect of Se on plant cultured cells has never been investigated. Thus, to
identify the most appropriate Se concentration to use in subsequent experiments, in preliminary
experiments we evaluated the effects of different Se concentrations on the accumulation of dead cells
induced by HS. Figure 1 shows that in cell cultures not subjected to HS, the percentage of dead cells
is very low and does not vary during the experiment. HS determines a progressive accumulation of
dead cells, already evident after 3 h of treatment. The results show that there is a progressive
protective effect on the appearance of dead cells induced by HS by increasing the concentration of Se
up to 1 mM. A further increase in Se concentration does not ameliorate the protective effect but rather
seems to reduce it. Therefore, the concentration of 1 mM Se was used in subsequent experiments. Figure 1. Effect of different Se concentrations on HS‐elicited accumulation of dead cells in the cultures. Means SD (n 9) are shown. Different letters show significant differences among treatments at each
time (Tukey HSD test, p ≤ 0.05). Figure 1. Effect of different Se concentrations on HS-elicited accumulation of dead cells in the cultures. Means ± SD (n ≥9) are shown. Different letters show significant differences among treatments at each
time (Tukey HSD test, p ≤0.05). Figure 1. Effect of different Se concentrations on HS‐elicited accumulation of dead cells in the cultures. Means SD (n 9) are shown. 1. Introduction In this work we investigated the effect of Se on the responses induced by HS. The
obtained results show that Se markedly reduces the effects of HS on cell vitality, cytoplasmic
shrinkage, superoxide anion production, membrane lipids peroxidation, activity of caspase‐3‐like
proteases, and the levels of some stress‐related proteins (Hsp90, BiP, 14‐3‐3s, cytochrome c). 2.1. HS and Se Effects on Cell Viability and Cytoplasmic Shrinkage
2.1. HS and Se Effects on Cell Viability and Cytoplasmic Shrinkage Effect of Se on HS-elicited cytoplasmic shrinkage. Means ± SD (n ≥9) are shown. Different
letters show significant differences among treatments at each time (Tukey HSD test, p ≤0.05). Figure 2. Effect of Se on HS‐elicited cytoplasmic shrinkage. Means SD (n 9) are shown. Different
letters show significant differences among treatments at each time (Tukey HSD test, p ≤ 0.05). Figure 2. Effect of Se on HS‐elicited cytoplasmic shrinkage. Means SD (n 9) are shown. Different
letters show significant differences among treatments at each time (Tukey HSD test, p ≤ 0.05). Figure 2. Effect of Se on HS-elicited cytoplasmic shrinkage. Means ± SD (n ≥9) are shown. Different
letters show significant differences among treatments at each time (Tukey HSD test, p ≤0.05). Figure 2. Effect of Se on HS‐elicited cytoplasmic shrinkage. Means SD (n 9) are shown. Different
letters show significant differences among treatments at each time (Tukey HSD test, p ≤ 0.05). 2.2. HS and Se Effects on Accumulation of O2.− and MDA and on Caspase‐3‐like Activity
2.2. HS and Se Effects on Accumulation of O2.−and MDA and on Caspase-3-like Activity
2.2. HS and Se Effects on Accumulation of O2.− and MDA and on Caspase‐3‐like Activity 2.2. HS and Se Effects on Accumulation of O2.− and MDA and on Caspase‐3‐like Activity
2.2. HS and Se Effects on Accumulation of O2.−and MDA and on Caspase-3-like Activity
2.2. HS and Se Effects on Accumulation of O2.− and MDA and on Caspase‐3‐like Activity O2.− is a highly reactive ROS, responsible for important oxidative damage [21]. Treatment with
HS (Figure 3) causes a progressive accumulation of O2.−. This accumulation is almost totally prevented
by Se, at least for the first experimental times (up to 4 h). O2.−is a highly reactive ROS, responsible for important oxidative damage [21]. Treatment with
HS (Figure 3) causes a progressive accumulation of O2.−. This accumulation is almost totally prevented
by Se, at least for the first experimental times (up to 4 h). O2.− is a highly reactive ROS, responsible for important oxidative damage [21]. Treatment with
HS (Figure 3) causes a progressive accumulation of O2.−. This accumulation is almost totally prevented
by Se, at least for the first experimental times (up to 4 h). Figure 3. HS and Se effects on O2− accumulation in the culture medium. Means SD (n 9) are shown. 2.1. HS and Se Effects on Cell Viability and Cytoplasmic Shrinkage
2.1. HS and Se Effects on Cell Viability and Cytoplasmic Shrinkage Different letters show significant differences among treatments at each
time (Tukey HSD test, p ≤ 0.05). Figure 1. Effect of different Se concentrations on HS-elicited accumulation of dead cells in the cultures. Means ± SD (n ≥9) are shown. Different letters show significant differences among treatments at each
time (Tukey HSD test, p ≤0.05). To better characterize the process of death induced by HS, we considered the appearance of cells
with shrinked cytoplasm. This morphological modification is presumably caused by destructuration
of the cytoskeleton, and in cultured cells is considered an index of PCD with apoptotic features [4,20]. Figure 2 shows that similarly to the percentage of dead cells the percentage of control cells with
To better characterize the process of death induced by HS, we considered the appearance of cells
with shrinked cytoplasm. This morphological modification is presumably caused by destructuration
of the cytoskeleton, and in cultured cells is considered an index of PCD with apoptotic features [4,20]. Figure 2 shows that, similarly to the percentage of dead cells, the percentage of control cells with
shrinked cytoplasm is very low, constant over time and not influenced by the presence of Se. The
treatment with HS leads to a considerable increase in the percentage of cells with shrinked cytoplasm
that is in part prevented by Se. At all experimental times it is observed that the percentage of dead
cells is slightly higher than that of cells with shrinked cytoplasm, suggesting the presence of different
Figure 2 shows that, similarly to the percentage of dead cells, the percentage of control cells with
shrinked cytoplasm is very low, constant over time and not influenced by the presence of Se. The
treatment with HS leads to a considerable increase in the percentage of cells with shrinked cytoplasm
that is in part prevented by Se. At all experimental times it is observed that the percentage of dead 4 of 10 Plants 2018, 7, 64 cells is slightly higher than that of cells with shrinked cytoplasm, suggesting the presence of different
forms of cell death [21]. Plants 2018, 7, x FOR PEER REVIEW
4 of 10
Plants 2018, 7, x FOR PEER REVIEW
4 of 10 Figure 2. Effect of Se on HS‐elicited cytoplasmic shrinkage. Means SD (n 9) are shown. Different
letters show significant differences among treatments at each time (Tukey HSD test, p ≤ 0.05). Figure 2. 2.1. HS and Se Effects on Cell Viability and Cytoplasmic Shrinkage
2.1. HS and Se Effects on Cell Viability and Cytoplasmic Shrinkage Different letters show significant differences among treatments at each time (Tukey HSD test, p ≤
0.05). Figure 3. HS and Se effects on O2− accumulation in the culture medium. Means SD (n 9) are shown. Different letters show significant differences among treatments at each time (Tukey HSD test, p ≤
0.05). Figure 3. HS and Se effects on O2−accumulation in the culture medium. Means ± SD (n ≥9) are
shown. Different letters show significant differences among treatments at each time (Tukey HSD test,
p ≤0.05). Figure 3. HS and Se effects on O2− accumulation in the culture medium. Means SD (n 9) are shown. Different letters show significant differences among treatments at each time (Tukey HSD test, p ≤
0.05). Figure 3. HS and Se effects on O2− accumulation in the culture medium. Means SD (n 9) are shown. Different letters show significant differences among treatments at each time (Tukey HSD test, p ≤
0.05). Figure 3. HS and Se effects on O2−accumulation in the culture medium. Means ± SD (n ≥9) are
shown. Different letters show significant differences among treatments at each time (Tukey HSD test,
p ≤0.05). At cell level, peroxidation of membrane lipids is one of the main damages induced by oxidative
stress and its degree was assessed by determining the level of malondialdehyde (MDA), a byproduct
At cell level, peroxidation of membrane lipids is one of the main damages induced by oxidative
stress and its degree was assessed by determining the level of malondialdehyde (MDA), a byproduct
of polyunsaturated fatty acids oxidation which typically originates after oxidative stress [21] Figure
At cell level, peroxidation of membrane lipids is one of the main damages induced by oxidative
stress and its degree was assessed by determining the level of malondialdehyde (MDA), a byproduct 5 of 10 Plants 2018, 7, 64 of polyunsaturated fatty acids oxidation, which typically originates after oxidative stress [21]. Figure 4
shows that the level of MDA of the control cells is low, constant, and not influenced by Se. At each
time, HS causes a considerable production of MDA, significantly decreased by Se. Plants 2018, 7, x FOR PEER REVIEW
5 of 10
Plants 2018, 7, x FOR PEER REVIEW
5 of 10 Figure 4. HS and Se effects on MDA accumulation. Means SD (n 9) are shown. 2.1. HS and Se Effects on Cell Viability and Cytoplasmic Shrinkage
2.1. HS and Se Effects on Cell Viability and Cytoplasmic Shrinkage Different letters
show significant differences among treatments at each time (Tukey HSD test, p ≤ 0.05). Figure 4. HS and Se effects on MDA accumulation. Means ± SD (n ≥9) are shown. Different letters
show significant differences among treatments at each time (Tukey HSD test, p ≤0.05). Figure 4. HS and Se effects on MDA accumulation. Means SD (n 9) are shown. Different letters
show significant differences among treatments at each time (Tukey HSD test, p ≤ 0.05). Figure 4. HS and Se effects on MDA accumulation. Means SD (n 9) are shown. Different letters
show significant differences among treatments at each time (Tukey HSD test, p ≤ 0.05). Figure 4. HS and Se effects on MDA accumulation. Means ± SD (n ≥9) are shown. Different letters
show significant differences among treatments at each time (Tukey HSD test, p ≤0.05). Figure 4. HS and Se effects on MDA accumulation. Means SD (n 9) are shown. Different letters
how significant differences among treatments at each time (Tukey HSD test, p ≤ 0.05). To characterize further the effect of Se on HS‐elicited cell death we analysed caspase3‐like
proteases activity, another typical PCD marker that often increases during plant PCD. Figure 5 shows
that Se strongly reduces the HS‐elicited marked increase in this activity. To characterize further the effect of Se on HS-elicited cell death we analysed caspase3-like
proteases activity, another typical PCD marker that often increases during plant PCD. Figure 5 shows
that Se strongly reduces the HS-elicited marked increase in this activity. To characterize further the effect of Se on HS‐elicited cell death we analysed caspase3‐like
proteases activity, another typical PCD marker that often increases during plant PCD. Figure 5 shows
that Se strongly reduces the HS‐elicited marked increase in this activity. Figure 5. HS and Se effects on caspase‐3‐like activity. Means SD (n 6) are shown. Different letters
show significant differences among treatments at each time (Tukey HSD test, p ≤ 0.05). 3 HS and Se Effects on Stress‐related Proteins
Figure 5. HS and Se effects on caspase‐3‐like activity. Means SD (n 6) are shown. Different letters
show significant differences among treatments at each time (Tukey HSD test, p ≤ 0.05). HS
d S Eff t
St
l t d P
t i
Figure 5. HS and Se effects on caspase-3-like activity. Means ± SD (n ≥6) are shown. 2.3. HS and Se Effects on Stress-related Proteins An arbitrary value of 100 was
assigned to the quantity of immunodecorated protein of the controls. Figure 6. HS and Se effects on the levels of stress-related proteins. (C) control; (Se) cells + 1 mM Se;
(HS) HS-treated cells; (HS + Se) HS-treated cells + 1 mM Se. Results of a typical experiment (n = 3) run
in duplicate are presented. 50 mg of proteins were run in each lane. An arbitrary value of 100 was
assigned to the quantity of immunodecorated protein of the controls. 2.3. HS and Se Effects on Stress-related Proteins Finally, we analysed the effect of HS and Se on the levels of some stress-related proteins by
gel blotting. Mitochondrial Hsp 90 are molecular chaperones that control the activity of different
substrates. BiP, an Hsp70 present in the endoplasmic reticulum, accumulates under different stress
conditions. The regulatory proteins 14-3-3s control many processes of plant cells, including cell death
and cytochrome c release from the mitochondrion to the cytosol, a marker of apoptotic death in animals
and plants [21]. Plants 2018, 7, x FOR PEER REVIEW
6 of 10 Figure 6 confirms the previously reported effects of HS on the examined proteins [21], and
shows that at both times Se diminishes the accumulation of microsomal BiP and almost completely
prevents the accumulation of cytosolic 14-3-3s, the reduction of mitochondrial Hsp90 and the release
of cytochrome c from mitochondria elicited by HS. Figure 6 confirms the previously reported effects of HS on the examined proteins [21], and shows
that at both times Se diminishes the accumulation of microsomal BiP and almost completely prevents
the accumulation of cytosolic 14‐3‐3s, the reduction of mitochondrial Hsp90 and the release of
cytochrome c from mitochondria elicited by HS. Figure 6. HS and Se effects on the levels of stress‐related proteins. (C) control; (Se) cells + 1 mM Se;
(HS) HS‐treated cells; (HS + Se) HS‐treated cells + 1 mM Se. Results of a typical experiment (n = 3) run
in duplicate are presented. 50 mg of proteins were run in each lane. An arbitrary value of 100 was
assigned to the quantity of immunodecorated protein of the controls. Figure 6. HS and Se effects on the levels of stress-related proteins. (C) control; (Se) cells + 1 mM Se;
(HS) HS-treated cells; (HS + Se) HS-treated cells + 1 mM Se. Results of a typical experiment (n = 3) run
in duplicate are presented. 50 mg of proteins were run in each lane. An arbitrary value of 100 was
assigned to the quantity of immunodecorated protein of the controls. Figure 6. HS and Se effects on the levels of stress‐related proteins. (C) control; (Se) cells + 1 mM Se;
(HS) HS‐treated cells; (HS + Se) HS‐treated cells + 1 mM Se. Results of a typical experiment (n = 3) run
in duplicate are presented. 50 mg of proteins were run in each lane. 3. Discussion
3. Discussion The influence of selenium on the HS‐elicited responses of tobacco cells was tested by measuring
in the absence and presence of Na‐selenate the following parameters: cell viability, cytoplasmic
shrinkage, superoxide anion production, membrane lipids peroxidation, activity of caspase‐3‐like
proteases, and the levels of some stress‐related proteins (Hsp90, BiP, 14‐3‐3s, cytochrome c). The influence of selenium on the HS-elicited responses of tobacco cells was tested by measuring in
the absence and presence of Na-selenate the following parameters: cell viability, cytoplasmic shrinkage,
superoxide anion production, membrane lipids peroxidation, activity of caspase-3-like proteases,
and the levels of some stress-related proteins (Hsp90, BiP, 14-3-3s, cytochrome c). 3.1. HS and Se Effects on Cell Viability and Cytoplasmic Shrinkage. 3.1. HS and Se Effects on Cell Viability and Cytoplasmic Shrinkage. 2.1. HS and Se Effects on Cell Viability and Cytoplasmic Shrinkage
2.1. HS and Se Effects on Cell Viability and Cytoplasmic Shrinkage Different letters
show significant differences among treatments at each time (Tukey HSD test, p ≤0.05). Figure 5. HS and Se effects on caspase‐3‐like activity. Means SD (n 6) are shown. Different letters
show significant differences among treatments at each time (Tukey HSD test, p ≤ 0.05). Figure 5. HS and Se effects on caspase‐3‐like activity. Means SD (n 6) are shown. Different letters
show significant differences among treatments at each time (Tukey HSD test, p ≤ 0.05). Figure 5. HS and Se effects on caspase-3-like activity. Means ± SD (n ≥6) are shown. Different letters
show significant differences among treatments at each time (Tukey HSD test, p ≤0.05). Plants 2018, 7, 64 6 of 10 2.3. HS and Se Effects on Stress-related Proteins 2.3. HS and Se Effects on Stress-related Proteins 2.3. HS and Se Effects on Stress-related Proteins 3.4. HS and Se Effects on the Levels of Hsp90, BiP and 14-3-3s. As widely reported, plants evolved a set of responses to deal with HS, that includes changes of
biochemical and physiological processes due to modifications of gene expression. These modifications
can result in acclimation or adaptation to stress [24]. In this investigation, we studied the HS and Se
effects on some stress-related proteins. Our data confirm the previously reported effects of HS on the
examined proteins [21] and show that Se diminishes the accumulation of BiP and almost completely
prevents the accumulation of 14-3-3s and the reduction of Hsp90 elicited by HS (Figure 6). To our knowledge, a genomic approach has scarcely been used to study the protective role of
Se against stresses. Sun and co-workers performed a comparative proteomics analysis on cucumber
plants treated with Cd [25]. Comparing 2-DE gels, these researchers observed several protein spots
changed by Se+Cd compared to Cd alone. By MALDI–TOF–MS mass spectrometry, they identified
proteins whose relative abundance was significantly reduced by Cd and restored by Se. Among the
others, ascorbate oxidase, glutathione-S-transferase and Hsp STI-like expression were strongly reduced
by Cd and reincreased by Se [25]. More studies were conducted on the Se effect on the proteome of
different plant species. For example, Wang and co-workers by 2-DE gels and MALDI-TOF/TOF mass
spectrometry performed a comparative proteomics analysis on the effect of different Se concentrations
on rice seedlings [19]. Their results showed that low (non-toxic) Se concentrations up-regulate proteins
involved in ROS detoxification and resistance to pathogens such as beta-1,3-glucanase and chitinase. The expression of the same proteins was down-regulated by high (toxic) Se concentrations [19]. Finally,
the Se hyperaccumulator Stanleya pinnata shows higher expression of genes involved in sulphur uptake
and assimilation, antioxidant activities and defense compared to the related secondary Se accumulator
Stanleya albescens [26]. In particular, the hyperaccumulator species shows a higher expression than the
related species for genes encoding Hsp and luminal chaperones such as BiP both in the absence and in
the presence of Se [26]. 3.3. HS and Se Effects on Caspase-3-like Activity and on Cytochrome c Release Specific cysteine proteases named caspases are required for the progression of animal apoptosis. In plant cells too there are proteins with similar activity called caspases-like or metacaspases [6]. Se
(Figure 5) largely prevents the HS-elicited increase in the activity of these enzymes, reported in our
previous work [21]. Another ubiquitous marker of apoptotic-like PCD related to caspases is the release
of cytochrome c from the mitochondrion [6]. We previously reported induction of cytochrome c release
by HS [6,21]. Here we show that this release is markedly reduced by Se (Figure 6). These protective
effects of Se can be due to its antioxidant effect (ROS are potent regulators of PCD) or to the effect of Se
on expression of genes implied in antioxidant activity and defense responses ([11] and see below for
further discussion). 3.1. HS and Se Effects on Cell Viability and Cytoplasmic Shrinkage.
3.1. HS and Se Effects on Cell Viability and Cytoplasmic Shrinkage. This work (Figures 1 and 2) shows that Se strongly reduces the previously reported eliciting
effect of HS on cell death and cytoplasmic shrinkage [6]. This work (Figures 1 and 2) shows that Se strongly reduces the previously reported eliciting effect
of HS on cell death and cytoplasmic shrinkage [6]. y
p
g [ ]
At proper concentrations, Se promotes growth, delays plant senescence, and precocious fruit
ripening induced by different abiotic stresses, HS included [13]. Leaf and fruit senescence are
processes involving programmed cell death, and when not precisely regulated can lead to important
decreases in productivity in several horticultural species. The protective effect of Se against
inappropriate senescence could be due to its reported ability to reduce respiratory intensity and
ethylene production in different plant species [13]. Interestingly, the inhibitor of ethylene production
Co2+ prevents cell death and cytoplasmic shrinkage induced by HS in tobacco cell cultures [6]. At proper concentrations, Se promotes growth, delays plant senescence, and precocious fruit
ripening induced by different abiotic stresses, HS included [13]. Leaf and fruit senescence are processes
involving programmed cell death, and when not precisely regulated can lead to important decreases
in productivity in several horticultural species. The protective effect of Se against inappropriate
senescence could be due to its reported ability to reduce respiratory intensity and ethylene production
in different plant species [13]. Interestingly, the inhibitor of ethylene production Co2+ prevents cell
death and cytoplasmic shrinkage induced by HS in tobacco cell cultures [6]. Plants 2018, 7, 64 7 of 10 7 of 10 3.2. HS and Se Effects on O2−. and Malondialdehyde Accumulations. 3.2. HS and Se Effects on O2−. and Malondialdehyde Accumulations. 3.2. HS and Se Effects on O2−. and Malondialdehyde Accumulations. The HS-elicited oxidative stress with accumulation of O2.−and malondialdehyde is largely
inhibited by Se (Figures 3 and 4). Similar results have been recently obtained in cucumber plants
under HS and in Zea mays exposed to water stress where stress-induced accumulations of O2.−and
MDA are prevented by Se [22,23]. These results are not surprizing. In fact, Se acts as an antioxidant
in different plant species under biotic and abiotic stresses. This protective effect of Se depends on
the induced higher activity of several antioxidant enzymes and on the increased content of some
antioxidant compounds (glutathione and flavonoids) [11,13]. 4.5. SDS-PAGE and Protein Gel Blots Cells were collected by gentle centrifugation, frozen in liquid nitrogen and homogenized for 5 min
at maximum speed with a Ultra-Turrax T25 device. The cell homogenate was differentially centrifuged
to obtain the different fractions (i.e., mitochondrial, microsomal and soluble) for SDS-PAGE analysis as
described [21]. Equal amounts of proteins were separated by discontinuous SDS-PAGE (4% stacking, 10%
resolving gel) as described [21]. Immunodecorations of cytochrome c, 14-3-3 proteins, Hsp 90 and BiP
were perfomed as described [21]. 4.6. Statistical Analyses GraphPad Prism 4 program from GraphPad Software, Inc., San Diego, CA, USA was used to
statistically analyse the results. Tukey HSD test, p ≤0.05, was used in the study. 5. Conclusions To summarize, Se can effectively reduce the effect of HS on cell death with apoptotic features,
oxidative stress and levels of stress-related proteins. This protective effect of Se can be due to its direct
antioxidant effect and/or to an effect on the expression of genes implied in antioxidant activity and
defense responses. In the future, molecular and genomic studies could be valuable to elucidate the
mechanisms associated with the protective effect of Se against heat stress in cell cultures as well as
in plants. Author Contributions: Conceptualization, M.M. and R.C.; Funding acquisition, R.C.; Investigation, M.M.;
Writing-original draft, M.M.; Writing-review & editing, R.C. Funding: This research received no external funding Funding: This research received no external funding Acknowledgments: Financial support of the University of Milano-Bicocca, Fondo d’Ateneo per la Ricerca,
is acknowledged. Acknowledgments: Financial support of the University of Milano-Bicocca, Fondo d’Ateneo per la Ricerca,
is acknowledged. Conflicts of Interest: The authors declare that they have no conflict of interest. Conflicts of Interest: The authors declare that they have no conflict of interest. 4.1. Cell Culture Growth and Experimental Conditions 4.1. Cell Culture Growth and Experimental Conditions Growth of tobacco BY-2 (Nicotiana tabacum L. cv Bright-Yellow 2) cells and heat treatment (5 min
at 50◦C) were performed as described [6]. Na-selenate (Na2SeO4) was supplied 10 min before HS. 8 of 10 Plants 2018, 7, 64 4.2. Cell Death and Cytoplasmic Shrinkage Assays 4.4. Proteases Activity and Membrane Lipid Peroxidation 4.4. Proteases Activity and Membrane Lipid Peroxidation Caspase3-like proteases activity was measured spectrophotometrically with a caspase-3
colorimetric activity assay kit following the manufacturer’s instructions (BioVision Research Products,
Mountain View, CA 94043, USA) as described [21]. The level of membrane lipid peroxidation was evaluated spectrophotometrically by measuring
the content of malondialdehyde, a secondary end product of the oxidation of polyunsaturated fatty
acids [21]. 4.3. O2.−Assay The O2.−anion generation was evaluated spectrophotometrically as reduction of XTT to XTT
formazan as described [21]. 4.2. Cell Death and Cytoplasmic Shrinkage Assays 4.2. Cell Death and Cytoplasmic Shrinkage Assays Cell death was estimated spectrophotometrically with the vital dye Evans Blue as in Reference [21]. The percentage of cells showing cytoplasmic shrinkage was determined as in Reference [6]. 4.3. O2.−Assay 4.3. O2.−Assay 4.3. O2.−Assay References Selenium as an integral part of factor 3 against dietary necrotic liver degeneration. J. Am. Chem. Soc. 1957, 70, 3292–3293. [CrossRef] 8. Tapiero, H.; Townsend, D.M.; Tew, K.D. The antioxidant role of selenium and seleno-compounds. Biomed. Pharmacother. 2003, 57, 134–144. [CrossRef] 9. Wu, Z.; Bañuelos, G.S.; Lin, Z.Q.; Liu, Y.; Yuan, L.; Yin, X.; Li, M. Biofortification and phytoremediation of
selenium in China. Front. Plant Sci. 2015, 6, 136. [CrossRef] [PubMed] 10. Roman, M.; Jitaru, P.; Barbante, C. Selenium biochemistry and its role for human health. Metallomics 2014, 6,
25–54. [CrossRef] [PubMed] 11. Gupta, M.; Gupta, S. An overview of selenium uptake, metabolism, and toxicity in plants. Front. Plant Sci. 2017, 7, 2074. [CrossRef] [PubMed] 12. Galeas, M.L.; Zhang, L.H.; Freeman, J.L.; Wegner, M.; Pilon-Smits, E.A.H. Seasonal fluctuations of selenium
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phytoremediation plant. Plant Soil Environ. 2012, 58, 105–110. [CrossRef] 17. Cartes, P.; Jara, A.A.; Pinilla, L.; Rosas, A.; Mora, M.L. Selenium improves the antioxidant ability against
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Negri, V.; Marconi, G.; Albertini, E.; et al. Soil selenium (Se) biofortification changes the physiological,
biochemical and epigenetic responses to water stress in Zea mays L. by inducing a higher drought tolerance. Front. Plant Sci. 2018, 9, 389. [CrossRef] [PubMed] 24. Hasanuzzaman, M.; Nahar, K.; Mahabub Alam, M.; Roychowdhury, R.; Fujita, M. Physiological, biochemical,
and molecular mechanisms of heat stress tolerance in plants. Int. J. Mol. Sci. 2013, 14, 9643–9684. [CrossRef]
[PubMed] 10 of 10 Plants 2018, 7, 64 25. Sun, H.; Dai, H.; Wang, X.; Wang, G. Physiological and proteomic analysis of selenium-mediated tolerance
to Cd stress in cucumber (Cucumis sativus L.). Ecotox. Environ. Safe. 2016, 133, 114–126. [CrossRef] [PubMed] © 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).
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Prediction of an arc-tunable Weyl Fermion metallic state in MoxW1−xTe2
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Nature communications
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ARTICLE Received 23 Sep 2015 | Accepted 7 Jan 2016 | Published 15 Feb 2016 1 Department of Physics, National Tsing Hua University, 30013 Hsinchu, Taiwan. 2 Laboratory for Topological Quantum Matter and Spectroscopy (B7),
Department of Physics, Princeton University, Princeton, 08544 New Jersey, USA. 3 Centre for Advanced 2D Materials and Graphene Research Centre,
National University of Singapore, 6 Science Drive 2, 117546 Singapore, Singapore. 4 Department of Physics, National University of Singapore, 2 Science Drive
3, 117542 Singapore, Singapore. 5 Department of Physics, Northeastern University, Boston, 02115 Massachusetts, USA. 6 Condensed Matter and Magnet
Science Group, Los Alamos National Laboratory, Los Alamos, 87545 New Mexico, USA. 7 Institute of Physics, Academia Sinica, 11529 Taipei, Taiwan.
8 Princeton Center for Complex Materials, Princeton Institute for the Science and Technology of Materials, Princeton University, Princeton, 08544 New Jersey,
USA. Correspondence and requests for materials should be addressed to S.-Y.X. (email: suyangxu@princeton.edu) or to H.L. (email: nilnish@gmail.com) or to
M.Z.H. (email: mzhasan@princeton.edu). Results Material system considerations. WTe2 crystalizes in an orthor-
hombic Bravais lattice, space group Pmn21 (No. 31). In this
structure, each tungsten layer is sandwiched by two tellurium
layers and forms strong ionic bonds. Figure 1a shows a top view
of the lattice. It can be seen that the tungsten atom is shifted away
from the centre of the hexagon formed by the tellurium atoms. This makes the in-plane lattice constant along the ^x direction a
longer than that of along the ^y direction b. The WTe2 sandwich
stacks along the out-of-plane ^z direction, with van der Waals
bonding between layers (Fig. 1b). We used the experimental
lattice constants reported by Brown36, a ¼ 6.282 Å, b ¼ 3.496 Å,
c ¼ 14.07 Å. The bulk BZ and the (001) surface BZ are shown in
Fig. 1c, where high symmetry points are noted. In Fig. 1d, we
show the bulk band structure of WTe2 along important high
symmetry directions. Our calculation shows that there is a
continuous energy gap near the Fermi level, but the conduction
and valence bands have a finite overlap in energy. The band gap
along the G Y direction is much smaller than that of along the
G X direction or G Z direction, consistent with the fact that
the lattice constant b is much smaller than a and c. At the Fermi
level, our calculation reveals a hole pocket and an electron
pocket along the G Y direction (Fig. 1d), which agrees with
previous calculation and photoemission results29,31–33. We also
calculated the band structure of MoTe2 by assuming that it is in
the same crystal structure. As shown in Fig. 1e, the general trend
is that the bands are ‘pushed’ closer to the Fermi level. For
example, in MoTe2, there are bands crossing the Fermi level even
along the G X and G Z directions. We emphasize that,
according to available literature36,37, MoTe2 has a different crystal
structure, either hexagonal37 or monoclinic36 both of which have
inversion
symmetry,
but
not
orthorhombic. Thus
in
our
calculation we assumed that MoTe2 has the orthorhombic
crystal structure as WTe2 and obtained the lattice constants and
atomic
coordinates
from
first-principle
calculations. Very
recently, a paper38 claimed that MoTe2 can be grown in the
orthorhombic structure. This still needs to be further confirmed. ARTICLE ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms10639 R
e
i
m R
esearch on Dirac fermions in graphene and topological
insulators have been one of the main themes in condensed
matter physics and materials science in the past decade. A particle that is also relevant for both condensed matter and
high-energy physics and that may give rise to even more exotic
physics is the Weyl fermion. The Weyl fermion was theore-
tically discovered by Weyl1 in 1929. He noted that the Dirac
equation takes a simple form if the mass term is set to zero:
i@LC ¼ cp rC, where c is constant, p is momentum, r is
conventional Pauli matrices and C is wavefunction. Such a
particle, the Weyl fermion, is massless but is associated with a
chirality. Weyl fermions may be thought of as the basic building
blocks for a Dirac fermion. For example, two Weyl fermions of
opposite chirality can combine to form a massless Dirac fermion. Weyl fermions have played a vital role in quantum field theory but
they have not been found as fundamental particles in vacuum. A
Weyl semimetal is a solid-state crystal that host Weyl fermions as
its low-energy quasiparticle excitations1–17. Weyl semimetals have
attracted intense research interest not only because they provide a
Weyl fermion in nature, but also because they allow topologically
nontrivial states in metals not insulators. In a Weyl semimetal, a
Weyl fermion is associated with an accidental degeneracy of the
band structure. Away from the degeneracy point, the bands
disperse linearly and the spin texture is chiral, giving rise to a
quasiparticle
with
a
two-component wavefunction,
a
fixed
chirality and a massless, linear dispersion. Weyl fermions have
distinct chiralities, either left-handed or right-handed. In a Weyl
semimetal crystal, the chiralities of the Weyl nodes give rise to
topological charges, which can be understood as monopoles and
anti-monopoles of Berry flux in momentum space. Remarkably,
the topological charges in a Weyl semimetal are protected only by
the translational invariance of the crystal6,8,17. The band structure
degeneracies in Weyl semimetals are uniquely robust against
disorder6,8,17, in contrast to the Dirac nodes in graphene,
topological insulators and Dirac semimetals, which depend on
additional symmetries beyond the translational symmetry13–18. As
a result, the Weyl fermion carriers are expected to transmit
electrical currents effectively. ARTICLE Moreover, the transport properties
of
Weyl
semimetals
are
predicted
to
show
many
exotic
phenomena including the negative magnetoresistance due to the
chiral anomaly known from quantum field theory, non-local
transport and quantum oscillations where electrons move in real
space between opposite sides of a sample surface10–12. These novel
properties suggest that Weyl semimetals are a flourishing field of
fundamental physics and future technology. The separation of the
opposite topological charges in momentum space leads to surface
state Fermi arcs which form an anomalous band structure
consisting of open curves that connect the projections of
opposite topological charges on the boundary of a bulk sample. Within band theory, the only way to destroy the topological Weyl
phase is to annihilate Weyl nodes with opposite charges by
bringing them together in the reciprocal space. Thus the length of
the Fermi arc provides a measure of the topological strength of a
Weyl state. For many years, research on Weyl semimetals has been held
back due to the lack of experimentally feasible candidate compensated semi-metallic ground state29,31–33. The coexistence
of inversion symmetry breaking and semi-metallic transport
behaviour resembles the properties of TaAs and hence suggests a
possible Weyl semimetal state. Here, we propose a tunable Weyl
metallic state in Mo-doped WTe2 via our first-principles
calculation, where the length of the Fermi arc and hence the
topological strength of the system can be adiabatically tuned as a
function of Mo doping. A very recent paper35 predicted the Weyl
state in pure WTe2, but the separation between Weyl nodes was
reported to be beyond spectroscopic experimental resolution. In this paper, we demonstrate that a 2% Mo doping is sufficient
to stabilize the Weyl metal state not only at low temperatures but
also at room temperatures. We show that, within a moderate
doping regime, the momentum space distance between the Weyl
nodes and hence the length of the Fermi arcs can be continuously
tuned from 0 to B3% of the Brillouin zone (BZ) size via changing
Mo concentration, thus increasing the topological strength of the
system. Our results present a tunable topological Weyl system,
which is not known to be possible in the TaAs class of Weyl
semimetals. NATURE COMMUNICATIONS | 7:10639 | DOI: 10.1038/ncomms10639 | www.nature.com/naturecommunications Prediction of an arc-tunable Weyl Fermion metallic
state in MoxW1 xTe2 Tay-Rong Chang1, Su-Yang Xu2, Guoqing Chang3,4, Chi-Cheng Lee3,4, Shin-Ming Huang3,4, BaoKai Wang3,4,5,
Guang Bian2, Hao Zheng2, Daniel S. Sanchez2, Ilya Belopolski2, Nasser Alidoust2, Madhab Neupane2,6,
Arun Bansil5, Horng-Tay Jeng1,7, Hsin Lin3,4 & M. Zahid Hasan2,8 A Weyl semimetal is a new state of matter that hosts Weyl fermions as emergent
quasiparticles. The Weyl fermions correspond to isolated points of bulk band degeneracy,
Weyl nodes, which are connected only through the crystal’s boundary by exotic Fermi arcs. The length of the Fermi arc gives a measure of the topological strength, because the only way
to destroy the Weyl nodes is to annihilate them in pairs in the reciprocal space. To date, Weyl
semimetals are only realized in the TaAs class. Here, we propose a tunable Weyl state in
MoxW1 xTe2 where Weyl nodes are formed by touching points between metallic pockets. We show that the Fermi arc length can be changed as a function of Mo concentration, thus
tuning the topological strength. Our results provide an experimentally feasible route to
realizing Weyl physics in the layered compound MoxW1 xTe2, where non-saturating
magneto-resistance and pressure-driven superconductivity have been observed. 1 Department of Physics, National Tsing Hua University, 30013 Hsinchu, Taiwan. 2 Laboratory for Topological Quantum Matter and Spectroscopy (B7),
Department of Physics, Princeton University, Princeton, 08544 New Jersey, USA. 3 Centre for Advanced 2D Materials and Graphene Research Centre,
National University of Singapore, 6 Science Drive 2, 117546 Singapore, Singapore. 4 Department of Physics, National University of Singapore, 2 Science Drive
3, 117542 Singapore, Singapore. 5 Department of Physics, Northeastern University, Boston, 02115 Massachusetts, USA. 6 Condensed Matter and Magnet
Science Group, Los Alamos National Laboratory, Los Alamos, 87545 New Mexico, USA. 7 Institute of Physics, Academia Sinica, 11529 Taipei, Taiwan. 8 Princeton Center for Complex Materials, Princeton Institute for the Science and Technology of Materials, Princeton University, Princeton, 08544 New Jersey,
USA. Correspondence and requests for materials should be addressed to S.-Y.X. (email: suyangxu@princeton.edu) or to H.L. (email: nilnish@gmail.com) or to
M.Z.H. (email: mzhasan@princeton.edu). 1 NATURE COMMUNICATIONS | 7:10639 | DOI: 10.1038/ncomms10639 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/ncomms10639 leads to a k space separation of the Weyl nodes that is similarly as
large as that of pure MoTe2. Therefore, we propose the Mo-doped
WTe2 as a better platform for studying Weyl physics. Doping dependency of the Weyl nodes. Figure 2e shows the
evolution of the k space distance between a pair of Weyl nodes as
a function of Mo concentration. Our calculation shows that a 2%
Mo doping is sufficient to stabilize the system in the Weyl metal
state. Also, the distance between the Weyl nodes increases rapidly
at the small doping regime. At a moderate doping x ¼ 20%, the
k space distance is found to be as large as 0.03 2p/a. As one
further increases the doping concentration, the distance seems
saturated. The distance is about 0.04 2p/a at x ¼ 40%. The energy
difference between the pair of Weyl nodes is shown in Fig. 2g. In
Fig. 2d we show the dispersion along the momentum space cut
that goes through the direct pair of Weyl nodes as defined in
Fig. 2b. It can be seen clearly that two singly generated bands, b2
and b3, cross each other and form the two Weyl nodes with
opposite chiralities. We name the Weyl node at lower energy as
W1 and the Weyl node at higher energy as W2. Another useful
quantity is the energy difference between the extrema of these two
bands. This characterizes the magnitude of the band inversion, as
shown in Fig. 2f. It is interesting to note that, in contrast to the k
space distance between the Weyl nodes (Fig. 2e), the energy
difference between the Weyl nodes (Fig. 2g) and the band
inversion energy (Fig. 2f) does not show signs of saturation as one
increases the Mo concentration x up to 40%. In Fig. 2h, we show a
schematic for the distribution of the Weyl nodes in Mo-doped
WTe2. We observe a pair of Weyl nodes in each quadrant of the y
p
y
y
We propose Mo-doped WTe2, MoxW1 xTe2, as an experi-
mental feasible platform to realize Weyl state in this compound. We have shown that pure WTe2 is very close to the phase-
transition boundary. Therefore, the k splitting between the Weyl
nodes would be beyond experimental resolution. On the other
hand, another very recent paper proposed a Weyl state in pure
MoTe2 (ref. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms10639 NATURE COMMUNICATIONS | DOI: 10.1038/ncomms10639 W/Mo
Te
x
y
a W/Mo
Te
x
y
a
Energy (eV)
Γ
d
Γ
Γ
e
f
z
c
(001) BZ
Γ
Γ
Γ
Γ
Γ
Γ
Γ
–1.5
–1.0
–0.5
0.0
0.5
1.0
1.5
–1.5
–1.0
–0.5
0.0
0.5
1.0
1.5
–1.5
–1.0
–0.5
0.0
0.5
1.0
1.5
b
Γ
X
M
Y
kz
U
R
T
X
kx
ky
Y
S
X S
Y S RU Z
RT Z
X S
YSR U Z
R T Z
X S
YSR U Z
RT Z
Figure 1 | Crystal structure and band structure. (a) A top view of WTe2 lattice. (b) A side view of WTe2 lattice. The silver and yellow balls represent W
and Te atoms, respectively. Rectanglar box denotes crystal unit cell. WTe2 crystalizes in an orthorhombic Bravais lattice, space group Pmn21 (No. 31). The
lattice constants are a ¼ 6.282 Å, b ¼ 3.496 Å, c ¼ 14.07 Å according to a previous X-ray diffraction measurement36. (c) The bulk and (001) surface BZ of
WTe2. (d) Bulk band structure of WTe2. (e) Bulk band structure of MoTe2 by assuming that it has the same crystal structure as WTe2. Note that, in fact,
according to available literature MoTe2 has two possible structures36,37, both of which are different from the crystal structure of WTe2. (f) Bulk band
structure of Mo0.2W0.8Te2. c
(001) BZ
Γ
Γ
X
M
Y
kz
U
R
T
X
kx
ky
Y
S b a c f
Γ
Γ
Γ
–1.5
–1.0
–0.5
0.0
0.5
1.0
1.5
X S
YSR U Z
RT Z Energy (eV)
Γ
d
Γ
Γ
–1.5
–1.0
–0.5
0.0
0.5
1.0
1.5
X S
Y S RU Z
RT Z e
Γ
Γ
Γ
–1.5
–1.0
–0.5
0.0
0.5
1.0
1.5
X S
YSR U Z
R T Z d f
1 e
1.5 e
1.5 Figure 1 | Crystal structure and band structure. (a) A top view of WTe2 lattice. (b) A side view of WTe2 lattice. The silver and yellow balls represent W
and Te atoms, respectively. Rectanglar box denotes crystal unit cell. WTe2 crystalizes in an orthorhombic Bravais lattice, space group Pmn21 (No. 31). The
lattice constants are a ¼ 6.282 Å, b ¼ 3.496 Å, c ¼ 14.07 Å according to a previous X-ray diffraction measurement36. (c) The bulk and (001) surface BZ of
WTe2. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms10639 (d) Bulk band structure of WTe2. (e) Bulk band structure of MoTe2 by assuming that it has the same crystal structure as WTe2. Note that, in fact,
according to available literature MoTe2 has two possible structures36,37, both of which are different from the crystal structure of WTe2. (f) Bulk band
structure of Mo0.2W0.8Te2. the results of Soluyanov et al.35 is due to the slightly different
values of the lattice constants36,39. The lattice constants used by
Soluyanov et al.35 were at low temperatures39. Thus the results35
better refelct the groundstate (T ¼ 0) of WTe2. We used the lattice
constants at room temperatures36, so our results correspond to
the state of WTe2 at elevated temperatures. The difference
between our results and the results of Soluyanov et al.35 shows
from another angle that WTe2 is very close to the phase boundary
between the Weyl state and the fully gapped state. For many
purposes, it is favourable to have the Weyl state in a material that
is robust at elevated (room) temperatures. Here, we use the room
temperature lattice constants for all of our calculations. We also
note that the very small difference of the lattice constant value
does not play a role except for undoped or very lightly doped
samples, that is, xr2%, where the separation of the Weyl nodes is
beyond experimental resolution anyway. the results of Soluyanov et al.35 is due to the slightly different
values of the lattice constants36,39. The lattice constants used by
Soluyanov et al.35 were at low temperatures39. Thus the results35
better refelct the groundstate (T ¼ 0) of WTe2. We used the lattice
constants at room temperatures36, so our results correspond to
the state of WTe2 at elevated temperatures. The difference
between our results and the results of Soluyanov et al.35 shows
from another angle that WTe2 is very close to the phase boundary
between the Weyl state and the fully gapped state. For many
purposes, it is favourable to have the Weyl state in a material that
is robust at elevated (room) temperatures. Here, we use the room
temperature lattice constants for all of our calculations. We also
note that the very small difference of the lattice constant value
does not play a role except for undoped or very lightly doped
samples, that is, xr2%, where the separation of the Weyl nodes is
beyond experimental resolution anyway. Results We now calculate the band structure of pure WTe2 throughout
the bulk BZ based on the lattice constants reported in the paper
by Brown36. Our results show that pure WTe2 has a continuous
energy gap throughout the bulk BZ without any Weyl nodes. The
k point that corresponds to the minimal gap is found to be close
to the G Y (Fig. 2a) axis. The minimal gap of WTe2 is 0.9 meV
(Fig 2c) We note that the discrepancy between our results and For many years, research on Weyl semimetals has been held
back due to the lack of experimentally feasible candidate
materials. Recently, a family of isostructural compounds, TaAs,
NbAs, TaP and NbP, was theoretically predicted and experimen-
tally discovered as the first Weyl semimetals19–28. So far, the TaAs
class of the four iso-electronic compounds remains to be the only
experimentally realized Weyl semimetals21–28. p
y
y
Tungsten ditelluride, WTe2, is a member of the transition
metal dichalcogenide materials that has recently drawn significant
interest because it shows very large, non-saturating magnetore-
sistance and pressure-induced superconductivity29–34. It has an
inversion symmetry breaking crystal structure, and exhibits a NATURE COMMUNICATIONS | 7:10639 | DOI: 10.1038/ncomms10639 | www.nature.com/naturecommunications 2 ARTICLE NATURE COMMUNICATIONS | 7:10639 | DOI: 10.1038/ncomms10639 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/ncomms10639 All nodes are
located on the kz ¼ 0 plane. kz ¼ 0 plane. Thus in total there are four pairs of Weyl nodes on
the kz ¼ 0 plane. perturbations. The existence of Weyl nodes and Fermi arcs are
guaranteed by the system’s topology, whereas the details of the
surface states can change under surface perturbations. In order
to do so, we change the surface on-site potentials of the system. Physically, the surface potentials can be changed by surface
deposition or applying an electric field on the surface. Figure 3b
shows
the
surface
band
structure
with
the
surface
on-site energy increased by 0.02 eV. We find that the normal
surface state moves further away from the Weyl nodes, whereas
the topological Fermi arc does not change significantly. Figure 3c shows the surface band structure with the surface
on-site energy decreased by 0.11 eV. The normal surface states
disappear. The Fermi arc also changes significantly. Instead of
directly connecting the two Weyl nodes in Fig. 3a,b, a surface
state stems from each Weyl node and disperses outside the
window. We note that the surface states in Fig. 3c are still
topological and are still arcs because they terminate directly
onto the projected Weyl nodes. We illustrate the two types of
Fermi arc connectivity in Fig. 3d,e. Figure 3d corresponds to the
case in Fig. 3a,b, where a Fermi arc directly connects the pair of
Weyl nodes in a quadrant. Figure 3e corresponds to Fig. 2d. In
this case, Fermi arcs connect Weyl nodes in two different
quadrants across the Y G Y line. The normal surface states
do not exist necessarily as they can be removed by tuning the
on-site energy as shown in Fig. 3a,b. The nontrivial topology in
a Weyl semimetal requires that there must be Fermi arc(s)
terminating onto each projected Weyl node with a non-zero
projected chiral charge and that the number of Fermi arcs
associated with a projected Weyl node must equal its projected
chiral charge. On the other hand, the pattern of connectivity can
vary depending on details of the surface. The observed different Tunable Fermi arc surface states. A critical signature of a Weyl
semimetal is the existence of Fermi arc surface states. We pre-
sent calculations of the (001) surface states in Fig. 3. We choose
the 20% Mo-doped system, Mo0.2W0.8Te2. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms10639 Cut
Min. gap
Cut
W2(+)
0.02
0.04
0.06
0.08
0.1
0.11
0.115
0.12
0.125
0.02
0.04
0.06
0.08
0.1
0.11
0.115
0.12
0.125
0.03
0.09
Energy (eV)
Energy (eV)
Momentum (2 / a)
Momentum (2 / a)
0.03
0.09
Energy (eV)
W1(–)
W2(+)
0.0
0.05
0.10
0.15
0.0
0.05
0.10
WPs distance (2 / a)
Mo doping (%)
10
20
30
40
0.02
0.04
0
Mo doping (%)
10
20
30
40
0
Mo doping (%)
10
20
30
40
0
0
0
10
30
Band inversion energy
(meV)
W2(+)
W1(+)
W1(+)
W2(+)
Γ
0
–10
–20
–0.2
–0.1
0.0
0.1
0.2
–0.1 –0.05 0.0
0.05
0.1
ky (2 / b)
ky (2 / b)
ky (2 / b)
kx (2 / a)
kx (2 / a)
W1(–)
b4
b3
b2
b1
b4
b3
b2
b1
40
20
W2(–)
W2(–)
W2(–)
W1(–)
kx (2 / a)
a
b
c
d
e
f
g
h
Cw = 0
Cw ≠ 0
Figure 2 | Tunable Weyl node separation (topological strength) in Mo-doped WTe2. (a) The k space location that corresponds to the minimal energy gap
of the pure (undoped) WTe2. (b) The k space locations of the pair of Weyl nodes within the first quadrant of the kz ¼ 0 plane for Mo0.2W0.8Te2. White and
black dots show the negative and positive chiral charges (Cw), respectively. (c) Band structures of WTe2 along momentum space cut as defined in a. The
four bands appeared in this panel are labelled as b1–b4, respectively. (d) Band structures of Mo0.2W0.8Te2 along momentum space cut as defined in b. We
find an inversion between the b2 and b3 bands, giving rise to the Weyl nodes W1 and W2 with opposite chiral charges Cw. (e) The k space separation
between the pair of Weyl nodes W1 and W2 as a function of Mo doping x. (f) The energy difference between the extrema of the b2 and b3 bands as a
function of Mo doping x. This characterizes the magnitude of the band inversion. (g) The energy offset between the pair of Weyl nodes W1 and W2 as a
function of Mo doping x. The blue dashed lines are guides to the eye. (h) A schematic for the distribution of the Weyl nodes within a bulk BZ. All nodes are
located on the kz ¼ 0 plane. Cut
Min. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms10639 gap
0.02
0.04
0.06
0.08
0.1
0.11
0.115
0.12
0.125
ky (2 / b)
kx (2 / a)
a 0.03
0.09
Energy (eV)
Momentum (2 / a)
Energy (eV)
0.0
0.05
0.10
0.15
b4
b3
b2
b1
c
d Cut
W2(+)
1
0.02
0.04
0.06
0.08
0.1
0.11
0.115
0.12
0.125
ky (2 / b)
kx (2 / a)
W1(–)
b 9
Momentum (2 / a)
0.03
0.09
Energy (eV)
W1(–)
W2(+)
0.0
0.05
0.10
b4
b3
b2
b1
d b d a c h WPs distance (2 / a)
Mo doping (%)
10
20
30
40
0.02
0.04
0
0
x
e f Mo doping (%)
10
20
30
40
0
Band inversion energy
(meV)
0
–10
–20
f
Cw = 0
Cw ≠ 0 Energy (eV)
Mo doping (%)
10
20
30
40
0
0
10
30
40
20
g W2(+)
W1(+)
W1(+)
W2(+)
Γ
–0.2
–0.1
0.0
0.1
0.2
–0.1 –0.05 0.0
0.05
0.1
ky (2 / b)
W2(–)
W2(–)
W2(–)
W1(–)
kx (2 / a)
h e h g g Mo doping (%) Figure 2 | Tunable Weyl node separation (topological strength) in Mo-doped WTe2. (a) The k space location that corresponds to the minimal energy gap
of the pure (undoped) WTe2. (b) The k space locations of the pair of Weyl nodes within the first quadrant of the kz ¼ 0 plane for Mo0.2W0.8Te2. White and
black dots show the negative and positive chiral charges (Cw), respectively. (c) Band structures of WTe2 along momentum space cut as defined in a. The
four bands appeared in this panel are labelled as b1–b4, respectively. (d) Band structures of Mo0.2W0.8Te2 along momentum space cut as defined in b. We
find an inversion between the b2 and b3 bands, giving rise to the Weyl nodes W1 and W2 with opposite chiral charges Cw. (e) The k space separation
between the pair of Weyl nodes W1 and W2 as a function of Mo doping x. (f) The energy difference between the extrema of the b2 and b3 bands as a
function of Mo doping x. This characterizes the magnitude of the band inversion. (g) The energy offset between the pair of Weyl nodes W1 and W2 as a
function of Mo doping x. The blue dashed lines are guides to the eye. (h) A schematic for the distribution of the Weyl nodes within a bulk BZ. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms10639 40), but, as shown above, the existence of the
orthorhombic MoTe2 needs to be confirmed. By contrast, we
show that the moderately Mo-doped WTe2 sample has a number
of advantages, making it experimentally feasible. First, pure
MoTe2 has many irrelevant bands crossing the Fermi level along
the G X and G Z directions, whereas the band structure
of moderately Mo-doped system is as clean as pure WTe2
(Fig. 1d–f). Second, as we will show below, a moderate Mo doping NATURE COMMUNICATIONS | 7:10639 | DOI: 10.1038/ncomms10639 | www.nature.com/naturecommunications 3 NATURE COMMUNICATIONS | DOI: 10.1038/ncomms10639 ARTICLE NATURE COMMUNICATIONS | 7:10639 | DOI: 10.1038/ncomms10639 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/ncomms10639 NATURE COMMUNICATIONS | DOI: 10.1038/ncomms10639 0.10
0.05
0.0
0.03
0.09
Energy (eV)
0.10
0.05
0.0
0.03
0.09
Energy (eV)
b
Normal
surface state
Topological
surface state
0.10
0.05
0.0
0.03
0.09
Energy (eV)
c
W2(+)
Topological
surface state
Topological
surface state
W2(+)
W2(+)
Topological
surface state
Normal
surface state
d
e
g
0.05
–0.05
0.1
–0.1
0.0
Energy (eV)
h
SS1
SS2
0.05
–0.05
0.1
–0.1
0.0
Energy (eV)
SS2
0.04 0.05 0.06 0.07
0.03
0.116
0.120
0.124
W2(+)
Topological
surface state
f
j
0.0
Energy (eV)
0.2
0.4
0.6
–0.6
′
′
′′
′′
′
′′
′
′′
W2(+)
W1(+)
W2(–)
W1(+)
W2(–)
W1(–)
W1(–)
W2(+)
Γ
–
W1(–)
W2(+)
M
–
M
–
X
–
X
–
Y
–
W2(–)
W1(+)
W2(–)
W1(+)
W1(–)
W2(+)
Γ
–
SS1
SS2
W1(–)
W2(+)
Γ
–
Momentum (2π/a)
Momentum (2π/a)
Momentum (2π/a)
W1(–)
W1(–)
W1(–)
W1(–)
ky (2π/b)
kx (2π/a)
–0.2
–0.4
a
i
3 | Tunable Fermi arc length and interconnectivity in MoxW1 xTe2. (a) Surface and bulk band structure of Mo0.2W0.8Te2 along t
cut that goes through a direct panel of Weyl nodes, W1( ) and W2( þ ). NATURE COMMUNICATIONS | DOI: 10.1038/ncomms10639 A topological Fermi arc surface state connects the Weyl 0.10
0.05
0.0
0.03
0.09
Energy (eV)
W2(+)
Topological
surface state
Normal
surface state
Momentum (2π/a)
W1(–)
a 0.10
0.05
0.0
0.03
0.09
Energy (eV)
b
Normal
surface state
Topological
surface state
W2(+)
Momentum (2π/a)
W1(–) 0.10
0.05
0.0
0.03
0.09
Energy (eV)
c
W2(+)
Topological
surface state
Topological
surface state
Momentum (2π/a)
W1(–) b c c a 0.10
0.05
0.0
0.03
0.09
Energy (eV)
0.10
0.05
0.0
0.03
0.09
Energy (eV)
Normal
surface state
Topological
surface state
0.10
0.05
0.0
0.03
0.09
Energy (eV)
W2(+)
Topological
surface state
Topological
surface state
W2(+)
W2(+)
Topological
surface state
Normal
surface state
d
e
g
0.05
–0.05
0.1
–0.1
0.0
Energy (eV)
h
SS1
SS2
0.05
–0.05
0.1
–0.1
0.0
Energy (eV)
SS2
0.04 0.05 0.06 0.07
0.03
0.116
0.120
0.124
W2(+)
Topological
surface state
f
j
0.0
Energy (eV)
0.2
0.4
0.6
–0.6
′
′
′′
′′
′
′′
′
′′
W2(+)
W1(+)
W2(–)
W1(+)
W2(–)
W1(–)
W1(–)
W2(+)
Γ
–
W1(–)
W2(+)
M
–
M
–
X
–
X
–
Y
–
W2(–)
W1(+)
W2(–)
W1(+)
W1(–)
W2(+)
Γ
–
SS1
SS2
W1(–)
W2(+)
Γ
–
Momentum (2π/a)
Momentum (2π/a)
Momentum (2π/a)
W1(–)
W1(–)
W1(–)
W1(–)
ky (2π/b)
kx (2π/a)
–0.2
–0.4
i
Figure 3 | Tunable Fermi arc length and interconnectivity in MoxW1 xTe2. (a) Surface and bulk band structure of Mo0.2W0.8Te2 along the momentum
space cut that goes through a direct panel of Weyl nodes, W1( ) and W2( þ ). A topological Fermi arc surface state connects the Weyl nodes W1 and
W2. A normal surface state avoids the Weyl nodes and merges into the bulk band continuum. (b) Same as in a but with the surface on-site energy
increased by 0.02 eV. (c) Same as in a but with the surface on-site energy decreased by 0.11 eV. (d) Schematic of Fermi arc connectivity pattern for a,b. (e) Schematic of Fermi arc connectivity pattern for c. (f) Surface band structure on a varying-energy (kx, ky) map. Specifically, we choose a different energy
on each (kx, ky) point, so that the there are no bulk states at all kx, ky except at the locations of the eight Weyl nodes. This is possible because we know that
the conduction and valence bands only cross at the eight Weyl nodes. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms10639 (g) Surface and bulk band structure along the k—space trajectory a b g d
defined in j. (h,i) Surface and bulk band structure along the k—space trajectories a0 b0 and a0 b0 defined in j. (j) Schematic of a quadrant of the surface
BZ, showing the k—space trajectories used in g–i. Momentum (2π/a) 0.04 0.05 0.06 0.07
0.03
0.116
0.120
0.124
W2(+)
Topological
surface state
f
W1(–)
ky (2π/b)
kx (2π/a) d
W2(+)
W1(+)
W2(–)
W1(+)
W2(–)
W1(–)
W1(–)
W2(+)
Γ
–
M
–
X
–
Y
– f e
W1(–)
W2(+)
M
–
X
–
W2(–)
W1(+)
W2(–)
W1(+)
W1(–)
W2(+)
Γ
– d d e e g
0.0
Energy (eV)
0.2
0.4
0.6
–0.6
SS1
SS2
–0.2
–0.4 g 0.0
0.2
0.4
0.6
–0.6
–0.2
–0.4 0.05
–0.05
0.1
–0.1
0.0
Energy (eV)
SS2
j
′′
′′
i j
′
′′
′
′′
W1(–)
W2(+)
Γ
– 0.05
–0.05
0.1
–0.1
0.0
Energy (eV)
h
SS1
SS2
′
′ j i i h Figure 3 | Tunable Fermi arc length and interconnectivity in MoxW1 xTe2. (a) Surface and bulk band structure of Mo0.2W0.8Te2 along the momentum
space cut that goes through a direct panel of Weyl nodes, W1( ) and W2( þ ). A topological Fermi arc surface state connects the Weyl nodes W1 and
W2. A normal surface state avoids the Weyl nodes and merges into the bulk band continuum. (b) Same as in a but with the surface on-site energy
increased by 0.02 eV. (c) Same as in a but with the surface on-site energy decreased by 0.11 eV. (d) Schematic of Fermi arc connectivity pattern for a,b. (e) Schematic of Fermi arc connectivity pattern for c. (f) Surface band structure on a varying-energy (kx, ky) map. Specifically, we choose a different energy
on each (kx, ky) point, so that the there are no bulk states at all kx, ky except at the locations of the eight Weyl nodes. This is possible because we know that
the conduction and valence bands only cross at the eight Weyl nodes. (g) Surface and bulk band structure along the k—space trajectory a b g d
defined in j. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms10639 Figure 3a shows the
surface energy dispersion along the momentum space cut that
goes through the direct pair of Weyl nodes, W1( ) and
W2( þ ), which arises from a single band inversion. Here ( þ )
and ( ) denote positive and negative chiral charge, respec-
tively. Our calculation (Fig. 3a) clearly shows the topological
Fermi arc surface state, which connects the direct pair of Weyl
nodes. The Fermi arc was found to terminate directly onto the
projected Weyl nodes. In addition, we also observe a normal
surface state, which avoids the Weyl node and merges into the
bulk band continuum. Because the W1 and W2 Weyl nodes
have different energies, and because W1 is a type II Weyl cone35,
constant energy maps always have finite Fermi surfaces. Type II
means that at the Weyl node energy, its constant energy contour
consists of an electron and a hole pocket touching at a point, the
Weyl node. Hence, visualizing Fermi arc connectivity in
constant energy maps is not straightforward. Instead of a
constant energy map, it is possible to use a varying-energy kx, ky
map, that is, Energy ¼ E(kx, ky), so that there are no bulk states
on this varying-energy map at all kx, ky points except the Weyl
nodes. Figure 3f shows the calculated surface and bulk electronic
structure on such a varying-energy kx, ky map in the vicinity of a
pair of Weyl nodes. A Fermi arc that connects the pair of Weyl
nodes can be clearly seen. We study the effect of surface NATURE COMMUNICATIONS | 7:10639 | DOI: 10.1038/ncomms10639 | www.nature.com/naturecommunications 4 4 ARTICLE NATURE COMMUNICATIONS | 7:10639 | DOI: 10.1038/ncomms10639 | www.nature.com/naturecommunications Discussions We discuss the tunability of the length of the Fermi arcs as a
function of Mo concentration x in our MoxW1 xTe2 system. The
undoped
x ¼ 0
sample
is
fully
gapped
according
to
our
calculations (Fig. 4g). A very small Mo concentration (B0.5%)
will drive the system to the critical point, where the conduction
and valence bands just touch each other Fig. 4h. The length of the
Fermi arc is zero, and hence the system is at the critical point. As
one further increases the Mo concentration x, the touching point
splits into a pair of Weyl nodes with opposite chiralities (Fig. 4i). The Weyl nodes are connected by a Fermi arc. A way to gap the
system without breaking any symmetry is to annihilate pairs of
Weyl nodes with opposite chiralities. In order to do so, one needs
to overcome the momentum space separation between the Weyl
nodes to bring them together in the k space. Thus the length of
the Fermi arc that directly connects the Weyl nodes by the
shortest distance provides a measure of the system’s topological
strength. Such a tunability is not known in the TaAs class of
Weyl system. We also compare our proposal to previous
candidates9,41–47, where analogous tunability was proposed. We
emphasize that the key difference is the experimental feasibility of
the proposals. For example, refs 41–43 used hypothetic crystal
structures which have never been synthesized. Reference
9
requires
a
ferromagnetic
normal
insulator
that
is
lattice
matched with the known topological insulators, which are
currently unknown. Reference 44 requires external magnetic
fields and hence ARPES cannot be done. References 46,47 require
external pressure and cannot be realized at ambient conditions. As stressed in the TaAs prediction19, in order to have a robust
material candidate, following aspects are crucial: first, the
proposal can be realized at ambient conditions with a realistic
crystal structure that has been successfully synthesized. Second,
there is no need to align the magnetic domains as in the
ferromagnetic
compounds;
Third,
the
proposed
candidate
does not need fine-tuning of the chemical composition (in our
case, any composition within the the wide range of 3 to 40%
works). Fourth, the separation of Weyl nodes is large enough
for experimental observation, which is not the case for WTe2. These aspects were crucial for the experimental discovery
of TaAs19,21 and are also satisfied by our prediction of
MoxW1 xTe2 here. ARTICLE By the same token, we can choose the loop
a00 b00 g d,
which
does
not
enclose
any
Weyl
node. Consistently, as shown in Fig. 3i, along a00 b00, SS1 does not
appear along this line and SS2 does not connect across the band
gap. Hence the net number of chiral edge mode is also zero along
this rectangular loop. shown in Fig. 4b. The surface state band keeps connecting the two
small pockets as one changes the energy. This evolution is shown
by real calculations in Fig. 4e,f. The orange line in Fig. 4b
connects the W1( ) and W2( þ ) Weyl nodes. At each energy,
the surface state band crosses the orange line at a specific k point. By picking up the crossing points at different energies, we can
reconstruct the Fermi arc that connects the W1( ) and W2( þ )
Weyl nodes shown in Fig. 4c. Therefore, from our systematic
studies above, we show that the Fermi arc connectivity means the
pattern in which the surface state connects the Weyl nodes. This
is defined on a varying-energy (kx, ky) map where the chosen
E(kx, ky) map crosses the bulk bands only at the Weyl nodes. If
there is no significant energy offset between Weyl nodes and if all
Weyl cones are normal rather than type II, then the connectivity
can also be seen in a constant energy contour. However, in our
case here, one needs to be careful with the simplified ideal picture,
that is, to study the Fermi arc connectivity from the constant
energy contour. Because of the energy offset between the Weyl
nodes and because of the existence of type II Weyl cones, how
surface bands connect different bulk pockets in a constant energy
contour
does
not
straightforwardly
show
the
Fermi
arc
connectivity. ARTICLE 3i, along a00 b00, SS1 does not
appear along this line and SS2 does not connect across the band
gap. Hence the net number of chiral edge mode is also zero along
this rectangular loop. chosen rectangular loop corresponds to a rectangular pipe along
the kz direction. Then topological band theory requires that the
net chiral charge of the Weyl nodes that are enclosed by the pipe
equals the Chern number on this manifold, which further equals
the net number of chiral edge modes along the loop. For example,
the rectangular loop a b g d in Fig. 3j encloses a W1( )
and a W2( þ ), which leads to a net chiral charge zero. The energy
dispersion along this rectangular loop is shown in Fig. 3g. It can
be seen that the bands are fully gapped without any surface states
along b g d a. Along a b, there are two surface states
(SS1 and SS2), both of which connect the band gap. Interestingly,
we note that these two surface bands are counter-propagating
although they seem to have the same sign of Fermi velocity. This is because the continuous energy gap a b is highly ‘tilted’. If we ‘tilt’ the energy gap back to being horizontal, then it can be
clearly seen that the two surface bands are counter-propagating,
which means that the net number of chiral edge modes is zero. Similarly, we can choose other loops. For example, we choose
another rectangular loop a0 b0 g d that encloses only the
W2( þ ) Weyl node. Because there are no surface states along the
two horizontal edges and the vertical edge to the right, we only
need to study the vertical edge to the left, that is the a0 b0. The enclosed net chiral charge is þ 1, which should equal the net
number of chiral edge mode along a0 b0. The band structure
along this line is shown in Fig. 3h. We see that while the surface
band SS1 still connects across the band gap, SS2 starts from and
ends at the conduction bands. Therefore, SS1 contributes one net
chiral edge mode, whereas SS2 contributes zero net chiral edge
mode. Hence there is one net chiral edge mode along the this
rectangular loop. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms10639 chosen rectangular loop corresponds to a rectangular pipe along
the kz direction. Then topological band theory requires that the
net chiral charge of the Weyl nodes that are enclosed by the pipe
equals the Chern number on this manifold, which further equals
the net number of chiral edge modes along the loop. For example,
the rectangular loop a b g d in Fig. 3j encloses a W1( )
and a W2( þ ), which leads to a net chiral charge zero. The energy
dispersion along this rectangular loop is shown in Fig. 3g. It can
be seen that the bands are fully gapped without any surface states
along b g d a. Along a b, there are two surface states
(SS1 and SS2), both of which connect the band gap. Interestingly,
we note that these two surface bands are counter-propagating
although they seem to have the same sign of Fermi velocity. This is because the continuous energy gap a b is highly ‘tilted’. If we ‘tilt’ the energy gap back to being horizontal, then it can be
clearly seen that the two surface bands are counter-propagating,
which means that the net number of chiral edge modes is zero. Similarly, we can choose other loops. For example, we choose
another rectangular loop a0 b0 g d that encloses only the
W2( þ ) Weyl node. Because there are no surface states along the
two horizontal edges and the vertical edge to the right, we only
need to study the vertical edge to the left, that is the a0 b0. The enclosed net chiral charge is þ 1, which should equal the net
number of chiral edge mode along a0 b0. The band structure
along this line is shown in Fig. 3h. We see that while the surface
band SS1 still connects across the band gap, SS2 starts from and
ends at the conduction bands. Therefore, SS1 contributes one net
chiral edge mode, whereas SS2 contributes zero net chiral edge
mode. Hence there is one net chiral edge mode along the this
rectangular loop. By the same token, we can choose the loop
a00 b00 g d,
which
does
not
enclose
any
Weyl
node. Consistently, as shown in Fig. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms10639 (h,i) Surface and bulk band structure along the k—space trajectories a0 b0 and a0 b0 defined in j. (j) Schematic of a quadrant of the surface
BZ, showing the k—space trajectories used in g–i. correspond to the case without additional changes to the on-site
energy. Specifically, we choose a closed loop in (kx, ky) space as
shown in Fig. 3j. As we mentioned above, the conduction and
valence bands only touch at the eight Weyl nodes. Thus as long as
the loop chosen does not go through these Weyl nodes, there is a
continuous bulk energy gap along the loop. In the bulk BZ, the Fermi arc connectivity patterns as a function of surface on-site
potential provide an explicit example of both the constraints
imposed and the freedoms allowed to the Fermi arc electronic
structure by the nontrivial topology in a Weyl semimetal. We further study the surface states via bulk boundary
correspondence. We note that except Fig. 3b–e, all other figures NATURE COMMUNICATIONS | 7:10639 | DOI: 10.1038/ncomms10639 | www.nature.com/naturecommunications 5 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms10639 NATURE COMMUNICATIONS | DOI: 10.1038/ncomms10639 W1(–)
W1(+)
W2(+)
W2(–)
Topological
surface state
Bulk states (E1)
Bulk state (E2)
Bulk state (E1)
Topo. ss (E1)
Topo. ss (E2)
Energy
W1(–)
W2(+)
Cut
Cut
W1(–)
W1(+)
W2(–)
W2(+)
X = 0
ky
ky
ky (2π / b)
kx (2π / a)
kx (2π / a)
X ~ 0.005
X = 0.2
Energy
Momentum
Momentum
Momentum
Mo doping (%)
Fermi arc length (2 / a)
Critical point
W1
W2
Critical point
W1
W2
b2
b3
b2
b2
b3
b3
Fermi arc
Fermi arc
Topological
surface state
Bulk
edge
W2(+)
Bulk edge
Normal
surface state
W1(–)
0.04
0.06
0.08
0.11
0.12
0.13
0.125
0.115
W2(+)
Bulk
edge
Topological
surface state
Normal
surface state
Bulk edge
W1(–)
0.04
0.06
0.08
Low
High
10
20
40
30
0.02
0.04
0
0
E1
E1
E1
E=E1
E=E2
E1
E2
E1
E2
E2
Y
Y
M
Y
M
M
Y
M
X
X
Γ
Γ
X
Γ
Y
M
X
Γ
Y
M
X
Γ
Γ
X
Figure 4 | Iso-energy contour interconnectivity on the surface of MoxW1 xTe2. (a) Schematic illustration of the surface and bulk electronic structure on
a constant energy E1. The shaded areas represent the projected bulk bands, whereas the red line show the surface states. (b) Schematic illustration of the
surface and bulk electronic structure on two energies E1 and E2. The band structure on E1 (E2) are shown by the solid (dotted) lines. The energies E1 and E2
are defined in c. (c) Band structure along a k space cut that goes through the direct pair of Weyl nodes, W1( ) and W2( þ ). The dotted lines denote the
energies E1 and E2 with respect to the W1 and W2 Weyl nodes. (d) Calculated surface and bulk electronic structure on at the energy E1 over the top half of
the surface BZ. (e) A zoomed-in view of d for the area highlighted by the blue box. (f) The same as e but at the energy E2. (g–i) Schematic band diagram to
show the evolution of the MoxW1 xTe2 system as a function of Mo concentration x. (j) The length of the Fermi arc as a function of Mo concentration x. The
arc length equals the k space separation of the Weyl nodes. W1(–)
W1(+)
W2(+)
W2(–)
Bulk state (E2)
Bulk state (E1)
Topo. ss (E1)
Topo. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms10639 ss (E2)
Energy
Cut
E1
E2
Y
M
Γ
X Topological
surface state
Bulk states (E1)
W1(–)
W1(+)
W2(–)
W2(+)
E1
Y
M
X
Γ Energy
W1(–)
W2(+)
Cut
E1
E2 ky
ky
E1
Y
M
X
Γ E kx (2π / a)
W2(+)
Bulk
edge
Topological
surface state
Normal
surface state
Bulk edge
W1(–)
0.04
0.06
0.08
Low
High
E=E2
E2 ky (2π / b)
kx (2π / a)
Topological
surface state
Bulk
edge
W2(+)
Bulk edge
Normal
surface state
W1(–)
0.04
0.06
0.08
0.11
0.12
0.13
0.125
0.115
E=E1
E1 X = 0.2
Momentum
W1
W2
W1
W2
b2
b3
Fermi arc
Fermi arc
Y
M
X
Γ Mo doping (%)
Fermi arc length (2 / a)
10
20
40
30
0.02
0.04
0
0 X = 0
Energy
Momentum
b2
b3
Y
M
X
Γ X ~ 0.005
Momentum
Critical point
b2
b3 X = 0
Energy
Momentum
b2
b3 Momentum
Critical point
Y
M
X
Γ Figure 4 | Iso-energy contour interconnectivity on the surface of MoxW1 xTe2. (a) Schematic illustration of the surface and bulk electronic structure on
a constant energy E1. The shaded areas represent the projected bulk bands, whereas the red line show the surface states. (b) Schematic illustration of the
surface and bulk electronic structure on two energies E1 and E2. The band structure on E1 (E2) are shown by the solid (dotted) lines. The energies E1 and E2
are defined in c. (c) Band structure along a k space cut that goes through the direct pair of Weyl nodes, W1( ) and W2( þ ). The dotted lines denote the
energies E1 and E2 with respect to the W1 and W2 Weyl nodes. (d) Calculated surface and bulk electronic structure on at the energy E1 over the top half of
the surface BZ. (e) A zoomed-in view of d for the area highlighted by the blue box. (f) The same as e but at the energy E2. (g–i) Schematic band diagram to
show the evolution of the MoxW1 xTe2 system as a function of Mo concentration x. (j) The length of the Fermi arc as a function of Mo concentration x. The
arc length equals the k space separation of the Weyl nodes. Methods
C
t ti between the two end points. Therefore, this approach is highly reliable and effective
for studying iso-valence substitution/doping. Previously, we used the same method to
investigate a similar iso-valence substitution in the BiTlSe1 xSx and predicted a
critical point of xc ¼ 0.48 that was in excellent agreement with experimental finding of
xc ¼ 0.5 (ref. 56). Also, since we mainly studied small Mo doping, for example,
Mo¼ 20% that is close enough to the end compound, a linear interpolation should be
a good approximation. The surface state electronic structure was calculated by the
surface Green’s function technique, which computes the spectral weight near the
surface of a semi-infinite system. Computational details. We computed the electronic structures using the projector
augmented wave method48,49 as implemented in the VASP package50 within the
generalized gradient approximation schemes51. For WTe2, experimental lattice
constants were used36. For MoTe2, we assumed that it has the same crystal
structure as WTe2 and calculated the lattice constants self-consistently (a¼ 6.328Å,
b ¼ 3.453 Å, c ¼ 13.506 Å). A 8 16 4 MonkhorstPack k-point mesh was used in
the computations. The spin-orbit coupling effects were included in calculations. In
order to systematically calculate the surface and bulk electronic structure, we
constructed a tight-binding Hamiltonian for both WTe2 and MoTe2, where the tight-
binding model matrix elements were calculated by projecting onto the Wannier
orbitals52–54, which used the VASP2WANNIER90 interface55. We used W (Mo) s
and d orbitals and Te p orbitals to construct Wannier functions without using the
maximizing localization procedure. The electronic structure of the samples with finite
dopings was calculated by a linear interpolation of tight-binding model matrix
elements of WTe2 and MoTe2. Since tight-binding parameters contained all
important information such as lattice constants and atomic bonding strength, an
interpolation presumably covered all systematic changes of the electronic structure NATURE COMMUNICATIONS | 7:10639 | DOI: 10.1038/ncomms10639 | www.nature.com/naturecommunications Discussions We hope that our prediction can provide
another material realization which is critically needed for this
rapidly developing field. g
We study the constant energy contours of the surface states. We emphasize that (i) there is a significant energy offset between
the W1 and W2 Weyl nodes, and that (ii) the W1 Weyl cones are
the type II Weyl cone35. These two properties are very different
from the ideal picture, where all Weyl cones are normal rather
than type II and their nodes are all at the same energy. We show
below that these two properties make the surface states’ constant
energy contours quite different from what one would expect
naively. Figure 4d shows the calculated constant energy contour
within the top half of the surface BZ at energy E1, which is
between the W1 and W2 nodes in energy. More constant energy
contours at other energies are shown in the Supplementary Fig. 1
and Supplementary Note 1. We see three bulk pockets. The
corresponding schematic is shown in Fig. 4a. Specifically, we see a
big pocket closer to the Y point, which encloses two W1 Weyl
nodes with opposite chiral charges. We also see two separate
small pockets closer to the G, each of which encloses a W2 Weyl
node. As for the surface states, from Fig. 4d,e we see a surface
state band that connects the two small pockets, each of which
encloses a W2 Weyl node. This is quite counter-intuitive because
we know that the Fermi arc connects the direct pair of Weyl
nodes, namely a W1( ) and a W2( þ ) or vice versa. We show
that there is no discrepancy. Specifically, we show that the surface
band seen in the constant energy contours is exactly the Fermi arc
that connects the W1( ) and the W2( þ ) Weyl nodes seen in
Fig. 4c. To do so, we consider the constant energy contours at two
different energies, E1 and E2. According to the energy dispersion
(Fig. 4c), we see that the big bulk pocket in the constant energy
map is electron-like, while the two small bulk pockets are hole-
like. Thus as we increase the energy from E1 to E2, the big pocket
should expand, whereas the two small pockets should shrink, as NATURE COMMUNICATIONS | 7:10639 | DOI: 10.1038/ncomms10639 | www.nature.com/naturecommunications 6 ARTICLE References
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(2015). Author contributions 33. Wu, Y. et al. Temperature induced Lifshitz transition in WTe2. Phys. Rev. Lett. 115, 166602 (2015). All authors contributed to the intellectual contents of this work. Preliminary
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were then performed by T.-R.C., G.C., C.-C.L., S.-M.H., B.W., A.B., H.-T.J. and H.L. All the authors analysed the data; T.-R.C., S.-Y.X., H.L. and M.Z.H. wrote the
manuscript; S.-Y.X., H.L. and M.Z.H. were responsible for the overall research
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37. Puotinen, D. & Newnham, R. E. The crystal structure of MoTe2. Acta Cryst. 14,
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semiconductors of the second kind. J. Low Temp. Phys. 5, 141–154
ð1972Þ: 7 ARTICLE & Miyake, T. Weyl
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at http://arxiv.org/abs/1508.03502 (2015). Supplementary Information accompanies this paper at http://www.nature.com/
naturecommunications 8 NATURE COMMUNICATIONS | 7:10639 | DOI: 10.1038/ncomms10639 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | 7:10639 | DOI: 10.1038/ncomms10639 | www.nature.com/naturecommunicat NATURE COMMUNICATIONS | DOI: 10.1038/ncomms10639 Competing financial interests: The authors declare no competing financial interests. This work is licensed under a Creative Commons Attribution 4.0
International License. The images or other third party material in this
article are included in the article’s Creative Commons license, unless indicated otherwise
in the credit line; if the material is not included under the Creative Commons license,
users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ Reprints and permission information is available online at http://npg.nature.com/
reprintsandpermissions/ How to cite this article: Chang, T.-R. et al. Prediction of an arc-tunable Weyl Fermion
metallic state in MoxW1 xTe2. Nat. Commun. 7:10639 doi: 10.1038/ncomms10639 (2016). NATURE COMMUNICATIONS | 7:10639 | DOI: 10.1038/ncomms10639 | www.nature.com/naturecommunications 9
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English
| null |
MERS-CoV
|
BMC infectious diseases
| 2,014
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cc-by
| 595
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MERS-CoV Marcel A Müller From International Symposium HIV and Emerging Infectious Diseases 2014
Marseille, France. 21-23 May 2014 From International Symposium HIV and Emerging Infectious Diseases 2014
Marseille, France. 21-23 May 2014 choice to control the current outbreak and prevent future
re-introductions into the human population. In 2002/03, a novel coronavirus (CoV) termed severe acute
respiratory syndrome (SARS)-CoV emerged in China and
caused an epidemic in which approximately 800 of 8000
cases died. Ten years later, a newly emerged Middle East
respiratory syndrome (MERS)-CoV has been confirmed in
189 patients of which 82 had a fatal outcome. All MERS
cases were linked to the Arabian Peninsula and to date
thirteen cases were imported into the European Union and
Africa. The clinical presentation resembles that of SARS-
CoV as both viruses affect mainly the lower respiratory
tract. As for other mammalian CoV, bats harbor a high
diversity of related viruses and are suspected to be the ani-
mal reservoir. Recent studies have revealed that dromedary
camels are the most likely intermediate host. MERS-CoV
has been circulating in dromedary camels for decades rais-
ing the question whether earlier human cases may have
remained undetected. Preliminary serological surveys in
Saudi Arabia could not determine increased seroprevalence
for MERS-CoV antibodies in the population. The possibi-
lity of human to human transmission has been demon-
strated but is clearly limited. The dipeptidyl peptidase-4
(DPP4) was identified as a cellular receptor for MERS-CoV
entry. DPP4 was found predominantly on non-ciliated
bronchial epithelial and alveolar cells in the lower parts of
the lungs. This might explain the lack of secondary infec-
tions because exposure to high doses of viruses may be
necessary. In-vitro studies showed that MERS-CoV inhib-
ited important cellular pathways in particular the interferon
(IFN) response. Confirmatory in-vivo experiments are still
limited due to the lack of a suitable animal model. Non-
human primates were shown to be susceptible but did not
develop a disease to an extent as humans. A combination
of pegylated IFN-alpha and ribavirin improved the clinical
symptoms confirming previous in-vitro experiments. The development of vaccines is ongoing and vaccination
programs in dromedary camels might be the optimal Published: 23 May 2014
doi:10.1186/1471-2334-14-S2-S22
Cite this article as: Müller: MERS-CoV. BMC Infectious Diseases 2014
14(Suppl 2):S22. Published: 23 May 2014 Published: 23 May 2014 doi:10.1186/1471-2334-14-S2-S22
Cite this article as: Müller: MERS-CoV. BMC Infectious Diseases 2014
14(Suppl 2):S22. Müller BMC Infectious Diseases 2014, 14(Suppl 2):S22
http://www.biomedcentral.com/1471-2334/14/S2/S22 Müller BMC Infectious Diseases 2014, 14(Suppl 2):S22
http://www.biomedcentral.com/1471-2334/14/S2/S22 MERS-CoV Submit your next manuscript to BioMed Central
and take full advantage of:
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and take full advantage of: Institute of Virology, University of Bonn Medical Center, Bonn, 53113,
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Portuguese
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Insuficiência renal aguda: experiência de um centro terciário de Nefrologia Pediátrica
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Brazilian Journal of Nephrology
| 2,024
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cc-by
| 5,773
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Autores Autores
Carolina Figueiredo1
Ana Margarida Rocha2
Liane Correia-Costa2,3,4,5
Maria do Sameiro Faria3,6
Teresa Costa3
Conceição Mota3 Carolina Figueiredo1
Ana Margarida Rocha2
Liane Correia-Costa2,3,4,5
Maria do Sameiro Faria3,6 Introduction: Acute kidney injury (AKI)
is an abrupt deterioration of kidney
function. The incidence of pediatric AKI
is increasing worldwide, both in critically
and non-critically ill settings. We aimed
to characterize the presentation, etiology,
evolution, and outcome of AKI in
pediatric patients admitted to a tertiary
care center. Methods: We performed a
retrospective observational single-center
study of patients aged 29 days to 17 years
and 365 days admitted to our Pediatric
Nephrology Unit from January 2012
to December 2021, with the diagnosis
of AKI. AKI severity was categorized
according to Kidney Disease Improving
Global Outcomes (KDIGO) criteria. The outcomes considered were death or
sequelae (proteinuria, hypertension, or
changes in renal function at 3 to 6 months
follow-up assessments). Results: Forty-six
patients with a median age of 13.0 (3.5–
15.5) years were included. About half
of the patients (n = 24, 52.2%) had an
identifiable risk factor for the development
of AKI. Thirteen patients (28.3%) were
anuric, and all of those were categorized as
AKI KDIGO stage 3 (p < 0.001). Almost
one quarter (n = 10, 21.7%) of patients
required
renal
replacement
therapy. Approximately 60% of patients (n = 26)
had at least one sequelae, with proteinuria
being the most common (n = 15, 38.5%;
median (P25–75) urinary protein-to-
creatinine ratio 0.30 (0.27–0.44) mg/mg),
followed by reduced glomerular filtration
rate (GFR) (n = 11, 27.5%; median (P25–
75) GFR 75 (62–83) mL/min/1.73 m2). Conclusions: Pediatric AKI is associated
with substantial morbidity, with potential
for proteinuria development and renal
function impairment and a relevant
impact on long-term prognosis. K
d
A
Kid
I j Introdução: Insuficiência renal aguda (IRA) é
uma deterioração abrupta da função renal. A
incidência de IRA pediátrica está aumentando
em todo o mundo, em ambientes críticos e
não críticos. Nosso objetivo foi caracterizar
apresentação, etiologia, evolução e desfechos
da IRA em pacientes pediátricos internados
em um centro de atendimento terciário. Métodos: Realizamos estudo retrospectivo
observacional de centro único de pacientes
com idade entre 29 dias a 17 anos e 365
dias internados em nossa Unidade de
Nefrologia Pediátrica, de janeiro de 2012
a dezembro de 2021, com diagnóstico de
IRA. A gravidade da IRA foi categorizada de
acordo com os critérios do Kidney Disease
Improving Global Outcomes (KDIGO). Artigo Original | Original Article Artigo Original | Original Article Data de submissão: 21/01/2024.
Data de aprovação: 12/03/2024.
Data de publicação: 29/04/2024. 6Universidade do Porto
e Universidade NOVA de
Lisboa, Unidade de Ciências
Biomoleculares Aplicadas
(UCIBIO), Lisboa, Portugal. Autores Os desfechos considerados foram óbito
ou sequelas (proteinúria, hipertensão ou
alterações na função renal em avaliações
de acompanhamento de 3 a 6 meses). Resultados: Incluímos 46 pacientes com idade
mediana de 13,0 (3,5–15,5) anos. Cerca de
metade (n = 24; 52,2%) apresentou um fator
de risco identificável para o desenvolvimento
de IRA. Treze pacientes (28,3%) eram
anúricos; todos foram classificados como
IRA KDIGO 3 (p < 0,001). Quase um quarto
(n = 10; 21,7%) dos pacientes necessitaram de
terapia renal substitutiva. Aproximadamente
60% (n = 26) apresentou pelo menos uma
sequela, sendo proteinúria a mais comum
(n = 15; 38,5%; mediana (P25–75) da relação
proteína/creatinina urinária 0,30 (0,27–
0,44) mg/mg), seguida de taxa de filtração
glomerular (TFG) reduzida (n = 11; 27,5%;
mediana (P25–75) da TFG 75 (62–83) mL/
min/1,73 m2). Conclusões: A IRA pediátrica
está associada à morbidade substancial,
com potencial para desenvolvimento de
proteinúria e comprometimento da função
renal e impacto relevante no prognóstico de
longo prazo. 1Hospital do Divino Espírito Santo
de Ponta Delgada, Serviço de
Pediatria, Ilha de São Miguel,
Portugal. 1Hospital do Divino Espírito Santo
de Ponta Delgada, Serviço de
Pediatria, Ilha de São Miguel,
Portugal. 2Universidade do Porto, Instituto
de Ciências Biomédicas Abel
Salazar, Porto, Portugal. 3Centro Hospitalar Universitário
de Santo António, Centro
Materno-Infantil do Norte,
Unidade de Nefrologia Pediátrica,
Porto, Portugal. 4Universidade do Porto, Instituto
de Saúde Pública (EPIUnit), Porto,
Portugal. 5Universidade do Porto,
Laboratório para a Investigação
Integrativa e Translacional em
Saúde Populacional (ITR), Porto,
Portugal. DOI: https://doi.org/10.1590/2175-
8239-JBN-2024-0012pt
Data de submissão: 21/01/2024. Data de aprovação: 12/03/2024. Data de publicação: 29/04/2024. Correspondência para:
Liane Correia-Costa. E-mail: lianecosta@icbas.up.pt DOI: https://doi.org/10.1590/2175-
8239-JBN-2024-0012pt
Correspondência para:
Liane Correia-Costa. E-mail: lianecosta@icbas.up.pt Descritores: Injúria Renal Aguda; Pediatria;
Etiologia; Fatores de Risco; Resultado do
Tratamento. Keywords:
Acute
Kidney
Injury;
Pediatrics;
Etiology;
Risk
Factors;
Treatment Outcome. Insuficiência renal aguda em crianças e adolescentes No presente estudo, nosso objetivo foi caracterizar
a apresentação, etiologia, evolução e desfechos de
todos os casos de IRA em pacientes pediátricos com
idade entre 29 dias e 17 anos e 365 dias internados
em um centro terciário de nefrologia pediátrica em
Portugal na última década. Introdução A insuficiência renal aguda (IRA) é uma deterioração
abrupta
da
função
renal1–3. O
espectro
de
manifestações é amplo, variando de alterações
analíticas sutis na função renal à falência do órgão4,5. De acordo com a literatura, a IRA afeta quase um
terço de crianças hospitalizadas, e sua incidência está
aumentando em todo o mundo6–11. Em ambiente não
crítico, um estudo recente realizado em um hospital
infantil de cuidados terciários com mais de dois mil
pacientes descreveu que a IRA foi observada em
pelo menos 5% deles4,10,12. A frequência de IRA é
particularmente elevada em pacientes críticos, pois a
mesma é considerada a complicação mais comum em
crianças internadas em unidades de terapia intensiva
pediátrica
(UTIP)13–15. Um
estudo
prospectivo
multinacional envolvendo quase cinco mil crianças e
adultos jovens com idades entre 3 meses e 25 anos
internados em UTIP relatou uma incidência de IRA
de 26,9%16. No entanto, a incidência geral de IRA na
população pediátrica é um pouco incerta, uma vez que
depende da população estudada. Um corpo relevante
de pesquisas tem se concentrado em pacientes de
alto risco, especialmente aqueles que foram expostos
a nefrotoxinas, submetidos a cirurgia cardíaca ou
internados em UTIP4,12. Desenho do Estudo e Amostra Desenho do Estudo e Amostra Realizamos um estudo de coorte retrospectivo
observacional de centro único de crianças e
adolescentes com idades entre 29 dias e 17 anos e 365
dias internados na Unidade de Nefrologia do Centro
Materno-Infantil do Norte por um período de 10 anos
consecutivos (de janeiro de 2012 a dezembro de 2021)
com o diagnóstico de IRA. Foram incluídos todos os
pacientes com diagnóstico de IRA no momento da alta
hospitalar, a menos que houvesse um diagnóstico prévio
de doença renal crônica (16 pacientes foram excluídos
da presente análise, uma vez que apresentavam doença
renal crônica nos estágios 2 a 4 e a lesão observada
foi considerada uma lesão renal aguda sobreposta à
doença renal crônica). Coleta de Dados e Definição de Variáveis Coleta de Dados e Definição de Variáveis Os dados clínicos foram recuperados de prontuários
clínicos eletrônicos dos pacientes incluídos. Diversos mecanismos fisiopatológicos podem
estar envolvidos na IRA. Atualmente, as etiologias
pré-renais são as mais comumente associadas à IRA
pediátrica, seguidas por distúrbios intrínsecos ou
renais, como a glomerulonefrite14,17,18. Uma vez que
poucas abordagens terapêuticas específicas e eficazes
estão disponíveis no momento, o conhecimento dos
fatores de risco para IRA é de suma importância1,19,20. Fatores como prematuridade ou comorbidades
crônicas e eventos como depleção de volume, exposição
a nefrotoxinas, sepse e cirurgias de grande porte
(cirurgias cardíacas, principalmente com circulação
extracorpórea) são os fatores preponderantes para o
desenvolvimento de IRA1,2,5. A gravidade da IRA foi avaliada usando os estágios
1 a 3 do Kidney Disease Improving Global Outcomes
(KDIGO), que foram definidos com base nos valores
de creatinina sérica basal e máximo registrados
durante a internação, da seguinte forma: IRA estágio
1 foi definido como um valor de creatinina sérica
(CrS) de 1,5 a 1,9 vezes o valor basal, ou aumento
de ≥0,3 mg/dL, ou volume urinário <0,5 mL/kg/h por
6 a 12 horas; IRA estágio 2 foi definido como um valor
de CrS de 2,0 a 2,9 vezes o valor basal, ou volume
urinário <0,5 mL/kg/h por ≥12 horas; IRA estágio 3
compreendia um valor de CrS 3,0 vezes o valor basal,
ou aumento na CrS para ≥4,0 mg/dL, ou início de
terapia renal substitutiva, ou diminuiçãoda taxa de
filtração glomerular (TFG) estimada para <35 mL/min
por 1,73 m2, ou volume urinário <0,3 mL/kg/h por
≥24 horas, ou anúria por ≥12 horas2. Com relação aos desfechos de curto prazo,
diversos estudos têm concluído que a IRA em
pacientes pediátricos hospitalizados pode levar à
ventilação mecânica prolongada, maior tempo de
internação e maior mortalidade3,7,18,21. Além disso,
a IRA pode estar associada ao desenvolvimento
posterior de proteinúria, hipertensão e doença renal
crônica3,7,9,18,19,22–24. O valor basal de CrS foi considerado o valor mais
baixo nos 6 meses anteriores à internação (incluindo
o valor no momento da internação); todas as
medições de creatinina foram realizadas pelo método Braz. J. Nephrol. (J. Bras. Nefrol.) 2024, 46(3):e20240012 2 Insuficiência renal aguda em crianças e adolescentes enzimático. A TFG foi calculada com base na fórmula
revisada
de
Schwartz,
k×(altura(cm)/creatinina
sérica(mg/dL)); usando uma constante k de 0,413. Universitário do Porto. Coleta de Dados e Definição de Variáveis Está em conformidade com
a Declaração de Helsinque, com as diretrizes para
a conduta ética de pesquisas médicas envolvendo
crianças e com a legislação nacional vigente. A proteinúria foi definida como uma relação
proteína/creatinina urinária (PU/CU) >0,2 mg/mg. A hematúria foi definida como ≥5 eritrócitos por
campo de grande aumento na análise de microscopia
da urina. Análise Estatística A análise estatística padrão foi realizada com o
software IBM SPSS Statistics for Macintosh, versão
28.0.1.0 (Armonk, NY: IBM Corp, EUA). As variáveis
são apresentadas como mediana e percentis 25 e 75
ou n (%), conforme apropriado. As diferenças entre
os grupos para variáveis contínuas foram avaliadas
com o teste de Mann-Whitney. O teste qui-quadrado
foi utilizado para comparação de proporções de
variáveis categóricas. Um valor de p inferior a 0,05
foi considerado significativo. As medidas de PA no hospital e no consultório
foram
avaliadas
com
esfigmomanômetros
oscilométricos validados utilizando um manguito de
tamanho adequado no braço direito, com a criança
em posição sentada e a fossa antecubital apoiada no
nível do coração, pelo menos duas vezes (idealmente
três vezes), com um intervalo de 1 minuto entre as
medições. O último valor disponível foi considerado
para análise. Para a classificação da PA, foram
considerados valores de referência de PAS e PAD
específicos para idade, sexo e altura, de acordo com
os valores de referência das diretrizes da Sociedade
Europeia de Hipertensão (hipertensão se os valores
sistólicos ou diastólicos estivessem dentro ou acima
do percentil 95)25. Resultados Foram incluídos na análise 46 pacientes pediátricos
com idade mediana (percentil 25–75, P25–75) de
13,0 (3,5–15,5) anos. As características demográficas
dos parâmetros clínicos e analíticos são mostradas
na Tabela 1, de acordo com os estágios KDIGO
[estágio 1, 10 (21,7%); estágio 2, 12 (26,1%); estágio
3, 24 (52,2%)]. O principal mecanismo patogênico
relatado foi o de causas renais intrínsecas (73,9%). As
etiologias mais comuns de IRA foram nefrite intersticial
aguda (23,9%), desidratação/choque (21,7%) e
glomerulonefrite aguda (19,6%). Aproximadamente
metade dos pacientes (n = 24; 52,2%) apresentou um
fator de risco identificável para o desenvolvimento de
IRA, sendo as comorbidades mais comuns (37,5%) as
patologias (renais: 1 síndrome nefrótica e 1 hematúria/
proteinúria em investigação; cardiovasculares: 1
hipertensão, 1 comunicação interventricular e 1
trombose da veia jugular interna; hemato-oncológicas:
1 osteoma osteoide; autoimune: 1 diabetes tipo 1 e
lúpus eritematoso sistêmico e 1 hepatite autoimune),
seguidas por exposição a nefrotoxinas (25,0%) e
pela presença de CAKUT (25,0%). Com relação à
classificação da IRA quanto ao débito urinário, 13
pacientes (28,3%) eram anúricos, 7 (15,2%) eram
oligúricos e 26 (56,5%) eram não oligúricos. Todos
os pacientes anúricos foram classificados como IRA
KDIGO 3. Nove (19,5%) pacientes apresentaram
sobrecarga hídrica e todos foram classificados como
IRA estágio 3 (considerando valores de creatinina
corrigidos para sobrecarga hídrica). A proporção
de pacientes com hiponatremia [KDIGO estágio 1 Em todos os pacientes, foi registrada a necessidade
de biópsia renal e terapia renal substitutiva. Foram
registrados dados sobre a admissão na unidade
de terapia intensiva, incluindo a necessidade de
ventilação mecânica e o uso de inotrópicos. O diagnóstico de nefrite intersticial aguda foi
baseado em critérios clínicos em todos os pacientes,
mas em quatro casos foi realizada uma biópsia
renal. Foram considerados os seguintes fatores de
risco: comorbidades, que incluíram doenças renais,
cardiovasculares, hemato-oncológicas ou autoimunes
anteriores; exposição a nefrotoxinas; prematuridade;
presença de anomalias congênitas do rim e do trato
urinário (CAKUT); e nefrolitíase. Os desfechos considerados foram sequelas e
óbito. As sequelas foram definidas como a presença
de pelo menos um dos eventos a seguir: proteinúria,
hipertensão ou TFG reduzida, definida como
TFG < 90 mL/min/1,73 m2, com base no monitoramento
clínico e analítico de 3 a 6 meses após a alta. Ética O projeto “Insuficiência renal aguda - experiência
de um centro terciário de Nefrologia Pediátrica” foi
aprovado pelo Departamento de Educação e Pesquisa
e pela Comissão de Ética do Centro Hospitalar Braz. J. Nephrol. (J. Bras. Ética Nefrol.) 2024, 46(3):e20240012 3 Insuficiência renal aguda em crianças e adolescentes Total
46
Estágio KDIGO
p
1
10 (21,7%)
2
12 (26,1%)
3
24 (52,2%)
Dados demográficos e antropométricos
Idade (anos)
12,96
(3,54–15,54)
13,50
(9,17–15,52)
14,21
(7,15–16,98)
10,54
(1,40–15,08)
0,109
Sexo masculino
24 (52,2%)
3 (30,0%)
7 (58,3%)
14 (58,3%)
0,284
Patogênese e diagnóstico da IRA
Pré-renal
9 (19,6%)
0 (0,0%)
2 (16,7%)
7 (29,2%)
0,122
Desidratação/choque
10 (21,7%)
1 (10,0%)
2 (16,7%)
7 (29,2%)
0,116
Renal
34 (73,9%)
8 (80,0%)
10 (83,3%)
16 (66,7%)
0,122
Nefrite intersticial aguda
11 (23,9%)
2 (20,0%)
3 (25,0%)
6 (25,0%)
0,116
Glomerulonefrite aguda
9 (19,6%)
2 (20,0%)
5 (41,7%)
2 (8,3%)
0,116
Síndrome hemolítico-urêmica
6 (13,0%)
0 (0,0%)
0 (0,0%)
6 (25,0%)
0,116
Nefrotoxicidade
4 (8,7%)
2 (20,0%)
1 (8,3%)
1 (4,2%)
0,116
Síndrome nefrótica
1 (2,2%)
0 (0,0%)
0 (0,0%)
1 (4,2%)
0,116
Pós-renal
3 (6,5%)
2 (20%)
0 (0,0%)
1 (4,2%)
0,122
Obstrução
3 (6,5%)
2 (20,0%)
0 (0,0%)
1 (4,2%)
0,116
Desconhecido
2 (4,3%)
1 (10,0%)
1 (8,3%)
0 (0,0%)
0,116
Fator de risco
24 (52,2%)
6 (60,0%)
7 (58,3%)
11 (45,8%)
0,665
Comorbidades
9 (37,5%)
1 (16,7%)
3 (42,9%)
5 (45,5%)
0,409
Nefrotóxico
6 (25,0%)
2 (33,3%)
3 (42,9%)
1 (9,1%)
0,409
Prematuridade
1 (4,2%)
1 (16,7%)
0 (0,0%)
0 (0,0%)
0,409
CAKUT
6 (25,0%)
1 (16,7%)
1 (14,3%)
4 (36,4%)
0,409
Nefrolitíase
2 (8,3%)
1 (16,7%)
0 (0,0%)
1 (9,1%)
0,409
Débito urinário
Anúria
13 (28,3%)
0 (0,0%)
0 (0,0%)
13 (54,2%)
<0,001
Oligúria
7 (15,2%)
1 (10,0%)
5 (41,7%)
1 (4,2%)
<0,001
Não oligúria
26 (56,5%)
9 (90,0%)
7 (58,3%)
10 (41,7%)
<0,001
Hipertensão
17 (37,0%)
3 (30,0%)
3 (25,0%)
11 (45,8%)
0,416
Parâmetros bioquímicos
Hiponatremia
9 (19,6%)
0 (0,0%)
1 (8,3%)
8 (33,3%)
0,043
Hipercalemia
9 (19,6%)
0 (0,0%)
1 (8,3%)
8 (33,3%)
0,043
Acidose metabólica
11 (23,9%)
1 (10,0%)
0 (0,0%)
10 (41,7%)
0,011
Proteinúria
29 (63,0%)
6 (60,0%)
7 (58,3%)
16 (66,7%)
0,865
Hematúria
20 (43,5%)
6 (60,0%)
5 (41,7%)
9 (37,5%)
0,478
Biópsia renal
10 (21,7%)
1 (10,0%)
4 (33,3%)
5 (20,8%)
0,413
Terapia renal substitutiva
10 (21,7%)
0 (0,0%)
0 (0,0%)
10 (41,7%)
0,003
Hemodiálise
2 (4,3%)
0 (0,0%)
0 (0,0%)
2 (8,3%)
Diálise peritoneal
6 (13,0%)
0 (0,0%)
0 (0,0%)
6 (25,0%)
Ambos
2 (4,3%)
0 (0,0%)
0 (0,0%)
2 (8,3%)
Necessidade de unidade de terapia intensiva
10 (21,7%)
1 (10,0%)
0 (0,0%)
9 (37,5%)
0,022
Ventilação mecânica
3 (6,5%)
0 (0,0%)
0 (0,0%)
3 (6,5%)
0,230
Ionotrópicos
2 (4,3%)
0 (0,0%)
0 (0,0%)
2 (8,3%)
0,384
IRA
Insuficiência renal aguda; CAKUT
Anomalias congênitas do rim e do trato urinário
Tabela 1
Parâmetros clínicos e analíticos de acordo com os estágios KDIGO de IRA Dados demográficos e antropométricos Braz. Os valores são relatados como mediana (percentil 25-percentil 75) ou n (%). IRA – Insuficiência renal aguda; CAKUT – Anomalias congênitas do rim e do trato urinário. ; CAKUT – Anomalias congênitas do rim e do trato urinário. os como mediana (percentil 25-percentil 75) ou n (%). IRA – Insuficiência renal aguda; CAKUT – Anomalias congênitas do rim e do trato urinário.
Os valores são relatados como mediana (percentil 25-percentil 75) ou n (%) Ética J. Nephrol. (J. Bras. Nefrol.) 2024, 46(3):e20240012 4 Insuficiência renal aguda em crianças e adolescentes Total
n = 44a
Estágio KDIGO
p
1
2
3
Sem qualquer sequela
18 (40,9%)
6 (60,0%)
6 (54,5%)
6 (26,1%)
0,108
PU/CU (mg/mg)
0,10 (0,06–0,15)
0,09 (0,06–0,15)
0,11 (0,07–0,16)
0,11 (0,06–0,15)
0,778
TFG (mL/min/1,73 m2)
108 (100–119)
93 (93-93)
107 (103–115)
115 (106–137)
0,035
Com sequela
26 (59,1%)
4 (40,0%)
5 (45,5%)
17 (73,9%)
0,108
Proteinúriab
15 (38,5%)
2 (22,2%)
2 (18,2%)
11 (57,9%)
0,051
PU/CU (mg/mg)
0,30 (0,27–0,44)
(0,29–0,36)c
(0,23–0,29)c
0,30 (0,27–0,47)
0,792
TFG reduzidad
11 (27,5%)
2 (28,6%)
2 (18,2%)
7 (31,8%)
0,709
TFG (mL/min/1,73 m2)
75 (62–83)
(71–77)c
(75–79)c
72 (59–80)
0,633
Hipertensãoe
4 (9,1%)
0 (0,0%)
2 (18,2%)
2 (8,7%)
0,349i
Tabela 2
Caracterização da sequela de acordo com os estágios KDIGO de IRA Tabela 2
Caracterização da sequela de acordo com os estágios KDIGO de IRA TFG – Taxa de filtração glomerular; KDIGO – Kidney Disease Improving Global Outcomes; PU/CU – Relação proteína/creatinina urinária. Os valores são relatados como n (%) e mediana (percentil 25–75). TFG – Taxa de filtração glomerular; KDIGO – Kidney Disease Improving Global Outcomes; PU/CU – Relação proteína/creatinina urinária. Os valores são relatados como n (%) e mediana (percentil 25–75). TFG – Taxa de filtração glomerular; KDIGO – Kidney Disease Improvi
Os valores são relatados como n (%) e mediana (percentil 25–75). i
cDevido ao baixo número de pacientes nessa categoria (menos de 3), os valores medianos (percentis 25–75) não puderam ser calculados e os
valores reais de cada paciente são apresentados. i
eHipertensão foi definida como valores sistólicos ou diastólicos acima do percentil 95 para idade, sexo e altura25. eHipertensão foi definida como valores sistólicos ou diastólicos acima do percentil 95 para idade, sexo e altura25. (n = 15, 38,5%; mediana (P25–75) da PU/CU 0,30
(0,27–0,44) mg/mg), seguida por TFG reduzida
(n = 11, 27,5%; mediana (P25–75) da TFG 75 (62–
83) mL/min/1,73 m2) e hipertensão (n = 4, 9,1%). Entre os pacientes sem sequelas no acompanhamento,
os valores medianos da PU/CU e da TFG foram 0,10
(0,06–0,15) mg/mg e 108 (100–119) mL/min/1,73 m2,
respectivamente. Embora dentro da faixa normal,
a mediana da TFG no acompanhamento aumentou
entre os estágios da IRA [KDIGO estágio 1 vs. estágio 2
vs. estágio 3: 93 (93-93) vs. 107 (103–115) vs. 115 (106–137) mL/min/1,73 m2, respectivamente,
p = 0,035]. Ética Doze dos 15 pacientes com proteinúria
iniciaram terapia com inibidor da enzima conversora
da angiotensina ou bloqueador do receptor da
angiotensina II durante o acompanhamento. vs. estágio 2 vs. estágio 3: 0 (0,0%) vs. 1 (8,3%) vs. 8 (33,3%), respectivamente, p = 0,043]; hipercalemia
[KDIGO estágio 1 vs. estágio 2 vs. estágio 3: 0 (0,0%)
vs. 1 (8,3%) vs. 8 (33,3%), respectivamente, p =
0,043] e acidose metabólica [KDIGO estágio 1 vs. estágio 2 vs. estágio 3: 1 (10,0%) vs. 0 (0,0%) vs. 10 (41,7%), respectivamente, p = 0,011] aumentou
entre os estágios de IRA. A proporção de pacientes
com hipertensão foi maior entre pacientes com IRA
estágio 3, mas a diferença não foi estatisticamente
significativa. Foi realizada biópsia renal em 10
pacientes, 5 dos quais apresentaram IRA estágio 3 (4
nefrite intersticial aguda). Quase um quarto (n = 10;
21,7%) dos pacientes necessitaram de terapia renal
substitutiva, a saber, diálise peritoneal, hemodiálise ou
ambas as técnicas, todos pertencentes ao grupo IRA
estágio 3. Dez (21,7%) pacientes foram internados
em uma unidade de terapia intensiva, 90% dos quais
apresentavam IRA estágio 3. Discussão A síndrome nefrótica,
por exemplo, é uma causa frequente de doença renal
em crianças e a IRA é descrita como uma complicação
potencial26,27. Embora a incidência de IRA em crianças
com síndrome nefrótica seja variável entre os estudos,
um estudo encontrou IRA em cerca de metade de sua
população27. No entanto, em nosso estudo, apenas
um paciente apresentou IRA com uma recidiva de
síndrome nefrótica. As doenças cardiovasculares,
como insuficiência cardíaca e cardiopatias congênitas,
também impõem um risco significativo de IRA28. A
IRA é particularmente comum em crianças submetidas
a cirurgia cardíaca, com estudos sugerindo uma
correlação significativa entre formas moderadas
a graves de lesão e mortalidade pós-operatória29,
o que está em conformidade com nossos achados. A IRA é uma comorbidade comum das doenças
hemato-oncológicas e relatos indicam que, nesses
pacientes, a IRA nos estágios 2 e 3 está associada a
uma maior mortalidade30. As doenças autoimunes
também podem levar ao desenvolvimento de IRA,
com potencial de progredir rapidamente para formas
graves de lesão31. Em nossa coorte, cerca de um terço
dos pacientes apresentou um histórico prévio de
doenças renais, cardiovasculares, hemato-oncológicas
ou autoimunes. Também é importante observar que a
maioria desses pacientes desenvolveu IRA moderada a
grave, correspondendo aos estágios do KDIGO 2 e 3,
portanto, consistente com relatos anteriores. Em nosso estudo, constatamos que a maioria
dos pacientes que necessitaram de terapia renal
substitutiva também foi admitida na UTIP durante o
curso da internação, destacando a gravidade inerente
à IRA estágio 3. Embora estudos anteriores tenham
encontrado uma correlação entre a gravidade da IRA
e a necessidade e duração da ventilação mecânica16,41,
não encontramos uma diferença estatisticamente
significativa na necessidade de ventilação mecânica
em diferentes estágios da IRA. Isso pode ser devido
ao pequeno número de pacientes em nossa população
que necessitaram de tratamento na UTIP e ventilação
mecânica. As baixas taxas de utilização de ventilação
mecânica e drogas vasoativas em nossa coorte de
estudo podem sugerir uma menor gravidade dos casos
em comparação com outras séries e podem contribuir
para a ausência de óbitos em nossa coorte. A oligoanúria foi relatada em aproximadamente
metade dos pacientes internados, onde quase todos
desenvolveram IRA nos estágios 2 e 3, sugerindo que
essas alterações no débito urinário podem representar
um risco para formas mais graves da doença e,
portanto, desfechos potencialmente piores, conforme
relatado anteriormente na literatura32. Discussão No presente estudo, relatamos a etiologia, gravidade
e desfechos da IRA entre pacientes internados em
uma Unidade de Nefrologia Pediátrica de um hospital
terciário durante a última década. A caracterização das sequelas de acordo com
os estágios da IRA é mostrada na Tabela 2. Dos
46 pacientes originais, 2 foram perdidos durante
o seguimento e, consequentemente, não foram
incluídos na análise. Não houve óbitos. A maioria
dos pacientes (n = 26; 59,1%) apresentou pelo menos
uma sequela de 3 a 6 meses após a alta. A frequência
de sequelas aumentou entre os estágios KDIGO de
IRA. As sequelas mais frequentes foram proteinúria A maioria dos casos de IRA foram associados
a causas renais intrínsecas, especialmente nefrite
intersticial aguda, glomerulonefrite aguda e síndrome
hemolítico-urêmica, seguidas por causas pré-renais,
nomeadamente desidratação/choque. Embora diversos
estudos relatem que a IRA pediátrica é derivada Braz. J. Nephrol. (J. Bras. Nefrol.) 2024, 46(3):e20240012 5 Insuficiência renal aguda em crianças e adolescentes principalmente
de
etiologias
pré-renais14,17,18,
a
predominância de causas renais pode estar relacionada
à natureza altamente diferenciada de nosso centro. Por
sermos o centro de referência em pacientes pediátricos
com doenças renais para toda a região norte do
país, a proporção de etiologias renais pode estar
sobrerrepresentada em nossa amostra. mais pronunciados e são consistentes com estudos
que sugerem uma associação entre anormalidades
eletrolíticas, principalmente acidose metabólica, e pior
prognóstico em crianças com IRA33,34. Relatamos que todos os pacientes que necessitaram
de terapia renal substitutiva foram categorizados
no estágio mais grave de IRA, o que está de acordo
com estudos anteriores18,35. A diálise peritoneal foi o
método de terapia renal substitutiva mais comumente
utilizado, o que é consistente com diversos estudos
que relatam que a diálise peritoneal é um método bem
tolerado, fácil de executar e com eficácia conhecida
no contexto da IRA pediátrica36–38. Além disso, a
hemodiálise requer um bom funcionamento do acesso
vascular e pacientes hemodinamicamente estáveis,
sendo, portanto, reservada para ambientes mais
específicos38. Embora as terapias renais substitutivas
contínuas tendam a ser a modalidade de escolha em
pacientes críticos e hemodinamicamente instáveis39,40,
a diálise peritoneal foi a terapia renal substitutiva
mais comumente utilizada para os pacientes na UTIP,
e nenhuma terapia contínua foi usada em nossa
população durante o período do estudo. Sabe-se que pacientes com comorbidades são
altamente suscetíveis à IRA1,2,5. Referências mais grave, parece contraintuitivo, mas pode representar
um estágio inicial de hiperfiltração em pacientes com
perda mais grave de néfrons durante o episódio de IRA,
como relatado anteriormente na literatura42–44. 1. Cho M. Pediatric acute kidney injury: focusing on diagnosis
and management. Child Kidney Dis. 2020;24(1):19–26. doi:
http://dx.doi.org/10.3339/jkspn.2020.24.1.19. 2. Kidney Disease: Improving Global Outcomes. KDIGO
clinical practice guideline for acute kidney injury: summary of
recommendation statements. Kidney Int Suppl. 2012;2(1):8–
12, 19–24. doi: http://dx.doi.org/10.1038/kisup.2012.7. Reconhecemos que nosso estudo teve algumas
limitações, particularmente o desenho retrospectivo
e a experiência de um único centro de atendimento
terciário. Apesar dessas limitações, acreditamos ter
descrito uma população bastante representativa de
pacientes pediátricos com IRA da região norte de
nosso país durante um longo período de tempo. Consideramos que o estudo apresentado contribui
para aumentar o conhecimento sobre a epidemiologia
da IRA, uma área que necessita de mais estudos para
promover a conscientização sobre as consequências
de longo prazo da IRA na pediatria. 3. Uber A, Sutherland S. Acute kidney injury in hospitalized
children: consequences and outcomes. Pediatr Nephrol. 2020;35(2):213–20. doi: http://dx.doi.org/10.1007/s00467-
018-4128-7. PubMed PMID: 30386936. 4. McGregor T, Jones D, Wang L, Danciu I, Bridges BC,
Fleming GM, et al. Acute kidney injury incidence in
noncritically III hospitalized children, adolescents, and young
adults: a retrospective observational study. Am J Kidney
Dis. 2016;67(3):384–90. doi: http://dx.doi.org/10.1053/j. ajkd.2015.07.019. PubMed PMID: 26319754. 5. Levey A, James M. Acute kidney injury. Ann Intern Med. 2017;167(9):ITC66–80. doi:
http://dx.doi.org/10.7326/
AITC201711070. PubMed PMID: 29114754. 6. Sethi S, Bunchman T, Chakraborty R, Raina R. Pediatric acute
kidney injury: new advances in the last decade. Kidney Res Clin
Pract. 2021;40(1):40–51. doi: http://dx.doi.org/10.23876/j. krcp.20.074. PubMed PMID: 33663033. Em conclusão, a IRA foi comum no ambiente
pediátrico,
principalmente
em
pacientes
com
comorbidades prévias, mas também afetou crianças
sem um fator de risco conhecido, enfatizando a
importância da suspeita precoce dessa condição. Também descobrimos que a maior gravidade da IRA
esteve associada a distúrbios eletrolíticos, à necessidade
de terapias renais substitutivas e à admissão na
UTIP. Nossos resultados sugerem que a IRA pode
estar associada a uma morbidade significativa,
especialmente ao desenvolvimento de proteinúria e à
redução da TFG e, portanto, ao comprometimento da
função renal. Isso destaca a necessidade de estudos
adicionais com foco no impacto de longo prazo da
IRA a fim de compreender melhor o potencial de
consequências transitórias ou permanentes, com
impacto importante no acompanhamento e manejo
de longo prazo desses pacientes. 7. Discussão Embora não tenha havido óbitos em nosso estudo,
destacamos o fato de que quase 60% dos pacientes
apresentaram pelo menos uma sequela de 3 a 6 meses
após a alta hospitalar, com mais de 25% apresentando
TFG reduzida no acompanhamento, portanto, sem
recuperar completamente a função renal normal. O achado de valores medianos da TFG que aumentam
na consulta de acompanhamento entre os estágios de
IRA, com valores mais elevados em pacientes com IRA Os pacientes com casos mais graves de IRA
foram aqueles com mais distúrbios nos parâmetros
bioquímicos, como hiponatremia, hipercalemia e acidose
metabólica. Esses achados sugerem que insultos renais
graves estão associados a distúrbios hidroeletrolíticos Braz. J. Nephrol. (J. Bras. Nefrol.) 2024, 46(3):e20240012 6 6 Insuficiência renal aguda em crianças e adolescentes Contribuição dos Autores Contribuição dos Autores 13. De Zan F, Amigoni F, Pozzato R, Pettenazzo A, Murer L, Vidal
E. Acute kidney injury in critically III children: a retrospective
analysis of risk factors. Blood Purif. 2020;49(1-2):1–7. doi:
http://dx.doi.org/10.1159/000502081. PubMed
PMID:
31382259. CF e AMR contribuíram igualmente para a criação
deste manuscrito e participaram da coleta de dados,
análise de dados e redação do manuscrito. LCC
conceituou e desenhou o estudo, supervisionou a
coleta de dados, participou e supervisionou a análise
dos dados, analisou e revisou o manuscrito e aprovou
o manuscrito final conforme submetido. TC, CM e
MSF participaram da conceptualização do estudo e
analisaram, revisaram e aprovaram o manuscrito final. 14. Nicolau B, Costa-Reis P. Advances in pediatric acute kidney
injury prevention and early diagnosis. Rev Port Nefrol
Hipertens. 2022;36(3):130–7. doi: http://dx.doi.org/10.32932/
pjnh.2022.08.196. 15. Bajracharya P, Kalra S, Dhingra S, Sood A, Yadav AK, Kanitkar
M. Acute kidney injury in the pediatric intensive care unit at
a tertiary care hospital of the Armed Forces: a cross-sectional
observational study. Med J Armed Forces India. 2020;76(1):84–
8. doi: http://dx.doi.org/10.1016/j.mjafi.2019.02.003. PubMed
PMID: 32020974. 16. Kaddourah A, Basu R, Bagshaw S, Goldstein S; AWARE
Investigators. Epidemiology of acute kidney injury in critically
III children and young adults. N Engl J Med. 2017;376(1):11–
20. doi: http://dx.doi.org/10.1056/NEJMoa1611391. PubMed
PMID: 27959707. Referências Devarajan P. Pediatric acute kidney injury: different from
acute renal failure but how and why. Curr Pediatr Rep. 2013;1(1):34–40. doi: http://dx.doi.org/10.1007/s40124-012-
0003-3. PubMed PMID: 23525203. 8. Askenazi D. Evaluation and management of critically III children
with acute kidney injury. Curr Opin Pediatr. 2011;23(2):201–
7. doi: http://dx.doi.org/10.1097/MOP.0b013e328342ff37. PubMed PMID: 21191296. 9. Wang F, Ding J. Pediatric acute kidney injury to the subsequent
CKD transition. Kidney Dis. 2021;7(1):10–3. doi: http://dx.doi. org/10.1159/000509935. PubMed PMID: 33614729. 10. Lameire N, van Biesen W, Vanholder R. Epidemiology of acute
kidney injury in children worldwide, including developing
countries. Pediatr Nephrol. 2017;32(8):1301–14. doi: http://
dx.doi.org/10.1007/s00467-016-3433-2. PubMed
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and renal recovery after acute kidney injury. Nephron
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Development of Anthraquinone Analogues as Phosphoglycerate Mutase 1 Inhibitors
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Received: 18 December 2018; Accepted: 28 January 2019; Published: 27 February 2019 Abstract: Phosphoglycerate mutase 1 (PGAM1) coordinates glycolysis and biosynthesis to promote
cancer cell proliferation, and is believed to be a promising target for cancer therapy. Herein, based on
the anthraquinone scaffold, we synthesized 31 anthraquinone derivatives and investigated the
structure−activity relationship (SAR). The 3-substitient of sulfonamide on the anthraquinone scaffold
was essential for maintaining potency and the modifications of the hydroxyl of alizarin would cause
a sharp decrease in potency. In the meantime, we determined the co-crystal structure of PGAM1
and one of the anthraquinone inhibitors 9i with IC50 value of 0.27 µM. The co-crystal structure
revealed that F22, K100 and R116 of PGAM1 were critical residues for the binding of inhibitors
which further validated the SAR. Consistent with the crystal structure, a competitive assay illustrated
that compound 9i was a noncompetitive inhibitor. In addition, compound 9i effectively restrained
different lung cancer cells proliferation in vitro. Taken together, this work provides reliable guide for
future development of PGAM1 inhibitors and compound 9i may act as a new leading compound for
further optimization. Keywords: phosphoglycerate mutase 1; anthraquinone inhibitors; co-crystal structure; cancer treatm Keywords: phosphoglycerate mutase 1; anthraquinone inhibitors; co-crystal structure; cancer treatment molecules molecules molecules Communication
Development of Anthraquinone Analogues as
Phosphoglycerate Mutase 1 Inhibitors
Ke Huang, Lulu Jiang, Huiti Li, Deyong Ye * and Lu Zhou *
Department of Medicinal Chemistry, School of Pharmacy, Fudan University, No. 826, Zhangheng Rd.,
Shanghai 201203, China; kehuang13@fudan.edu.cn (K.H.); 18111030012@fudan.edu.cn (L.J.);
17211030005@fudan.edu.cn (H.L.)
* Correspondence: dyye@shmu.edu.cn (D.Y.); zhoulu@fudan.edu.cn (L.Z.); Tel.: +86-21-5198-0117 (D.Y.);
+86-21-5198-0125 (L.Z.) Development of Anthraquinone Analogues as
Phosphoglycerate Mutase 1 Inhibitors Ke Huang, Lulu Jiang, Huiti Li, Deyong Ye * and Lu Zhou * Department of Medicinal Chemistry, School of Pharmacy, Fudan University, No. 826, Zhangheng Rd.,
Shanghai 201203, China; kehuang13@fudan.edu.cn (K.H.); 18111030012@fudan.edu.cn (L.J.);
17211030005@fudan.edu.cn (H.L.)
* Correspondence: dyye@shmu.edu.cn (D.Y.); zhoulu@fudan.edu.cn (L.Z.); Tel.: +86-21-5198-0117 (D.Y.);
+86-21-5198-0125 (L.Z.)
www.mdpi.com/journal/molecules Molecules 2019, 24, 845; doi:10.3390/molecules24050845 molecules molecules 1. Introduction In 1924, Warburg discovered that cancer cells tended to metabolize glucose through aerobic
glycolysis rather than oxidative phosphorylation, even if there was sufficient oxygen [1,2]. This character of cancer cells was distinct from normal differentiated cells and the phenomenon
was thereafter named the “Warburg effect”. However, the reason why cancer cells adopt such an
altered metabolism remained unclear. In the past decades, reprogramming energy metabolism was
considered as an emerging hallmark of cancer cells [3] and the study of cancer metabolism has
drawn great interest [4–8]. During aerobic glycolysis of cancer cells, adenosine 5′-triphosphate (ATP)
is produced inefficiently in this metabolic pattern, whereas building blocks such as nucleotides,
and amino acids are generated in large amounts, satisfying the demands of anabolic biosynthesis
which are essential for rapid cell proliferation [9–12]. Nowadays, various enzymes playing critical
roles in cancer metabolism have been reported, including isocitrate dehydrogenase [13–15]. Inhibitors
targeting these enzymes have been approved by Food and Drug Administration [14,15] which further
demonstrated the potential of modulating cancer metabolism for cancer therapy. Phosphoglycerate mutase 1 (PGAM1) is a key enzyme in the 8th step of glycolysis pathway
converting 3-phosphoglycerate (3PG) to 2-phosphoglycerate (2PG) [16]. Previously we found
that PGAM1 could balance the intracellular concentrations of its substrate 3PG and its product
2PG to control tumor growth [17]. In detail, 3PG inhibits 6-phosphogluconate dehydrogenase in Molecules 2019, 24, 845; doi:10.3390/molecules24050845 www.mdpi.com/journal/molecules 2 of 20
2PG to
entose Molecules 2019, 24, 845
PGAM1 could bala
control tumor grow pentose phosphate pathway (PPP) and 2PG activates 3-phosphoglycerate dehydrogenase in serine
synthesis [17]. When PGAM1 is inhibited by a small molecule or shRNA, intracellular level of 3PG
increased and 2PG decreased which led to the blockade of PPP, serine synthesis, glycolysis and
subsequently impaired tumor growth. Since PGAM1 regulates both glycolysis and biosynthesis,
developing inhibitors targeting PGAM1 is considered as a way of dual inhibition of catabolism
and anabolism for cancer therapy [18]. Furthermore, PGAM1 is upregulated in many kinds of
tumors and the expression of PGAM1 is correlated with cancer patients’ prognosis [19–21]. Recently,
more functions of PGAM1 in cancer metabolism were described. Posttranslational modulations of
PGAM1 such as Y26 phosphorylation [22] and deacetylation of K100 [23] would promote cancer
cell proliferation and tumor growth. Besides, it is essential for homologous recombination repair by
regulating the deoxyribonucleotide triphosphate nucleoside (dNTP) pool [24] and it facilitates cancer
cell migration independent of its metabolic function [25]. 1. Introduction Taken together, PGAM1 is a promising target
for cancer treatment. [17]. When PGAM1 is inhibited by a small molecule or shRNA, intracellular level of 3PG increased
and 2PG decreased which led to the blockade of PPP, serine synthesis, glycolysis and subsequently
impaired tumor growth. Since PGAM1 regulates both glycolysis and biosynthesis, developing
inhibitors targeting PGAM1 is considered as a way of dual inhibition of catabolism and anabolism
for cancer therapy [18]. Furthermore, PGAM1 is upregulated in many kinds of tumors and the
expression of PGAM1 is correlated with cancer patients’ prognosis [19–21]. Recently, more
functions of PGAM1 in cancer metabolism were described. Posttranslational modulations of
PGAM1 such as Y26 phosphorylation [22] and deacetylation of K100 [23] would promote cancer cell
proliferation and tumor growth. Besides, it is essential for homologous recombination repair by
regulating the deoxyribonucleotide triphosphate nucleoside (dNTP) pool [24] and it facilitates
cancer cell migration independent of its metabolic function [25]. Taken together, PGAM1 is a
promising target for cancer treatment. However, up to now, only four representative PGAM1 inhibitors were reported (Figure 1). The However, up to now, only four representative PGAM1 inhibitors were reported (Figure 1). The first
PGAM1 inhibitor 1 (MJE3) was discovered by the Cravatt group in 2005 [26]. It was identified as
a covalent inhibitor labeling the K100 of PGAM1 by spiroepoxides [27]. Another PGAM1 inhibitor
described these days was 2((−)-epigallocatechin-3-gallate (EGCG)), a natural product from green
tea [28]. It demonstrated strong inhibition activity towards PGAM1 [28] but it was reported to display
extensive potency on multiple targets [29–31]. We discovered the other two inhibitors, one of which was
anthraquinone derivative 3 (PGMI-004A) [17] and the other was the xanthone derivative 4 (12r) [32]. Both of them showed moderate inhibition activity on PGAM1 and cancer cell proliferation. first PGAM1 inhibitor 1 (MJE3) was discovered by the Cravatt group in 2005 [26]. It was identified
as a covalent inhibitor labeling the K100 of PGAM1 by spiroepoxides [27]. Another PGAM1
inhibitor described these days was 2((−)-epigallocatechin-3-gallate (EGCG)), a natural product from
green tea [28]. It demonstrated strong inhibition activity towards PGAM1 [28] but it was reported to
display extensive potency on multiple targets [29–31]. We discovered the other two inhibitors, one
of which was anthraquinone derivative 3 (PGMI-004A) [17] and the other was the xanthone
derivative 4 (12r) [32]. Both of them showed moderate inhibition activity on PGAM1 and cancer cell
proliferation. Figure 1. 1. Introduction Reported phosphoglycerate mutase 1 （PGAM1） inhibitors. Figure 1. Reported phosphoglycerate mutase 1 (PGAM1) inhibitors. Figure 1. Reported phosphoglycerate mutase 1 （PGAM1） inhibitor
Figure 1. Reported phosphoglycerate mutase 1 (PGAM1) inhibitors. Since only a limited amount of anthraquinone inhibitors were reported, the structure−activity
relationship (SAR) of anthraquinone derivatives and PGAM1 was not well studied yet. Besides, up
to these days, the molecular interactions of PGAM1 and anthraquinone inhibitor were not observed
in crystal structure. However, anthraquinones exhibit a variety of pharmacological activities
including anticancer [33–35], antifungal [36], and antibacterial properties [37] and so on. What’s
more, there are many clinically approved anthraquinone drugs such as doxorubicin and
mitoxantrone with strong potency in suppressing cancer cell proliferation [38]. Here we designed
and synthesized a series of new anthraquinone compounds and evaluated their biological activity
on PGAM1 and cancer cell proliferation. In addition, we solved the co-crystal structure of PGAM1
and compound 9i which interpreted the molecular mechanism of compound 9i interacting with
PGAM1 and further confirmed the SAR we investigated. Combined with the crystal structure,
additional competitive assay demonstrated the noncompetitive binding of compound 9i with the
Since only a limited amount of anthraquinone inhibitors were reported, the structure−activity
relationship (SAR) of anthraquinone derivatives and PGAM1 was not well studied yet. Besides, up to
these days, the molecular interactions of PGAM1 and anthraquinone inhibitor were not observed in
crystal structure. However, anthraquinones exhibit a variety of pharmacological activities including
anticancer [33–35], antifungal [36], and antibacterial properties [37] and so on. What’s more, there are
many clinically approved anthraquinone drugs such as doxorubicin and mitoxantrone with strong
potency in suppressing cancer cell proliferation [38]. Here we designed and synthesized a series
of new anthraquinone compounds and evaluated their biological activity on PGAM1 and cancer
cell proliferation. In addition, we solved the co-crystal structure of PGAM1 and compound 9i
which interpreted the molecular mechanism of compound 9i interacting with PGAM1 and further
confirmed the SAR we investigated. Combined with the crystal structure, additional competitive assay
demonstrated the noncompetitive binding of compound 9i with the 3PG substrate. 2.1. Chemistry
Four classes of anthraquinone derivatives were synthesized, with 1,2-substituted, 3-substituted,
2,4-substituted, and 2,3-substituted anthraquinone core scaffolds (Figure 2). 2. Results and
2.1. Chemistry Reagents and conditions: (a) BrCH2COOEt, K2CO3, DMF, 80
°C; (b) LiOH, MeOH, H2O, 30 °C; (c)NHMe2·HCl, EDCI, HOBt, DIPEA, DMF. Scheme 1. Synthesis of compounds 6a–e. Reagents and conditions: (a) BrCH2COOEt, K2CO3, DMF,
80 ◦C; (b) LiOH, MeOH, H2O, 30 ◦C; (c)NHMe2·HCl, EDCI, HOBt, DIPEA, DMF. Scheme 1. Synthesis of compounds 6a–e. Reagents and conditions: (a) BrCH2COOEt, K2CO3, DMF, 80
°C; (b) LiOH, MeOH, H2O, 30 °C; (c)NHMe2·HCl, EDCI, HOBt, DIPEA, DMF. Scheme 1 Synthesis of compounds 6a e Reagents and conditions: (a) BrCH2COOEt K2CO3 DMF 80
Scheme 1. Synthesis of compounds 6a–e. Reagents and conditions: (a) BrCH2COOEt, K2CO3, DMF, 80
°C; (b) LiOH, MeOH, H2O, 30 °C; (c)NHMe2·HCl, EDCI, HOBt, DIPEA, DMF. Scheme 1. Synthesis of compounds 6a–e. Reagents and conditions: (a) BrCH2COOEt, K2CO3, DMF,
80 ◦C; (b) LiOH, MeOH, H2O, 30 ◦C; (c)NHMe2·HCl, EDCI, HOBt, DIPEA, DMF. Scheme 1. Synthesis of compounds 6a–e. Reagents and conditions: (a) BrCH2COOEt, K2CO3, DMF, 80
°C; (b) LiOH, MeOH, H2O, 30 °C; (c)NHMe2·HCl, EDCI, HOBt, DIPEA, DMF. Scheme 1 Synthesis of compounds 6a e Reagents and conditions: (a) BrCH2COOEt K2CO3 DMF 80
Scheme 1. Synthesis of compounds 6a–e. Reagents and conditions: (a) BrCH2COOEt, K2CO3, DMF, 80
°C; (b) LiOH, MeOH, H2O, 30 °C; (c)NHMe2·HCl, EDCI, HOBt, DIPEA, DMF. Scheme 1. Synthesis of compounds 6a–e. Reagents and conditions: (a) BrCH2COOEt, K2CO3, DMF,
80 ◦C; (b) LiOH, MeOH, H2O, 30 ◦C; (c)NHMe2·HCl, EDCI, HOBt, DIPEA, DMF. Scheme 1. Synthesis of compounds 6a–e. Reagents and conditions: (a) BrCH2COOEt, K2CO3, DMF, 80
°C; (b) LiOH, MeOH, H2O, 30 °C; (c)NHMe2·HCl, EDCI, HOBt, DIPEA, DMF. °C; (b) LiOH, MeOH, H2O, 30 °C; (c)NHMe2·HCl, EDCI, HOBt, DIPEA, DMF. In Scheme 2, firstly, alizarin was nitrated by HNO3 and then reduced by Sn and HCl to afford
the key amine intermediate 8 which was reacted with diverse sulfonyl chlorides to give the target
In Scheme 2, firstly, alizarin was nitrated by HNO3 and then reduced by Sn and HCl to afford
the key amine intermediate 8, which was reacted with diverse sulfonyl chlorides to give the target
compounds 9a–q. In Scheme 2, firstly, alizarin was nitrated by HNO3 and then reduced by Sn and HCl to afford
the key amine intermediate 8, which was reacted with diverse sulfonyl chlorides to give the target
compounds 9a–q. 2. Results and
2.1. Chemistry 2.1. Chemistry
Four classes of anthraquinone derivatives were synthesized, with 1,2-substituted, 3-substituted,
2,4-substituted, and 2,3-substituted anthraquinone core scaffolds (Figure 2). Molecules 2019, 24, 845
Four classes
b
d
3 substituted, 2,4
3 substituted, 2,4 3 of 20
ituted,
re 2). e
). 3 of 20
ituted,
re 2). e
). Figure 2. The optimization strategy and SAR exploration of the anthraquinone derivatives against
Figure 2. The optimization strategy and SAR exploration of the anthraquinone derivatives against PGAM1. Figure 2. The optimization strategy and SAR exploration of the anthraquinone derivatives against
PGAM1. Figure 2. The optimization strategy and SAR exploration of the anthraquinone derivatives against
PGAM1. none derivatives against
none derivatives against Figure 2. The optimization strategy and SAR exploration of the anthraquinone derivatives against
Figure 2. The optimization strategy and SAR exploration of the anthraquinone derivatives against PGAM1. PGAM1. PGAM1. Figure 2. The optimization strategy and SAR exploration of the anthraquinone derivatives against
Figure 2. The optimization strategy and SAR exploration of the anthraquinone derivatives against PGAM1. PGAM1. PGAM1. PGAM1. As shown in Scheme 1, starting from commercially available alizarin, compounds 6a–e were
As shown in Scheme 1, starting from commercially available alizarin, compounds 6a–e were
obtained by substitution with ethyl bromoacetate, hydrolysis of the ester, and amidation. As shown in Scheme 1, starting from commercially available alizarin, compounds 6a–e were
obtained by substitution with ethyl bromoacetate, hydrolysis of the ester, and amidation. As shown in Scheme 1, starting from commercially available alizarin, compounds 6a–e were
obtained by substitution with ethyl bromoacetate, hydrolysis of the ester, and amidation. btained by substitution with ethyl bromoacetate, hydrolysis of the ester, and amidation. Scheme 1 Synthesis of compounds 6a e Reagents and conditions: (a) BrCH2COOEt K2CO3 DMF 80
Scheme 1. Synthesis of compounds 6a–e. Reagents and conditions: (a) BrCH2COOEt, K2CO3, DMF, 80
°C; (b) LiOH, MeOH, H2O, 30 °C; (c)NHMe2·HCl, EDCI, HOBt, DIPEA, DMF. Scheme 1. Synthesis of compounds 6a–e. Reagents and conditions: (a) BrCH2COOEt, K2CO3, DMF,
80 ◦C; (b) LiOH, MeOH, H2O, 30 ◦C; (c)NHMe2·HCl, EDCI, HOBt, DIPEA, DMF. Scheme 1. Synthesis of compounds 6a–e. Reagents and conditions: (a) BrCH2COOEt, K2CO3, DMF, 80
°C; (b) LiOH, MeOH, H2O, 30 °C; (c)NHMe2·HCl, EDCI, HOBt, DIPEA, DMF. tion with ethyl bromoacetate, hydrolysis of the este Scheme 1 Synthesis of compounds 6a–e Reagents and conditions: (a) BrCH2COOEt K2CO3 DMF 80
Scheme 1. Synthesis of compounds 6a–e. 2. Results and
2.1. Chemistry In Scheme 2, firstly, alizarin was nitrated by HNO3 and then reduced by Sn and HCl to afford
the key amine intermediate 8, which was reacted with diverse sulfonyl chlorides to give the target
compounds 9a–q. y
,
y
g
g
compounds 9a–q. Scheme 2. Synthesis of compounds 9a–q. Reagents and conditions: (a) HNO3, CH3COOH, 50 °C; (b)
Scheme 2. Synthesis of compounds 9a–q. Reagents and conditions: (a) HNO3, CH3COOH, 50 °C; (b)
Sn, SnCl2·H2O, HCl, EtOH; (c) R3SO2Cl, Py. Scheme 2. Synthesis of compounds 9a–q. Reagents and conditions: (a) HNO3, CH3COOH, 50 °C; (b)
Sn, SnCl2·H2O, HCl, EtOH; (c) R3SO2Cl, Py. Scheme 2. Synthesis of compounds 9a–q. Reagents and conditions: (a) HNO3, CH3COOH, 50 ◦C;
(b) Sn, SnCl2·H2O, HCl, EtOH; (c) R3SO2Cl, Py. Scheme 2 Synthesis of compounds 9a–q Reagents and conditions: (a) HNO3 CH3COOH 50 °C; (b)
Scheme 2. Synthesis of compounds 9a–q. Reagents and conditions: (a) HNO3, CH3COOH, 50 °C; (b)
Sn, SnCl2·H2O, HCl, EtOH; (c) R3SO2Cl, Py. Scheme 2. Synthesis of compounds 9a–q. Reagents and conditions: (a) HNO3, CH3COOH, 50 °C; (b)
Sn, SnCl2·H2O, HCl, EtOH; (c) R3SO2Cl, Py. Scheme 2. Synthesis of compounds 9a–q. Reagents and conditions: (a) HNO3, CH3COOH, 50 ◦C;
(b) Sn, SnCl2·H2O, HCl, EtOH; (c) R3SO2Cl, Py. Scheme 2 Synthesis of compounds 9a–q Reagents and conditions: (a) HNO3 CH3COOH 50 °C; (b)
Scheme 2. Synthesis of compounds 9a–q. Reagents and conditions: (a) HNO3, CH3COOH, 50 °C; (b)
Sn, SnCl2·H2O, HCl, EtOH; (c) R3SO2Cl, Py. Scheme 2. Synthesis of compounds 9a–q. Reagents and conditions: (a) HNO3, CH3COOH, 50 °C; (b)
Sn, SnCl2·H2O, HCl, EtOH; (c) R3SO2Cl, Py. Scheme 2. Synthesis of compounds 9a–q. Reagents and conditions: (a) HNO3, CH3COOH, 50 ◦C;
(b) Sn, SnCl2·H2O, HCl, EtOH; (c) R3SO2Cl, Py. Scheme 2 Synthesis of compounds 9a–q Reagents and conditions: (a) HNO3 CH3COOH 50 °C; (b)
Scheme 2. Synthesis of compounds 9a–q. Reagents and conditions: (a) HNO3, CH3COOH, 50 °C; (b)
Sn, SnCl2·H2O, HCl, EtOH; (c) R3SO2Cl, Py. Scheme 2. Synthesis of compounds 9a–q. Reagents and conditions: (a) HNO3, CH3COOH, 50 °C; (b)
Sn, SnCl2·H2O, HCl, EtOH; (c) R3SO2Cl, Py. Scheme 2. Synthesis of compounds 9a–q. Reagents and conditions: (a) HNO3, CH3COOH, 50 ◦C;
(b) Sn, SnCl2·H2O, HCl, EtOH; (c) R3SO2Cl, Py. Scheme 2. Synthesis of compounds 9a–q. Reagents and conditions: (a) HNO3, CH3COOH, 50 °C; (b)
Scheme 2. Synthesis of compounds 9a–q. 2. Results and
2.1. Chemistry Due to
the limited amount of compounds and the lack of chemical diversity, the SAR of anthraquinone
derivatives and PGAM1 remained poorly understood. Here we synthesized four classes of
compounds on the base of bioisosterism to study the SAR of anthraquinone derivatives against
PGAM1 in detail (Figure 2), using an optimized enzymatic assay in which PGMI-004A served as a
control with an IC50 value of 1.2 ± 0.3 μM. Firstly, we found that different modifications of the
hydroxyl of alizarin (6a–e) showed weak PGAM1 inhibition activity at 10 μM (Table 1)
Previously we reported several anthraquinone compounds as PGAM1 inhibitors [17]. Due to the
limited amount of compounds and the lack of chemical diversity, the SAR of anthraquinone derivatives
and PGAM1 remained poorly understood. Here we synthesized four classes of compounds on the base
of bioisosterism to study the SAR of anthraquinone derivatives against PGAM1 in detail (Figure 2),
using an optimized enzymatic assay in which PGMI-004A served as a control with an IC50 value of
1.2 ± 0.3 µM. Firstly, we found that different modifications of the hydroxyl of alizarin (6a–e) showed
weak PGAM1 inhibition activity at 10 µM (Table 1). hydroxyl of alizarin (6a–e) showed weak PGAM1 inhibition activity at 10 μM (Table 1). Table 1. Inhibition activity of compounds 6a–e towards PGAM1. Compound
R 1
R 2
Inhibition of PGAM1 Activity at 10 μM 1
6a
−0.6 ± 1%
hydroxyl of alizarin (6a e) showed weak PGAM1 inhibition activity at 10 μM (Table 1). Table 1. Inhibition activity of compounds 6a–e towards PGAM1. Compound
R 1
R 2
Inhibition of PGAM1 Activity at 10 μM 1
6a
−0.6 ± 1%
In order to explore the SAR of 3-substituents of the anthraquinone scaffold against PGAM1,
compounds 9a–q were synthesized by reversing the sulfonamide group of PGMI-004A. Compounds
9a–e with different substituents on the phenyl ring displayed comparable inhibition activity towards
PGAM1, with IC50 values of 0.5 µM or so (Table 2). They were much more potent than the compounds
without the sulfonamide group, suggesting the sulfonamide group was essential for maintaining
potency. Then, we studied the impact of substituents of different halogens on the phenyl ring (9f–q) on
the inhibition activity towards PGAM1 (Table 2). Generally, compounds with Cl, Br and I substituents
(9i–q) with IC50 values below 0.5 µM performed better than F substituents (9f–h). 2. Results and
2.1. Chemistry Finally, compound 9i was employed as a starting material, and compounds 14a–g were
obtained by substitution with different reagents and further hydrolysis of the ester or amidation of
the carboxyl group (Scheme 4). Scheme 4. Synthesis of compounds 14a–g. Reagents and conditions: (a) K2CO3, DMF, corresponding
substituted reagent; (b) methanesulfonyl chloride, DIPEA, Py; (c) LiOH, MeOH, H2O, 30 °C; (d)
NH2NH2·H2O, EtOH, reflux. Scheme 4. Synthesis of compounds 14a–g. Reagents and conditions: (a) K2CO3, DMF, corresponding
substituted reagent; (b) methanesulfonyl chloride, DIPEA, Py; (c) LiOH, MeOH, H2O, 30 °C; (d)
NH2NH2·H2O, EtOH, reflux. Scheme 4. Synthesis of compounds 14a–g. Reagents and conditions: (a) K2CO3, DMF, corresponding
substituted reagent; (b) methanesulfonyl chloride, DIPEA, Py; (c) LiOH, MeOH, H2O, 30 ◦C;
(d) NH2NH2·H2O, EtOH, reflux. Scheme 4. Synthesis of compounds 14a–g. Reagents and conditions: (a) K2CO3, DMF, corresponding
substituted reagent; (b) methanesulfonyl chloride, DIPEA, Py; (c) LiOH, MeOH, H2O, 30 °C; (d)
NH2NH2·H2O, EtOH, reflux. Scheme 4. Synthesis of compounds 14a–g. Reagents and conditions: (a) K2CO3, DMF, corresponding
substituted reagent; (b) methanesulfonyl chloride, DIPEA, Py; (c) LiOH, MeOH, H2O, 30 °C; (d)
NH2NH2·H2O, EtOH, reflux. Scheme 4. Synthesis of compounds 14a–g. Reagents and conditions: (a) K2CO3, DMF, corresponding
substituted reagent; (b) methanesulfonyl chloride, DIPEA, Py; (c) LiOH, MeOH, H2O, 30 ◦C;
(d) NH2NH2·H2O, EtOH, reflux. 2 2 SAR Exploration of the Anthraquinone Derivatives Against PGAM1
2.2. SAR Exploration of the Anthraquinone Derivatives Against PGAM1
2.2. SAR Exploration of the Anthraquinone Derivatives against PGAM1 2 2 SAR Exploration of the Anthraquinone Derivatives Against PGAM1
2.2. SAR Exploration of the Anthraquinone Derivatives Against PGAM1
2.2. SAR Exploration of the Anthraquinone Derivatives against PGAM1 2.2. SAR Exploration of the Anthraquinone Derivatives Against PGAM1
Previously we reported several anthraquinone compounds as PGAM1 inhibitors [17]. Due to
the limited amount of compounds and the lack of chemical diversity, the SAR of anthraquinone
derivatives and PGAM1 remained poorly understood. Here we synthesized four classes of
compounds on the base of bioisosterism to study the SAR of anthraquinone derivatives against
PGAM1 in detail (Figure 2), using an optimized enzymatic assay in which PGMI-004A served as a
control with an IC50 value of 1.2 ± 0.3 μM. Firstly, we found that different modifications of the
h d
l f li
i
(6
) h
d
k PGAM1 i hibiti
ti it
t 10
M (T bl 1)
Previously we reported several anthraquinone compounds as PGAM1 inhibitors [17]. 2. Results and
2.1. Chemistry Reagents and conditions: (a) HNO3, CH3COOH, 50 °C; (b)
Sn, SnCl2·H2O, HCl, EtOH; (c) R3SO2Cl, Py. Scheme 2. Synthesis of compounds 9a–q. Reagents and conditions: (a) HNO3, CH3COOH, 50 °C; (b)
Sn, SnCl2·H2O, HCl, EtOH; (c) R3SO2Cl, Py. Scheme 2. Synthesis of compounds 9a–q. Reagents and conditions: (a) HNO3, CH3COOH, 50 ◦C;
(b) Sn, SnCl2·H2O, HCl, EtOH; (c) R3SO2Cl, Py. y
p
q
g
( )
( )
Sn, SnCl2·H2O, HCl, EtOH; (c) R3SO2Cl, Py. As shown in Scheme 3, the 4-nitro group was reduced to an amino group after the nitrification of
compound 6c, followed by sulfonylation of the amino group and hydrolysis of the ester as described above. Molecules 2019, 24, 845
of compound 6c, f
d
ib d b
of compound 6c,
described above 4 of 20
ter as
ster as Scheme 3. Synthesis of compounds 12 and 13. Reagents and conditions: (a) HNO3, CH3COOH, 50 °C;
(b) Sn, SnCl2·H2O, HCl, EtOH; (c) 4-chlorobenzenesulfonyl chloride, Py; (d) LiOH, MeOH, H2O, 30 °C. Scheme 3. Synthesis of compounds 12 and 13. Reagents and conditions: (a) HNO3, CH3COOH, 50 ◦C;
(b) Sn, SnCl2·H2O, HCl, EtOH; (c) 4-chlorobenzenesulfonyl chloride, Py; (d) LiOH, MeOH, H2O, 30 ◦C. Scheme 3. Synthesis of compounds 12 and 13. Reagents and conditions: (a) HNO3, CH3COOH, 50 °C;
(b) Sn, SnCl2·H2O, HCl, EtOH; (c) 4-chlorobenzenesulfonyl chloride, Py; (d) LiOH, MeOH, H2O, 30 °C. Scheme 3. Synthesis of compounds 12 and 13. Reagents and conditions: (a) HNO3, CH3COOH, 50 °C;
(b) Sn, SnCl2·H2O, HCl, EtOH; (c) 4-chlorobenzenesulfonyl chloride, Py; (d) LiOH, MeOH, H2O, 30 °C. Scheme 3. Synthesis of compounds 12 and 13. Reagents and conditions: (a) HNO3, CH3COOH, 50 ◦C;
(b) Sn, SnCl2·H2O, HCl, EtOH; (c) 4-chlorobenzenesulfonyl chloride, Py; (d) LiOH, MeOH, H2O, 30 ◦C. Scheme 3. Synthesis of compounds 12 and 13. Reagents and conditions: (a) HNO3, CH3COOH, 50 °C;
(b) Sn, SnCl2·H2O, HCl, EtOH; (c) 4-chlorobenzenesulfonyl chloride, Py; (d) LiOH, MeOH, H2O, 30 °C. Finally, compound 9i was employed as a starting material, and compounds 14a–g were
obtained by substitution with different reagents and further hydrolysis of the ester or amidation of
the carboxyl group (Scheme 4). Finally, compound 9i was employed as a starting material, and compounds 14a–g were obtained
by substitution with different reagents and further hydrolysis of the ester or amidation of the carboxyl
group (Scheme 4). 2. Results and
2.1. Chemistry Generally, compound
d I substituents (9i–q) with IC50 values below 0.5 μM performed better than F subst
mong them, we discovered one of the most potent PGAM1 inhibitors 9i with an IC5
M and we solved the co-crystal structure of compound 9i with PGAM1 which wo
d below. The data are presented as mean ±s.d. The data are p
H
1 The data are presented as mean ±s.d. 1 The data are presented as mean ± s.d. (n = 2). h b
f
d
d
y
p The data are presented as mean ±s.d. The data are p
H
1 The data are presented as mean ±s.d. 1 The data are presented as mean ± s.d. (n = 2). In order to explore the SAR of 3-substituents of the anthraquinone scaffold a
pounds 9a–q were synthesized by reversing the sulfonamide group of PGMI-004
with different substituents on the phenyl ring displayed comparable inhibition a
M1, with IC50 values of 0.5 μM or so (Table 2). They were much more p
pounds without the sulfonamide group, suggesting the sulfonamide group w
taining potency. Then, we studied the impact of substituents of different h
yl ring (9f–q) on the inhibition activity towards PGAM1 (Table 2). Generally, co
r and I substituents (9i–q) with IC50 values below 0.5 μM performed better than
h). Among them, we discovered one of the most potent PGAM1 inhibitors 9i wi
27 μM and we solved the co-crystal structure of compound 9i with PGAM1 w
ussed below. Table 2. Inhibition activity of compounds 9a–q towards PGAM1. O
O
OH
NHS
O
O
R3
OH
Compound
R 3
PGAM1 IC50 (μM) 1
9a
0.54 ± 0.04
9b
0.47 ± 0.08
9c
0.90 ± 0.07
9d
2.2 ± 0.5
In order to explore the SAR of 3-substituents of the anth
compounds 9a–q were synthesized by reversing the sulfonamid
9a–e with different substituents on the phenyl ring displayed co
PGAM1, with IC50 values of 0.5 μM or so (Table 2). They
compounds without the sulfonamide group, suggesting the su
maintaining potency. Then, we studied the impact of substit
phenyl ring (9f–q) on the inhibition activity towards PGAM1 (T
Cl, Br and I substituents (9i–q) with IC50 values below 0.5 μM p
(9f–h). Among them, we discovered one of the most potent PG
of 0.27 μM and we solved the co-crystal structure of compoun
discussed below. Table 2. 2. Results and
2.1. Chemistry Inhibition activity of compounds 9a–q
O
O
OH
NHS
O
O
R3
OH
Compound
R 3
PGAM1
9a
0.54
9b
0.47
9c
0.90
9d
2.2
6e
H
20.2 ± 2.2%
1 The data are presented as mean ±s.d. (n = 2). In order to explore the SAR of 3-substituents of the anthraquinone scaffold a
pounds 9a–q were synthesized by reversing the sulfonamide group of PGMI-004
with different substituents on the phenyl ring displayed comparable inhibition a
M1, with IC50 values of 0.5 μM or so (Table 2). They were much more p
pounds without the sulfonamide group, suggesting the sulfonamide group w
taining potency. Then, we studied the impact of substituents of different h
yl ring (9f–q) on the inhibition activity towards PGAM1 (Table 2). Generally, co
r and I substituents (9i–q) with IC50 values below 0.5 μM performed better than
h). Among them, we discovered one of the most potent PGAM1 inhibitors 9i wi
27 μM and we solved the co-crystal structure of compound 9i with PGAM1 w
ussed below. Table 2. Inhibition activity of compounds 9a–q towards PGAM1. O
O
OH
NHS
O
O
R3
OH
Compound
R 3
PGAM1 IC50 (μM) 1
9a
0.54 ± 0.04
9b
0 47 ± 0 08
In order to explore the SAR of 3-substituents of the anthraquinone scaffold a
pounds 9a–q were synthesized by reversing the sulfonamide group of PGMI-004
with different substituents on the phenyl ring displayed comparable inhibition a
M1, with IC50 values of 0.5 μM or so (Table 2). They were much more p
pounds without the sulfonamide group, suggesting the sulfonamide group w
taining potency. Then, we studied the impact of substituents of different h
yl ring (9f–q) on the inhibition activity towards PGAM1 (Table 2). Generally, co
r and I substituents (9i–q) with IC50 values below 0.5 μM performed better than
). Among them, we discovered one of the most potent PGAM1 inhibitors 9i wi
27 μM and we solved the co-crystal structure of compound 9i with PGAM1 w
ussed below. Table 2. Inhibition activity of compounds 9a–q towards PGAM1. O
O
OH
NHS
O
O
R3
OH
Compound
R 3
PGAM1 IC50 (μM) 1
9a
0.54 ± 0.04
9b
0.47 ± 0.08
9c
0.90 ± 0.07
9d
2 2 ± 0 5
Table 2. Inhibition activity of compounds 9a–q towards PGAM1. Table 2. Inhibition activity of compounds 9a–q towards PGAM1. 2. Results and
2.1. Chemistry Among them,
we discovered one of the most potent PGAM1 inhibitors 9i with an IC50 value of 0.27 µM and we
solved the co-crystal structure of compound 9i with PGAM1 which would be discussed below. 5 of 20
s of the
A served
cations of
in which
hat differ
s [17]. Du
nthraquin Molecules 2019, 24, 845
hydroxyl of alizarin
PGAM1 in d
control with
Previou
the limited a
6b Table 1. Inhibition activity of compounds 6a–e towards PGAM1. Table 1. Inhibition activity of compounds 6a–e towards PGAM1. Table 1 Inhibition activity of compounds 6a e towards PGA
Table 1 Inhibition activity of compo
p
y
y
on the base of bioisosterism to study the SAR of anthraqu
H
0 ± 1.8%
H
0 ± 1 8%
H
0 ± 1 8%
H
0 ± 1 8%
H
0
1 8% Table 1. Inhibition activity of compounds 6a–e towards PGAM1. Table 1. Inhibition activity of compounds 6a–e towards PGAM1. Table 1 Inhibition activity of compounds 6a e towards PGAM1
Table 1 Inhibition activity of compounds 6a e towards PGA
p
y
y
n the base of bioisosterism to study the SAR of anthraquinone derivatives aga
H
0 ± 1.8%
H
0 ± 1 8%
H
0 ± 1 8%
H
0
1 8%
H
0
1 8% ibition activity of compounds 6a–e towards PGAM1. Table 1. Inhibition activity of compounds 6a–e towards PG
ing an optimized enzymatic assay in which PGMI-004A served
1.2 ± 0.3 μM. Firstly, we found that different modifications o
wed weak PGAM1 inhibition activity at 10 μM (Table 1). b
f
d
d
GA
H
5.5 ± 0.03%
H
20 2 ± 2 2%
H
0 ± 1.8%
H
5.5 ± 0.03%
H
0 ± 1.8%
H
5.5 ± 0.03%
H
0 ± 1.8%
H
5.5 ± 0.03%
H
0 ± 1.8%
H
5.5 ± 0.03%
H
0 ± 1.8%
H
5.5 ± 0.03% hibition activity of compou
Table 1. Inhib
ing an optimized enzym
1.2 ± 0.3 μM. 2. Results and
2.1. Chemistry Firstly, w
wed weak PGAM1 inhib
hibi i
i i
f
H
H
H
H
H
H
H
H
H
H
H
H Compound
R 1
R 2
Inhibition of PGAM1 Activity at 10 μM 1
6a
−0.6 ± 1%
Compound
R 1
R 2
Inhibition of PGAM1
Activity at 10 µM 1
6a
Compound
R 1
R 2
Inhibition of PGAM1 Activity at 10 μM
6a
−0.6 ± 1%
Compound
R 1
R 2
Inhibition of PG
6a
−0.6 ± 1%
6b
ecules 2019, 24, 845
6b
14.3 ± 5.9%
6c
H
0 ± 1.8%
6d
H
5.5 ± 0.03%
6e
H
20.2 ± 2.2%
1 The data are presented as mean ±s.d. (n = 2). Molecules 2019, 24, 845
6b
1
6c
H
6d
H
5
6e
H
2
1 The data are presented as mean ±s.d. (n
14.3 ± 5.9%
6c
y
(
)
y
μ
(
)
Table 1. Inhibition activity of compounds 6a–e towards PGAM1. Compound
R 1
R 2
Inhibition of PGAM1 Activity at 10 μM
6a
−0.6 ± 1%
H
0 ± 1.8%
6d
ecules 2019, 24, 845
6b
14.3 ± 5.9%
6c
H
0 ± 1.8%
6d
H
5.5 ± 0.03%
H
5.5 ± 0.03%
6e
ecules 2019, 24, 845
6b
14.3 ± 5.9%
6c
H
0 ± 1.8%
6d
H
5.5 ± 0.03%
6e
H
20.2 ± 2.2%
1 The data are presented as mean ±s.d. (n = 2). H
20.2 ± 2.2%
1 The data are presented as mean ± s.d. (n = 2). 6e
H
20.2 ± 2.2%
1 The data are presented as mean ±s.d. (n = 2). der to explore the SAR of 3-substituents of the anthraquinone scaffold against PG
ds 9a–q were synthesized by reversing the sulfonamide group of PGMI-004A. Compo
different substituents on the phenyl ring displayed comparable inhibition activity tow
with IC50 values of 0.5 μM or so (Table 2). They were much more potent tha
ds without the sulfonamide group, suggesting the sulfonamide group was essenti
ng potency. Then, we studied the impact of substituents of different halogens o
g (9f–q) on the inhibition activity towards PGAM1 (Table 2). 2. Results and
2.1. Chemistry Generally, compounds
I substituents (9i–q) with IC50 values below 0.5 μM performed better than F substit
mong them, we discovered one of the most potent PGAM1 inhibitors 9i with an IC50
M and we solved the co-crystal structure of compound 9i with PGAM1 which wou
below. bl
I h b
f
d
d
A
6e
H
20.2 ± 2.2%
1 The data are presented as mean ±s.d. (n = 2). rder to explore the SAR of 3-substituents of the anthraquinone scaffold against PG
nds 9a–q were synthesized by reversing the sulfonamide group of PGMI-004A. Comp
h different substituents on the phenyl ring displayed comparable inhibition activity to
with IC50 values of 0.5 μM or so (Table 2). They were much more potent th
nds without the sulfonamide group, suggesting the sulfonamide group was essen
ing potency. Then, we studied the impact of substituents of different halogens
ng (9f–q) on the inhibition activity towards PGAM1 (Table 2). Generally, compound
d I substituents (9i–q) with IC50 values below 0.5 μM performed better than F subst
mong them, we discovered one of the most potent PGAM1 inhibitors 9i with an IC5
M and we solved the co-crystal structure of compound 9i with PGAM1 which wo
d below. 6e
H
20.2 ± 2.2%
1 The data are presented as mean ±s.d. (n = 2). rder to explore the SAR of 3-substituents of the anthraquinone scaffold against PG
nds 9a–q were synthesized by reversing the sulfonamide group of PGMI-004A. Comp
h different substituents on the phenyl ring displayed comparable inhibition activity to
with IC50 values of 0.5 μM or so (Table 2). They were much more potent th
nds without the sulfonamide group, suggesting the sulfonamide group was essen
ing potency. Then, we studied the impact of substituents of different halogens
ng (9f–q) on the inhibition activity towards PGAM1 (Table 2). Generally, compound
d I substituents (9i–q) with IC50 values below 0.5 μM performed better than F subst
mong them, we discovered one of the most potent PGAM1 inhibitors 9i with an IC5
M and we solved the co-crystal structure of compound 9i with PGAM1 which wo
d below. 6e
H
20.2 ± 2.2%
1 The data are presented as mean ±s.d. (n = 2). rder to explore the SAR of 3-substituents of the anthraquinone scaffold against PG
nds 9a–q were synthesized by reversing the sulfonamide group of PGMI-004A. 2. Results and
2.1. Chemistry Comp
h different substituents on the phenyl ring displayed comparable inhibition activity to
with IC50 values of 0.5 μM or so (Table 2). They were much more potent th
nds without the sulfonamide group, suggesting the sulfonamide group was essen
ing potency. Then, we studied the impact of substituents of different halogens
ng (9f–q) on the inhibition activity towards PGAM1 (Table 2). Generally, compound
d I substituents (9i–q) with IC50 values below 0.5 μM performed better than F subst
mong them, we discovered one of the most potent PGAM1 inhibitors 9i with an IC5
M and we solved the co-crystal structure of compound 9i with PGAM1 which wo
d below. 6e
H
20.2 ± 2.2%
1 The data are presented as mean ±s.d. (n = 2). rder to explore the SAR of 3-substituents of the anthraquinone scaffold against PG
nds 9a–q were synthesized by reversing the sulfonamide group of PGMI-004A. Comp
h different substituents on the phenyl ring displayed comparable inhibition activity to
with IC50 values of 0.5 μM or so (Table 2). They were much more potent th
nds without the sulfonamide group, suggesting the sulfonamide group was essen
ing potency. Then, we studied the impact of substituents of different halogens
ng (9f–q) on the inhibition activity towards PGAM1 (Table 2). Generally, compound
d I substituents (9i–q) with IC50 values below 0.5 μM performed better than F subst
mong them, we discovered one of the most potent PGAM1 inhibitors 9i with an IC5
M and we solved the co-crystal structure of compound 9i with PGAM1 which wo
d below. 6e
H
20.2 ± 2.2%
1 The data are presented as mean ±s.d. (n = 2). rder to explore the SAR of 3-substituents of the anthraquinone scaffold against PG
nds 9a–q were synthesized by reversing the sulfonamide group of PGMI-004A. Comp
h different substituents on the phenyl ring displayed comparable inhibition activity to
with IC50 values of 0.5 μM or so (Table 2). They were much more potent th
nds without the sulfonamide group, suggesting the sulfonamide group was essen
ing potency. Then, we studied the impact of substituents of different halogens
ng (9f–q) on the inhibition activity towards PGAM1 (Table 2). 2. Results and
2.1. Chemistry Generally, compound
d I substituents (9i–q) with IC50 values below 0.5 μM performed better than F subst
mong them, we discovered one of the most potent PGAM1 inhibitors 9i with an IC5
M and we solved the co-crystal structure of compound 9i with PGAM1 which wo
d below. Compound
R 1
R 2
Inhibition of PGAM1 Activity at 10 μM 1
6a
−0.6 ± 1%
Compound
R 1
R 2
Inhibition of PGAM1
Activity at 10 µM 1
6a
Compound
R 1
R 2
Inhibition of PGAM1 Activity at 10 μM
6a
−0.6 ± 1%
Compound
R 1
R 2
Inhibition of PG
6a
−0.6 ± 1%
6b
ecules 2019, 24, 845
6b
14.3 ± 5.9%
6c
H
0 ± 1.8%
6d
H
5.5 ± 0.03%
6e
H
20.2 ± 2.2%
1 The data are presented as mean ±s.d. (n = 2). Molecules 2019, 24, 845
6b
1
6c
H
6d
H
5
6e
H
2
1 The data are presented as mean ±s.d. (n
14.3 ± 5.9%
6c
Table 1. Inhibition activity of compounds 6a–e towards PGAM1. Compound
R 1
R 2
Inhibition of PGAM1 Activity at 10 μM
6a
−0.6 ± 1%
H
0 ± 1.8%
6d
ecules 2019, 24, 845
6b
14.3 ± 5.9%
6c
H
0 ± 1.8%
6d
H
5.5 ± 0.03%
H
5.5 ± 0.03%
6e
ecules 2019, 24, 845
6b
14.3 ± 5.9%
6c
H
0 ± 1.8%
6d
H
5.5 ± 0.03%
6e
H
20.2 ± 2.2%
1 The data are presented as mean ±s.d. (n = 2). H
20.2 ± 2.2%
1 The data are presented as mean ± s.d. (n = 2). 6e
H
20.2 ± 2.2%
1 The data are presented as mean ±s.d. (n = 2). der to explore the SAR of 3-substituents of the anthraquinone scaffold against PG
s 9a–q were synthesized by reversing the sulfonamide group of PGMI-004A. Compo
different substituents on the phenyl ring displayed comparable inhibition activity tow
with IC50 values of 0.5 μM or so (Table 2). They were much more potent tha
s without the sulfonamide group, suggesting the sulfonamide group was essenti
ng potency. Then, we studied the impact of substituents of different halogens o
g (9f–q) on the inhibition activity towards PGAM1 (Table 2). 2. Results and
2.1. Chemistry Generally, compounds
I substituents (9i–q) with IC50 values below 0.5 μM performed better than F substit
mong them, we discovered one of the most potent PGAM1 inhibitors 9i with an IC50
M and we solved the co-crystal structure of compound 9i with PGAM1 which wou
below. T bl 2 I hibi i
i i
f
d 9
d PGAM1
6e
H
20.2 ± 2.2%
1 The data are presented as mean ±s.d. (n = 2). rder to explore the SAR of 3-substituents of the anthraquinone scaffold against PG
nds 9a–q were synthesized by reversing the sulfonamide group of PGMI-004A. Comp
h different substituents on the phenyl ring displayed comparable inhibition activity to
with IC50 values of 0.5 μM or so (Table 2). They were much more potent th
nds without the sulfonamide group, suggesting the sulfonamide group was essen
ing potency. Then, we studied the impact of substituents of different halogens
ng (9f–q) on the inhibition activity towards PGAM1 (Table 2). Generally, compound
d I substituents (9i–q) with IC50 values below 0.5 μM performed better than F subst
mong them, we discovered one of the most potent PGAM1 inhibitors 9i with an IC5
M and we solved the co-crystal structure of compound 9i with PGAM1 which wo
d below. 6e
H
20.2 ± 2.2%
1 The data are presented as mean ±s.d. (n = 2). rder to explore the SAR of 3-substituents of the anthraquinone scaffold against PG
nds 9a–q were synthesized by reversing the sulfonamide group of PGMI-004A. Comp
h different substituents on the phenyl ring displayed comparable inhibition activity to
with IC50 values of 0.5 μM or so (Table 2). They were much more potent th
nds without the sulfonamide group, suggesting the sulfonamide group was essen
ing potency. Then, we studied the impact of substituents of different halogens
ng (9f–q) on the inhibition activity towards PGAM1 (Table 2). Generally, compound
d I substituents (9i–q) with IC50 values below 0.5 μM performed better than F subst
mong them, we discovered one of the most potent PGAM1 inhibitors 9i with an IC5
M and we solved the co-crystal structure of compound 9i with PGAM1 which wo
d below. 6e
H
20.2 ± 2.2%
1 The data are presented as mean ±s.d. (n = 2). rder to explore the SAR of 3-substituents of the anthraquinone scaffold against PG
nds 9a–q were synthesized by reversing the sulfonamide group of PGMI-004A. 2. Results and
2.1. Chemistry Comp
h different substituents on the phenyl ring displayed comparable inhibition activity to
with IC50 values of 0.5 μM or so (Table 2). They were much more potent th
nds without the sulfonamide group, suggesting the sulfonamide group was essen
ing potency. Then, we studied the impact of substituents of different halogens
ng (9f–q) on the inhibition activity towards PGAM1 (Table 2). Generally, compound
d I substituents (9i–q) with IC50 values below 0.5 μM performed better than F subst
mong them, we discovered one of the most potent PGAM1 inhibitors 9i with an IC5
M and we solved the co-crystal structure of compound 9i with PGAM1 which wo
d below. 6e
H
20.2 ± 2.2%
1 The data are presented as mean ±s.d. (n = 2). rder to explore the SAR of 3-substituents of the anthraquinone scaffold against PG
ds 9a–q were synthesized by reversing the sulfonamide group of PGMI-004A. Comp
h different substituents on the phenyl ring displayed comparable inhibition activity to
with IC50 values of 0.5 μM or so (Table 2). They were much more potent th
ds without the sulfonamide group, suggesting the sulfonamide group was essen
ing potency. Then, we studied the impact of substituents of different halogens
ng (9f–q) on the inhibition activity towards PGAM1 (Table 2). Generally, compound
d I substituents (9i–q) with IC50 values below 0.5 μM performed better than F subst
mong them, we discovered one of the most potent PGAM1 inhibitors 9i with an IC5
M and we solved the co-crystal structure of compound 9i with PGAM1 which wo
d below. 6e
H
20.2 ± 2.2%
1 The data are presented as mean ±s.d. (n = 2). rder to explore the SAR of 3-substituents of the anthraquinone scaffold against PG
nds 9a–q were synthesized by reversing the sulfonamide group of PGMI-004A. Comp
h different substituents on the phenyl ring displayed comparable inhibition activity to
with IC50 values of 0.5 μM or so (Table 2). They were much more potent th
nds without the sulfonamide group, suggesting the sulfonamide group was essen
ing potency. Then, we studied the impact of substituents of different halogens
ng (9f–q) on the inhibition activity towards PGAM1 (Table 2). 2. Results and
2.1. Chemistry O
O
OH
NHS
O
O
R3
OH
Compound
R 3
PGAM1 IC50 (μM) 1
9a
0.54 ± 0.04
9b
0.47 ± 0.08
9c
0.90 ± 0.07
9d
2.2 ± 0.5
9e
0.61 ± 0.12
Compound
R 3
PGAM1 IC50 (µM) 1
9a
Table 2. Inhibition activity of compounds 9a–q towards PGAM1. O
O
OH
NHS
O
O
R3
OH
Compound
R 3
PGAM1 IC50 (μM) 1
9a
0.54 ± 0.04
9b
0.47 ± 0.08
9c
0.90 ± 0.07
9d
2.2 ± 0.5
9e
0.61 ± 0.12
0.54 ± 0.04
9b
Table 2. Inhibition activity of compounds 9a–q towards PGAM1. O
O
OH
NHS
O
O
R3
OH
Compound
R 3
PGAM1 IC50 (μM) 1
9a
0.54 ± 0.04
9b
0.47 ± 0.08
9c
0.90 ± 0.07
9d
2.2 ± 0.5
9e
0.61 ± 0.12
0.47 ± 0.08
9c
Table 2. Inhibition activity of compounds 9a–q towards PGAM1. O
O
OH
NHS
O
O
R3
OH
Compound
R 3
PGAM1 IC50 (μM) 1
9a
0.54 ± 0.04
9b
0.47 ± 0.08
9c
0.90 ± 0.07
9d
2.2 ± 0.5
9e
0.61 ± 0.12
0.90 ± 0.07
9d
Table 2. Inhibition activity of compounds 9a–q towards PGAM1. O
O
OH
NHS
O
O
R3
OH
Compound
R 3
PGAM1 IC50 (μM) 1
9a
0.54 ± 0.04
9b
0.47 ± 0.08
9c
0.90 ± 0.07
9d
2.2 ± 0.5
9e
0.61 ± 0.12
2.2 ± 0.5
9e
Table 2. Inhibition activity of compounds 9a–q towards PGAM1. O
O
OH
NHS
O
O
R3
OH
Compound
R 3
PGAM1 IC50 (μM) 1
9a
0.54 ± 0.04
9b
0.47 ± 0.08
9c
0.90 ± 0.07
9d
2.2 ± 0.5
9e
0.61 ± 0.12 0.61 ± 0.12
9f
24, 845
Table 2. Cont. 9f
0.54 ± 0.07
9g
0.79 ± 0.04
9h
0.89 ± 0.13
0.54 ± 0.07
9g
24, 845
Table 2. Cont. 9f
0.54 ± 0.07
9g
0.79 ± 0.04
9h
0.89 ± 0.13
0.79 ± 0.04
9h
24, 845
Table 2. Cont. 2. Results and
2.1. Chemistry 0.35 ± 0.005
9p
9h
0.89 ± 0.13
9i
0.27 ± 0.04
9j
0.28 ± 0.07
9k
0.89 ± 0.33
9l
0.90 ± 0.26
9m
0.26 ± 0.02
9n
0.20 ± 0.04
9o
0.35 ± 0.005
9p
0.47 ± 0.07
9q
0.26 ± 0.03
1 The data are presented as mean ± s.d. (n = 3). 0.47 ± 0.07
9q
9h
0.89 ± 0.13
9i
0.27 ± 0.04
9j
0.28 ± 0.07
9k
0.89 ± 0.33
9l
0.90 ± 0.26
9m
0.26 ± 0.02
9n
0.20 ± 0.04
9o
0.35 ± 0.005
9p
0.47 ± 0.07
9q
0.26 ± 0.03
1 The data are presented as mean ± s.d. (n = 3). 0.26 ± 0.03
1 The data are presented as mean ± s.d. (n = 3). a
ide to the C4 positio
of ali a i
by
id
t
th
C4
iti
f
li
i
b
id
h
C4
i i
f
li
i
b
p
(
)
he data are presented as mean ± s.d. (n 3). he data are presented as mean ± s.d. (n = 3). he data are presented as mean ± s.d. (n = 3). he data are presented as mean ± s.d. (n = 3). 1 The data are presented as mean ± s.d. (n = 3). We next moved the sulfonamide to the C4 position of alizarin by substituting the hydroxyl,
ollowed by nitrification, reduction, and sulfonylation. Probably due to the modification of the
hydroxyl and replacement of the sulfonamide, compounds 12 and 13 failed to maintain potency,
decreasing 10% and 34% of PGAM1 activity at 10 μM, respectively. Finally, considering the good
solubility and potency of inhibitor 9i, we kept the 4-chlorobenzene group and synthesized
compounds 14a–g. However, diverse modifications of the hydroxyl caused a loss of inhibition
activity toward PGAM1 to different extents (Table 3). In order to exclude the inhibition of the other three enzymes (enolase-pyruvate kinase-LDH) in
We next moved the sulfonamide to the C4 position of alizarin by substituting the hydroxyl,
ollowed by nitrification, reduction, and sulfonylation. Probably due to the modification of the
hydroxyl and replacement of the sulfonamide, compounds 12 and 13 failed to maintain potency,
decreasing 10% and 34% of PGAM1 activity at 10 μM, respectively. Finally, considering the good
solubility and potency of inhibitor 9i, we kept the 4-chlorobenzene group and synthesized
compounds 14a–g. 2. Results and
2.1. Chemistry However, diverse modifications of the hydroxyl caused a loss of inhibition
activity toward PGAM1 to different extents (Table 3). In order to exclude the inhibition of the other three enzymes (enolase-pyruvate kinase-LDH) in
We next moved the sulfonamide to the C4 position of alizarin by substituting the hydroxyl,
ollowed by nitrification, reduction, and sulfonylation. Probably due to the modification of the
hydroxyl and replacement of the sulfonamide, compounds 12 and 13 failed to maintain potency,
decreasing 10% and 34% of PGAM1 activity at 10 μM, respectively. Finally, considering the good
solubility and potency of inhibitor 9i, we kept the 4-chlorobenzene group and synthesized
compounds 14a–g. However, diverse modifications of the hydroxyl caused a loss of inhibition
activity toward PGAM1 to different extents (Table 3). In order to exclude the inhibition of the other three enzymes (enolase-pyruvate kinase-LDH) in
We next moved the sulfonamide to the C4 position of alizarin by substituting the hydroxyl,
followed by nitrification, reduction, and sulfonylation. Probably due to the modification of the
hydroxyl and replacement of the sulfonamide, compounds 12 and 13 failed to maintain potency,
decreasing 10% and 34% of PGAM1 activity at 10 μM, respectively. Finally, considering the good
solubility and potency of inhibitor 9i, we kept the 4-chlorobenzene group and synthesized
compounds 14a–g. However, diverse modifications of the hydroxyl caused a loss of inhibition
activity toward PGAM1 to different extents (Table 3). In order to exclude the inhibition of the other three enzymes (enolase-pyruvate kinase-LDH) in
We next moved the sulfonamide to the C4 position of alizarin by substituting the hydroxyl,
ollowed by nitrification, reduction, and sulfonylation. Probably due to the modification of the
hydroxyl and replacement of the sulfonamide, compounds 12 and 13 failed to maintain potency,
decreasing 10% and 34% of PGAM1 activity at 10 μM, respectively. Finally, considering the good
olubility and potency of inhibitor 9i, we kept the 4-chlorobenzene group and synthesized
compounds 14a–g. However, diverse modifications of the hydroxyl caused a loss of inhibition
activity toward PGAM1 to different extents (Table 3). In order to exclude the inhibition of the other three enzymes (enolase pyruvate kinase LDH) in
We next moved the sulfonamide to the C4 position of alizarin by substituting the hydroxyl,
ollowed by nitrification, reduction, and sulfonylation. 2. Results and
2.1. Chemistry Probably due to the modification of the
hydroxyl and replacement of the sulfonamide, compounds 12 and 13 failed to maintain potency,
decreasing 10% and 34% of PGAM1 activity at 10 μM, respectively. Finally, considering the good
olubility and potency of inhibitor 9i, we kept the 4-chlorobenzene group and synthesized
compounds 14a–g. However, diverse modifications of the hydroxyl caused a loss of inhibition
activity toward PGAM1 to different extents (Table 3). I
o de to e
lude the i hibitio
of the othe th ee e
y
e (e ola e
y u ate ki a e LDH) i
We next moved the sulfonamide to the C4 position of alizarin by substituting the hydroxyl,
ollowed by nitrification, reduction, and sulfonylation. Probably due to the modification of the
hydroxyl and replacement of the sulfonamide, compounds 12 and 13 failed to maintain potency,
decreasing 10% and 34% of PGAM1 activity at 10 μM, respectively. Finally, considering the good
olubility and potency of inhibitor 9i, we kept the 4-chlorobenzene group and synthesized
compounds 14a–g. However, diverse modifications of the hydroxyl caused a loss of inhibition
activity toward PGAM1 to different extents (Table 3). I
d
l d
h
h b
f h
h
h
l
k
We next moved the sulfonamide to the C4 position of alizarin by substituting the hydroxyl,
ollowed by nitrification, reduction, and sulfonylation. Probably due to the modification of the
hydroxyl and replacement of the sulfonamide, compounds 12 and 13 failed to maintain potency,
decreasing 10% and 34% of PGAM1 activity at 10 μM, respectively. Finally, considering the good
solubility and potency of inhibitor 9i, we kept the 4-chlorobenzene group and synthesized
compounds 14a–g. However, diverse modifications of the hydroxyl caused a loss of inhibition
activity toward PGAM1 to different extents (Table 3). We next moved the sulfonamide to the C4 position of alizarin by substituting the hydroxyl,
ollowed by nitrification, reduction, and sulfonylation. Probably due to the modification of the
hydroxyl and replacement of the sulfonamide, compounds 12 and 13 failed to maintain potency,
decreasing 10% and 34% of PGAM1 activity at 10 μM, respectively. Finally, considering the good
olubility and potency of inhibitor 9i, we kept the 4-chlorobenzene group and synthesized
compounds 14a–g. However, diverse modifications of the hydroxyl caused a loss of inhibition
activity toward PGAM1 to different extents (Table 3). 2. Results and
2.1. Chemistry We next moved the sulfonamide to the C4 position of alizarin by substituting the hydroxyl,
followed by nitrification, reduction, and sulfonylation. Probably due to the modification of the
hydroxyl and replacement of the sulfonamide, compounds 12 and 13 failed to maintain potency,
decreasing 10% and 34% of PGAM1 activity at 10 µM, respectively. Finally, considering the good
solubility and potency of inhibitor 9i, we kept the 4-chlorobenzene group and synthesized compounds
14a–g. However, diverse modifications of the hydroxyl caused a loss of inhibition activity toward
PGAM1 to different extents (Table 3). y
py
the coupled assay, we performed counter screening of selected compounds with IC50 value below 1
μM on PGAM1. y
(
py
)
the coupled assay, we performed counter screening of selected compounds with IC50 value below 1
μM on PGAM1. y
(
py
)
the coupled assay, we performed counter screening of selected compounds with IC50 value below 1
μM on PGAM1. In order to exclude the inhibition of the other three enzymes (enolase pyruvate kinase LDH) in
the coupled assay, we performed counter screening of selected compounds with IC50 value below 1
μM on PGAM1. In order to exclude the inhibition of the other three enzymes (enolase-pyruvate kinase-LDH) in
he coupled assay, we performed counter screening of selected compounds with IC50 value below 1
μM on PGAM1. In order to exclude the inhibition of the other three enzymes (enolase-pyruvate kinase-LDH) in
he coupled assay, we performed counter screening of selected compounds with IC50 value below 1
μM on PGAM1. In order to exclude the inhibition of the other three enzymes (enolase-pyruvate kinase-LDH) in
he coupled assay, we performed counter screening of selected compounds with IC50 value below 1
μM on PGAM1. In order to exclude the inhibition of the other three enzymes (enolase-pyruvate kinase-LDH) in
he coupled assay, we performed counter screening of selected compounds with IC50 value below 1
μM on PGAM1. In order to exclude the inhibition of the other three enzymes (enolase-pyruvate kinase-LDH) in
he coupled assay, we performed counter screening of selected compounds with IC50 value below 1
μM on PGAM1. In order to exclude the inhibition of the other three enzymes (enolase-pyruvate kinase-LDH) in
the coupled assay, we performed counter screening of selected compounds with IC50 value below
1 µM on PGAM1. Table 3. Inhibition activity of compounds 14a–14g towards PGAM1. Table 3. Inhibition activity of compounds 14a–14g towards PGAM1. 2. Results and
2.1. Chemistry 9f
0.54 ± 0.07
9g
0.79 ± 0.04
9h
0.89 ± 0.13 0.89 ± 0.13 o explore the SAR of 3-substituents of the anthraquinone
–q were synthesized by reversing the sulfonamide group of
In order to explore the SAR of 3-substituents o
compounds 9a–q were synthesized by reversing the s
6e
H
20.2 ± 2.2
o explore the SAR of 3-substituents of the anthraquinone
–q were synthesized by reversing the sulfonamide group of
Table 2. Inhibition activity of compounds 9a–q towards PGAM1. ab e . I
ibi io
ac i i y o co
pou
s 9a q o
a
s
GAM
Table 2. Inhibition activity of compounds 9a–q towards PGAM1. Table 2. Inhibition activity of compounds 9a–q towards PGAM1. Table 2. Inhibition activity of compounds 9a–q towards PGAM1. Table 2. Inhibition activity of compounds 9a–q towards PGAM1. Table 2. Inhibition activity of compounds 9a–q towards PGAM1. to explore the SAR of 3-substituents of the anthraquinone
a–q were synthesized by reversing the sulfonamide group of
In order to explore the SAR of 3-substituents o
compounds 9a–q were synthesized by reversing the s
6e
H
20.2 ± 2.2
to explore the SAR of 3-substituents of the anthraquinone
a–q were synthesized by reversing the sulfonamide group of
Table 2. Inhibition activity of compounds 9a–q towards PGAM1. a
e
I
i i io
a i i y o
o
pou
a q o
a
A
Table 2. Inhibition activity of compounds 9a–q towards PGAM1. Table 2. Inhibition activity of compounds 9a–q towards PGAM1. Table 2. Inhibition activity of compounds 9a–q towards PGAM1. Table 2. Inhibition activity of compounds 9a–q towards PGAM1. Table 2. Inhibition activity of compounds 9a–q towards PGAM1. 6 of 20 Molecules 2019, 24, 845 Table 2. Cont. Compound
R 3
PGAM1 IC50 (µM) 1
9i
9h
0.89 ± 0.13
9i
0.27 ± 0.04
9j
0.28 ± 0.07
9k
0.89 ± 0.33
9l
0.90 ± 0.26
9m
0.26 ± 0.02
9n
0.20 ± 0.04
9o
0.35 ± 0.005
9p
0.47 ± 0.07
9q
0.26 ± 0.03
1 The data are presented as mean ± s.d. (n = 3). 2. Results and
2.1. Chemistry ext moved the sulfonamide to the C4 position of alizarin by substituting the hyd
0.27 ± 0.04
9j
9h
0.89 ± 0.13
9i
0.27 ± 0.04
9j
0.28 ± 0.07
9k
0.89 ± 0.33
9l
0.90 ± 0.26
9m
0.26 ± 0.02
9n
0.20 ± 0.04
9o
0.35 ± 0.005
9p
0.47 ± 0.07
9q
0.26 ± 0.03
1 The data are presented as mean ± s.d. (n = 3). ext moved the sulfonamide to the C4 position of alizarin by substituting the hyd
0.28 ± 0.07
9k
9h
0.89 ± 0.13
9i
0.27 ± 0.04
9j
0.28 ± 0.07
9k
0.89 ± 0.33
9l
0.90 ± 0.26
9m
0.26 ± 0.02
9n
0.20 ± 0.04
9o
0.35 ± 0.005
9p
0.47 ± 0.07
9q
0.26 ± 0.03
1 The data are presented as mean ± s.d. (n = 3). t
d th
lf
id
t
th
C4
iti
f
li
i
b
b tit ti
th
h d
0.89 ± 0.33
9l
9h
0.89 ± 0.13
9i
0.27 ± 0.04
9j
0.28 ± 0.07
9k
0.89 ± 0.33
9l
0.90 ± 0.26
9m
0.26 ± 0.02
9n
0.20 ± 0.04
9o
0.35 ± 0.005
9p
0.47 ± 0.07
9q
0.26 ± 0.03
1 The data are presented as mean ± s.d. (n = 3). d h
lf
d
h
f
l
b
b
h
h d
0.90 ± 0.26
9m
9h
0.89 ± 0.13
9i
0.27 ± 0.04
9j
0.28 ± 0.07
9k
0.89 ± 0.33
9l
0.90 ± 0.26
9m
0.26 ± 0.02
9n
0.20 ± 0.04
9o
0.35 ± 0.005
9p
0.47 ± 0.07
9q
0.26 ± 0.03
1 The data are presented as mean ± s.d. (n = 3). 0.26 ± 0.02
9n
9h
0.89 ± 0.13
9i
0.27 ± 0.04
9j
0.28 ± 0.07
9k
0.89 ± 0.33
9l
0.90 ± 0.26
9m
0.26 ± 0.02
9n
0.20 ± 0.04
9o
0.35 ± 0.005
9p
0.47 ± 0.07
9q
0.26 ± 0.03
1 The data are presented as mean ± s.d. (n = 3). 0.20 ± 0.04
9o
9h
0.89 ± 0.13
9i
0.27 ± 0.04
9j
0.28 ± 0.07
9k
0.89 ± 0.33
9l
0.90 ± 0.26
9m
0.26 ± 0.02
9n
0.20 ± 0.04
9o
0.35 ± 0.005
9p
0.47 ± 0.07
9q
0.26 ± 0.03
1 The data are presented as mean ± s.d. (n = 3). 2. Results and
2.1. Chemistry H
>10 µM
1 The data are presented as mean ± s.d. (n = 3). p
(
)
data are presented as mean ±s.d. (n = 3). data are presented as mean ±s.d. (n = 3). data are presented as mean ±s.d. (n = 3). data are presented as mean ±s.d. (n = 3). 1 The data are presented as mean ± s.d. (n = 3). wn in Table 4, the compounds displayed almost no inhibition on the three do
0.5 μM which further confirmed the on-target effect of the compounds. n Table 4, the compounds displayed almost no inhibition on the three downs
M
hi h f
th
fi
d th
t
t ff
t f th
d
n Table 4, the compounds displayed almost no inhibition on the three downs
M
hi h f
h
fi
d h
ff
f h
d
n Table 4, the compounds displayed almost no inhibition on the three downs
μM which further confirmed the on target effect of the compounds
n Table 4, the compounds displayed almost no inhibition on the three downs
μM which further confirmed the on target effect of the compounds
Table 4. The inhibition in the control linked enzyme assay of selected compounds. hown in Table 4, the compounds displayed almost no inhibition on the three down
at 0.5 μM which further confirmed the on-target effect of the compounds. Table 4. The inhibition in the control linked enzyme assay of selected compounds. ompound
PGAM1 IC50 (μM)
Inhibition in the Control Linked Enzyme Assay (0.5 μM)
9a
0.54 ± 0.04
8.0 ± 5.0%
9b
0.47 ± 0.08
−2.9 ± 0.3%
9c
0.90 ± 0.07
3.7 ± 1.8%
9e
0.61 ± 0.12
6.7 ± 0.2%
9f
0.54 ± 0.07
5.7 ± 0.7%
9g
0.79 ± 0.04
10.2 ± 3.0%
9h
0.89 ± 0.13
0.06 ± 0.4%
9i
0.27 ± 0.04
−6.4 ± 5.0%
9j
0.28 ± 0.07
−2.0 ± 5.1%
9k
0.89 ± 0.33
0.5 ± 3.2%
9l
0.90 ± 0.26
0.2 ± 1.7%
9m
0.26 ± 0.02
−0.1 ± 2.5%
9n
0.20 ± 0.04
6.1 ± 0.8%
9o
0.35 ± 0.005
7.7 ± 0.2%
9p
0.47 ± 0.07
1.9 ± 0.1%
wn in Table 4, the compounds displayed almost no inhibition on the three downstr
.5 μM which further confirmed the on-target effect of the compounds. Table 4. The inhibition in the control linked enzyme assay of selected compounds. 2. Results and
2.1. Chemistry ound
PGAM1 IC50 (μM)
Inhibition in the Control Linked Enzyme Assay (0.5 μM) 1
a
0.54 ± 0.04
8.0 ± 5.0%
b
0.47 ± 0.08
−2.9 ± 0.3%
c
0.90 ± 0.07
3.7 ± 1.8%
e
0.61 ± 0.12
6.7 ± 0.2%
f
0.54 ± 0.07
5.7 ± 0.7%
g
0.79 ± 0.04
10.2 ± 3.0%
h
0.89 ± 0.13
0.06 ± 0.4%
i
0.27 ± 0.04
−6.4 ± 5.0%
j
0.28 ± 0.07
−2.0 ± 5.1%
k
0.89 ± 0.33
0.5 ± 3.2%
l
0.90 ± 0.26
0.2 ± 1.7%
m
0.26 ± 0.02
−0.1 ± 2.5%
n
0.20 ± 0.04
6.1 ± 0.8%
o
0.35 ± 0.005
7.7 ± 0.2%
p
0.47 ± 0.07
1.9 ± 0.1%
wn in Table 4, the compounds displayed almost no inhibition on the three downstr
.5 μM which further confirmed the on-target effect of the compounds. Table 4. The inhibition in the control linked enzyme assay of selected compounds. ound
PGAM1 IC50 (μM)
Inhibition in the Control Linked Enzyme Assay (0.5 μM) 1
a
0.54 ± 0.04
8.0 ± 5.0%
b
0.47 ± 0.08
−2.9 ± 0.3%
c
0.90 ± 0.07
3.7 ± 1.8%
e
0.61 ± 0.12
6.7 ± 0.2%
f
0.54 ± 0.07
5.7 ± 0.7%
g
0.79 ± 0.04
10.2 ± 3.0%
h
0.89 ± 0.13
0.06 ± 0.4%
i
0.27 ± 0.04
−6.4 ± 5.0%
j
0.28 ± 0.07
−2.0 ± 5.1%
k
0.89 ± 0.33
0.5 ± 3.2%
l
0.90 ± 0.26
0.2 ± 1.7%
m
0.26 ± 0.02
−0.1 ± 2.5%
n
0.20 ± 0.04
6.1 ± 0.8%
o
0.35 ± 0.005
7.7 ± 0.2%
p
0.47 ± 0.07
1.9 ± 0.1%
wn in Table 4, the compounds displayed almost no inhibition on the three downstr
.5 μM which further confirmed the on-target effect of the compounds. Table 4. The inhibition in the control linked enzyme assay of selected compounds. 2. Results and
2.1. Chemistry ound
PGAM1 IC50 (μM)
Inhibition in the Control Linked Enzyme Assay (0.5 μM) 1
a
0.54 ± 0.04
8.0 ± 5.0%
b
0.47 ± 0.08
−2.9 ± 0.3%
c
0.90 ± 0.07
3.7 ± 1.8%
e
0.61 ± 0.12
6.7 ± 0.2%
f
0.54 ± 0.07
5.7 ± 0.7%
g
0.79 ± 0.04
10.2 ± 3.0%
h
0.89 ± 0.13
0.06 ± 0.4%
i
0.27 ± 0.04
−6.4 ± 5.0%
j
0.28 ± 0.07
−2.0 ± 5.1%
k
0.89 ± 0.33
0.5 ± 3.2%
l
0.90 ± 0.26
0.2 ± 1.7%
m
0.26 ± 0.02
−0.1 ± 2.5%
n
0.20 ± 0.04
6.1 ± 0.8%
o
0.35 ± 0.005
7.7 ± 0.2%
p
0.47 ± 0.07
1.9 ± 0.1%
wn in Table 4, the compounds displayed almost no inhibition on the three downstr
.5 μM which further confirmed the on-target effect of the compounds. Table 4. The inhibition in the control linked enzyme assay of selected compounds. ound
PGAM1 IC50 (μM)
Inhibition in the Control Linked Enzyme Assay (0.5 μM) 1
a
0.54 ± 0.04
8.0 ± 5.0%
b
0.47 ± 0.08
−2.9 ± 0.3%
c
0.90 ± 0.07
3.7 ± 1.8%
e
0.61 ± 0.12
6.7 ± 0.2%
f
0.54 ± 0.07
5.7 ± 0.7%
g
0.79 ± 0.04
10.2 ± 3.0%
h
0.89 ± 0.13
0.06 ± 0.4%
i
0.27 ± 0.04
−6.4 ± 5.0%
j
0.28 ± 0.07
−2.0 ± 5.1%
k
0.89 ± 0.33
0.5 ± 3.2%
l
0.90 ± 0.26
0.2 ± 1.7%
m
0.26 ± 0.02
−0.1 ± 2.5%
n
0.20 ± 0.04
6.1 ± 0.8%
o
0.35 ± 0.005
7.7 ± 0.2%
p
0.47 ± 0.07
1.9 ± 0.1%
Table 4. The inhibition in the control linked enzyme assay of selected compounds. 9q
0.26 ± 0.03
1 The data are presen
9q
0.26 ± 0.03
1 The data are presented
9q
0.26 ± 0.03
1 The data are presented
9q
0.26 ± 0.03
1 The data are presented
9q
0.26 ± 0.03
1 The data are presented
2.3. Binding Mode of Compound 9i with PGAM1 2.3. Binding Mode of Compound 9i with PGAM1
To further understand the molecular mechanism of the anthraquinone derivatives interacting
with PGAM1, we determined the X-ray structure of PGAM1 in complex with compound 9i at
resolution of 1.98 Å (Table 5). Compound 9i occupied a novel allosteric site adjacent to substrate
binding site with nice electron density (Figure 3A and Figure 3B). The allosteric pocket was
surrounded by the residues of F22, R90, K100, R116 and R191. In detail, the anthraquinone scaffold
and sulfonamide of compound 9i interacted with the main chain carbonyl of K100 through water
p
(
)
Binding Mode of Compound 9i with PGAM1
To further understand the molecular mechanism of the anthraquinone derivatives interacting
h PGAM1, we determined the X-ray structure of PGAM1 in complex with compound 9i at
olution of 1.98 Å (Table 5). Compound 9i occupied a novel allosteric site adjacent to substrate
ding site with nice electron density (Figure 3A and Figure 3B). The allosteric pocket was
rounded by the residues of F22, R90, K100, R116 and R191. In detail, the anthraquinone scaffold
p
(
)
Binding Mode of Compound 9i with PGAM1
To further understand the molecular mechanism of the anthraquinone derivatives interacting
h PGAM1, we determined the X-ray structure of PGAM1 in complex with compound 9i at
olution of 1.98 Å (Table 5). Compound 9i occupied a novel allosteric site adjacent to substrate
ding site with nice electron density (Figure 3A and Figure 3B). The allosteric pocket was
rounded by the residues of F22, R90, K100, R116 and R191. In detail, the anthraquinone scaffold
Binding Mode of Compound 9i with PGAM1
To further understand the molecular mechanism of the anthraquinone derivatives interacting
h PGAM1, we determined the X-ray structure of PGAM1 in complex with compound 9i at
olution of 1.98 Å (Table 5). Compound 9i occupied a novel allosteric site adjacent to substrate
ding site with nice electron density (Figure 3A and Figure 3B). The allosteric pocket was
rounded by the residues of F22, R90, K100, R116 and R191. 2. Results and
2.1. Chemistry Compound
PGAM1 IC50 (µM)
Inhibition in the Control Linked
Enzyme Assay (0.5 µM) 1
9a
0.54 ± 0.04
8.0 ± 5.0%
9b
0.47 ± 0.08
−2.9 ± 0.3%
9c
0.90 ± 0.07
3.7 ± 1.8%
9e
0.61 ± 0.12
6.7 ± 0.2%
9f
0.54 ± 0.07
5.7 ± 0.7%
9g
0.79 ± 0.04
10.2 ± 3.0%
9h
0.89 ± 0.13
0.06 ± 0.4%
9i
0.27 ± 0.04
−6.4 ± 5.0%
9j
0.28 ± 0.07
−2.0 ± 5.1%
9k
0.89 ± 0.33
0.5 ± 3.2%
9l
0.90 ± 0.26
0.2 ± 1.7%
9m
0.26 ± 0.02
−0.1 ± 2.5%
9n
0.20 ± 0.04
6.1 ± 0.8%
9o
0.35 ± 0.005
7.7 ± 0.2%
9p
0.47 ± 0.07
1.9 ± 0.1%
9q
0.26 ± 0.03
−2.3 ± 2.4%
1 The data are presented as mean ± s.d. (n = 2). 2. Results and
2.1. Chemistry Table 3. Inhibition activity of compounds 14a–14g towards PGAM1. Table 3. Inhibition activity of compounds 14a–14g towards PGAM1. Table 3. Inhibition activity of compounds 14a–14g towards PGAM1. Table 3. Inhibition activity of compounds 14a–14g towards PGAM1. Table 3. Inhibition activity of compounds 14a–14g towards PGAM1. Table 3. Inhibition activity of compounds 14a–14g towards PGAM1. Table 3. Inhibition activity of compounds 14a–14g towards PGAM1. As shown in Table 4, the compounds displayed almost no inhibition on the three downstream
enzymes at 0.5 µM which further confirmed the on-target effect of the compounds. 7 of 20 Molecules 2019, 24, 845 Table 3. Inhibition activity of compounds 14a–14g towards PGAM1. Table 3. Inhibition activity of compounds 14a–14g towards PGAM1. Compound
R 5
R 6
PGAM1 IC50 (μM) 1
14a
H
>10 μM
14b
H
>10 μM
14c
CH3
H
2.92 ± 0.11
14d
CH3
CH3
7.01 ± 0.92
14e
-CH2-
-CH2-
>10 μM
14f
H
2.0 ± 0.2
14g
H
>10 μM
1 The data are presented as mean ±s.d. (n = 3). bl
h
d d
l
d l
h b
h
h
d
Compound
R 5
R 6
PGAM1 IC50 (µM) 1
14a
4, 845
7 of
Compound
R 5
R 6
PGAM1 IC50 (μM) 1
14a
H
>10 μM
14b
H
>10 μM
14c
CH3
H
2.92 ± 0.11
14d
CH3
CH3
7.01 ± 0.92
14e
-CH2-
-CH2-
>10 μM
14f
H
2.0 ± 0.2
14g
H
>10 μM
1 The data are presented as mean ±s.d. (n = 3). H
>10 µM
14b
, 845
7 of
Compound
R 5
R 6
PGAM1 IC50 (μM) 1
14a
H
>10 μM
14b
H
>10 μM
14c
CH3
H
2.92 ± 0.11
14d
CH3
CH3
7.01 ± 0.92
14e
-CH2-
-CH2-
>10 μM
14f
H
2.0 ± 0.2
14g
H
>10 μM
1 The data are presented as mean ±s.d. (n = 3). H
>10 µM
14c
CH3
H
2.92 ± 0.11
14d
CH3
CH3
7.01 ± 0.92
14e
-CH2-
-CH2-
>10 µM
14f
,
Compound
R 5
R 6
PGAM1 IC50 (μM) 1
14a
H
>10 μM
14b
H
>10 μM
14c
CH3
H
2.92 ± 0.11
14d
CH3
CH3
7.01 ± 0.92
14e
-CH2-
-CH2-
>10 μM
14f
H
2.0 ± 0.2
14g
H
>10 μM
1 The data are presented as mean ±s.d. (n = 3). 2. Results and
2.1. Chemistry H
2.0 ± 0.2
14g
, 845
7 of
Compound
R 5
R 6
PGAM1 IC50 (μM) 1
14a
H
>10 μM
14b
H
>10 μM
14c
CH3
H
2.92 ± 0.11
14d
CH3
CH3
7.01 ± 0.92
14e
-CH2-
-CH2-
>10 μM
14f
H
2.0 ± 0.2
14g
H
>10 μM
1 The data are presented as mean ±s.d. (n = 3). H
>10 µM
1 The data are presented as mean ± s.d. (n = 3). Table 3. Inhibition activity of compounds 14a–14g towards PGAM1. Compound
R 5
R 6
PGAM1 IC50 (μM) 1
14a
H
>10 μM
14b
H
>10 μM
14c
CH3
H
2.92 ± 0.11
14d
CH3
CH3
7.01 ± 0.92
14e
-CH2-
-CH2-
>10 μM
14f
H
2.0 ± 0.2
14g
H
>10 μM
1 The data are presented as mean ±s.d. (n = 3). Compound
R 5
R 6
PGAM1 IC50 (µM) 1
14a
Compound
R 5
R 6
PGAM1 IC50 (μM) 1
14a
H
>10 μM
14b
H
>10 μM
14c
CH3
H
2.92 ± 0.11
14d
CH3
CH3
7.01 ± 0.92
14e
-CH2-
-CH2-
>10 μM
14f
H
2.0 ± 0.2
14g
H
>10 μM
1 The data are presented as mean ±s.d. (n = 3). H
>10 µM
14b
Compound
R 5
R 6
PGAM1 IC50 (μM) 1
14a
H
>10 μM
14b
H
>10 μM
14c
CH3
H
2.92 ± 0.11
14d
CH3
CH3
7.01 ± 0.92
14e
-CH2-
-CH2-
>10 μM
14f
H
2.0 ± 0.2
14g
H
>10 μM
1 The data are presented as mean ±s.d. (n = 3). H
>10 µM
14c
CH3
H
2.92 ± 0.11
14d
CH3
CH3
7.01 ± 0.92
14e
-CH2-
-CH2-
>10 µM
14f
Compound
R 5
R 6
PGAM1 IC50 (μM) 1
14a
H
>10 μM
14b
H
>10 μM
14c
CH3
H
2.92 ± 0.11
14d
CH3
CH3
7.01 ± 0.92
14e
-CH2-
-CH2-
>10 μM
14f
H
2.0 ± 0.2
14g
H
>10 μM
1 The data are presented as mean ±s.d. (n = 3). H
2.0 ± 0.2
14g
Compound
R 5
R 6
PGAM1 IC50 (μM) 1
14a
H
>10 μM
14b
H
>10 μM
14c
CH3
H
2.92 ± 0.11
14d
CH3
CH3
7.01 ± 0.92
14e
-CH2-
-CH2-
>10 μM
14f
H
2.0 ± 0.2
14g
H
>10 μM
1 The data are presented as mean ±s.d. (n = 3). 9q
0.26 ± 0.03
1 The data are presen
9q
0.26 ± 0.03
1 The data are presented
9q
0.26 ± 0.03
1 The data are presented
9q
0.26 ± 0.03
1 The data are presented
9q
0.26 ± 0.03
1 The data are presented
2.3. Binding Mode of Compound 9i with PGAM1 In detail, the anthraquinone scaffold
d
lf
id
f
d 9i i t
t d
ith th
i
h i
b
l
f K100 th
h
t
Binding Mode of Compound 9i with PGAM1
To further understand the molecular mechanism of the anthraquinone derivatives interacting
h PGAM1, we determined the X-ray structure of PGAM1 in complex with compound 9i at
olution of 1.98 Å (Table 5). Compound 9i occupied a novel allosteric site adjacent to substrate
ding site with nice electron density (Figure 3A and Figure 3B). The allosteric pocket was
rounded by the residues of F22, R90, K100, R116 and R191. In detail, the anthraquinone scaffold
d
lf
id
f
d 9i i
d
i h h
i
h i
b
l
f K100 h
h
To further understand the molecular mechanism of the anthraquinone derivatives interacting with
PGAM1, we determined the X-ray structure of PGAM1 in complex with compound 9i at resolution of
1.98 Å (Table 5). Compound 9i occupied a novel allosteric site adjacent to substrate binding site with
nice electron density (Figure 3A,B). The allosteric pocket was surrounded by the residues of F22, R90,
K100, R116 and R191. In detail, the anthraquinone scaffold and sulfonamide of compound 9i interacted
with the main chain carbonyl of K100 through water bridges (Figure 3C). In addition, a hydrophobic
interaction was observed between F22 and chlorine-substituted phenyl ring of compound 9i (Figure 3C). 8 of 20 Molecules 2019, 24, 845 Compound 9i also engaged in a π-cation interaction with R116 (Figure 3C), which explains why
modifications of the hydroxyl group led to decreased potency [39]. To validate the binding mode
revealed by the co-crystal structure, we tested the activity of PGAM1 mutants (Supplementary Data,
Figure S1) and the inhibition activity of compound 9i on different mutations of PGAM1. Compound
9i failed to inhibit mutations of PGAM1 (F22A, R116H and R191H) as effectively as the wild type at
concentration of 5 µM which agreed with the results from crystal structure. Furthermore, a substrate
competitive assay demonstrated that compound 9i held a non-competitive property with substrate 3PG
which was also consistent with the binding mode revealed by X-ray structure. The co-crystal structure
together with the molecular biological assays illustrated the binding mode of the anthraquinone
inhibitor with PGAM1 and provided useful information for further optimization. 9q
0.26 ± 0.03
1 The data are presen
9q
0.26 ± 0.03
1 The data are presented
9q
0.26 ± 0.03
1 The data are presented
9q
0.26 ± 0.03
1 The data are presented
9q
0.26 ± 0.03
1 The data are presented
2.3. Binding Mode of Compound 9i with PGAM1 Compound 9i also engaged in a π-cation interaction with R116 (Figure 3C), which explains why
modifications of the hydroxyl group led to decreased potency [39]. To validate the binding mode
revealed by the co-crystal structure, we tested the activity of PGAM1 mutants (Supplementary Data,
Figure S1) and the inhibition activity of compound 9i on different mutations of PGAM1. Compound
9i failed to inhibit mutations of PGAM1 (F22A, R116H and R191H) as effectively as the wild type at
concentration of 5 µM which agreed with the results from crystal structure. Furthermore, a substrate
competitive assay demonstrated that compound 9i held a non-competitive property with substrate 3PG
which was also consistent with the binding mode revealed by X-ray structure. The co-crystal structure
together with the molecular biological assays illustrated the binding mode of the anthraquinone
inhibitor with PGAM1 and provided useful information for further optimization. Table 5. Data collection and refinement statistics. Property
Value
Data Collection
Space group
P 21 21 21
Cell constants
a, b, c (Å)
82.54, 82.76, 104.20
α, β, γ (◦)
90.00, 90.00, 90.00
Resolution *(Å)
50–1.98 (2.05–1.98)
% Data completeness
100.0 (100.0)
No. Observations
330639
No. Unique Reflections
50593 (4986)
Redundancy
6.5 (6.6)
Rmerge
0.06 (0.88)
< I/σ(I) >
2.05 (at 1.98Å)
CC1/2
0.996 (0.728)
Refinement
Resolution (Å)
38.89–1.98 (2.02–1.98)
% Data completeness
100.0 (100.0)
No. reflections
50362 (2636)
Rwork/Rfree
0.197/0.225
Rfree test set
2569 reflections (5.08%)
Wilson B-factor (Å2)
39.4
Fo, Fc correlation
0.96
Total number of atoms
4046
Average B, all atoms (Å2)
41.0
Ramachandran favored (%)
98.7
Ramachandran allowed (%)
1.3
Ramachandran outliers (%)
0.0
Rmsd bond lengths (Å)
0.007
Rmsd bond angles (◦)
1.044
PDB Accession Code
6ISN Table 5. Data collection and refinement statistics. 9 of 20
hibitor Molecules 2019, 24, 845
with the molecular
with PGAM1 and p Figure 3. Binding mode of anthraquinone inhibitor 9i with PGAM1. (a) Chemical structure of
compound 9i and Fo–Fc electron density of compound 9i contoured at 2.0σ; (b) Overlay of
compound 9i (PBD: 6ISN) and 3PG (PBD:2F90) in PGAM1; (c) Interactions of compound 9i and the
critical residues of PGAM1 in the co-crystal structure; (d) Inhibition of compounds 9i on wild-type
and mutations of PGAM1 at concentration of 5 μM; (e) Noncompetitive property of compound 9i
with substrate 3PG. The data are presented as mean ±s.d. Figure 3. Binding mode of anthraquinone inhibitor 9i with PGAM1. 9q
0.26 ± 0.03
1 The data are presen
9q
0.26 ± 0.03
1 The data are presented
9q
0.26 ± 0.03
1 The data are presented
9q
0.26 ± 0.03
1 The data are presented
9q
0.26 ± 0.03
1 The data are presented
2.3. Binding Mode of Compound 9i with PGAM1 (a) Chemical structure of
compound 9i and Fo–Fc electron density of compound 9i contoured at 2.0σ; (b) Overlay of compound
9i (PBD: 6ISN) and 3PG (PBD:2F90) in PGAM1; (c) Interactions of compound 9i and the critical residues
of PGAM1 in the co-crystal structure; (d) Inhibition of compounds 9i on wild-type and mutations of
PGAM1 at concentration of 5 µM; (e) Noncompetitive property of compound 9i with substrate 3PG. The data are presented as mean ± s.d. Figure 3. Binding mode of anthraquinone inhibitor 9i with PGAM1. (a) Chemical structure of
compound 9i and Fo–Fc electron density of compound 9i contoured at 2.0σ; (b) Overlay of
compound 9i (PBD: 6ISN) and 3PG (PBD:2F90) in PGAM1; (c) Interactions of compound 9i and the
critical residues of PGAM1 in the co-crystal structure; (d) Inhibition of compounds 9i on wild-type
and mutations of PGAM1 at concentration of 5 μM; (e) Noncompetitive property of compound 9i
with substrate 3PG. The data are presented as mean ±s.d. Figure 3. Binding mode of anthraquinone inhibitor 9i with PGAM1. (a) Chemical structure of
compound 9i and Fo–Fc electron density of compound 9i contoured at 2.0σ; (b) Overlay of compound
9i (PBD: 6ISN) and 3PG (PBD:2F90) in PGAM1; (c) Interactions of compound 9i and the critical residues
of PGAM1 in the co-crystal structure; (d) Inhibition of compounds 9i on wild-type and mutations of
PGAM1 at concentration of 5 µM; (e) Noncompetitive property of compound 9i with substrate 3PG. The data are presented as mean ± s.d. Table 5 Data collection and refinement statistic
2.4. Inhibition Activity of Selected Compounds on Cancer Cell Proliferation Table 5 Data collection and refinement statistic
2.4. Inhibition Activity of Selected Compounds on Cancer Cell Proliferation Property
Value
Data Collection
Space group
P 21 21 21
Cell constants
a, b, c (Å)
82.54, 82.76, 104.20
α, β, γ (°)
90.00, 90.00, 90.00
Resolution *(Å)
50–1.98 (2.05–1.98)
% Data completeness
100.0 (100.0)
Given PGAM1 plays a crucial part in cancer metabolism, inhibition of PGAM1 by small molecules
is supposed to suppress cancer cell proliferation. We selected the compounds with IC50 values
below 10 µM toward PGAM1 to inhibit H1299 cells proliferation. Then the potent inhibitors with
IC50 values below 20 µM on H1299 cells were further tested on A549 and PC9 cells. In general,
proliferation inhibition of the compounds was correlated with PGAM1 inhibition and the inhibitors
performed similarly among different cancer cells, with IC50 values ranging from approximately
6–50 µM (Tables 6 and 7). Notably, compound 9i effectively suppressed the three different cancer cells
proliferation which was also potent towards PGAM1. 10 of 20 Molecules 2019, 24, 845
cells proliferation w
w. w. e o
e
e o
w. d we solved the co c
w. d we solved the co-c
w Table 6. Inhibition activity of compounds 9a–q on cancer cell proliferation. y
p
q
p
ibition activity of compounds 9a–q towards PGAM1. ibition activity of compounds 9a–q towards PGAM1. ibition activity of compounds 9a–q towards PGAM1. ibition activity of compounds 9a–q towards PGAM1. ibition activity of compounds 9a–q towards PGAM1. NHS
O
O
R3
OH
NHS
O
O
R3
OH
NHS
O
R3
OH
NHS
O
R3
OH
NHS
O
R3
OH Compound
R3
H1299 IC50 (μM)
A549 IC50 (μM)
PC9 IC50 (μM) 1
9a
20.7 ± 0.6
n.d. n.d. 9b
25.8 ± 2.2
n.d. n.d. 9c
16.6 ± 1.8
8.0 ± 0.8
10.2 ± 0.7
9d
13.9 ± 3.6
17.0 ± 4.9
44.2 ± 1.5
9e
26.3 ± 4.5
n.d. n.d. Compound
R3
H1299 IC50 (µM)
A549 IC50 (µM)
PC9 IC50 (µM) 1
9a
O
NHS
O
O
R3
OH
Compound
R 3
PGAM1 IC50 (μM) 1
9a
0.54 ± 0.04
9b
0.47 ± 0.08
9c
0.90 ± 0.07
9d
2.2 ± 0.5
9e
0.61 ± 0.12
20.7 ± 0.6
n.d. n.d. Table 5 Data collection and refinement statistic
2.4. Inhibition Activity of Selected Compounds on Cancer Cell Proliferation 9b
O
NHS
O
O
R3
OH
Compound
R 3
PGAM1 IC50 (μM) 1
9a
0.54 ± 0.04
9b
0.47 ± 0.08
9c
0.90 ± 0.07
9d
2.2 ± 0.5
9e
0.61 ± 0.12
25.8 ± 2.2
n.d. n.d. 9c
O
NHS
O
O
R3
OH
Compound
R 3
PGAM1 IC50 (μM) 1
9a
0.54 ± 0.04
9b
0.47 ± 0.08
9c
0.90 ± 0.07
9d
2.2 ± 0.5
9e
0.61 ± 0.12
16.6 ± 1.8
8.0 ± 0.8
10.2 ± 0.7
9d
O
NHS
O
O
R3
OH
Compound
R 3
PGAM1 IC50 (μM) 1
9a
0.54 ± 0.04
9b
0.47 ± 0.08
9c
0.90 ± 0.07
9d
2.2 ± 0.5
9e
0.61 ± 0.12
13.9 ± 3.6
17.0 ± 4.9
44.2 ± 1.5
9e
O
NHS
O
O
R3
OH
Compound
R 3
PGAM1 IC50 (μM) 1
9a
0.54 ± 0.04
9b
0.47 ± 0.08
9c
0.90 ± 0.07
9d
2.2 ± 0.5
9e
0.61 ± 0.12
26.3 ± 4.5
n.d. n.d. 9f
6 of 19
Table 2. Cont. 9f
0.54 ± 0.07
9g
0.79 ± 0.04
9h
0.89 ± 0.13
9i
0.27 ± 0.04
9j
0.28 ± 0.07
9k
0.89 ± 0.33
9l
0.90 ± 0.26
9m
0.26 ± 0.02
9n
0.20 ± 0.04
9o
0.35 ± 0.005
9p
0.47 ± 0.07
9q
0.26 ± 0.03
1 The data are presented as mean ± s.d. (n = 3). the sulfonamide to the C4 position of alizarin by substituting the hydroxyl,
24.2 ± 2.6
n.d. n.d. 9g
6 of 19
Table 2. Cont. 9f
0.54 ± 0.07
9g
0.79 ± 0.04
9h
0.89 ± 0.13
9i
0.27 ± 0.04
9j
0.28 ± 0.07
9k
0.89 ± 0.33
9l
0.90 ± 0.26
9m
0.26 ± 0.02
9n
0.20 ± 0.04
9o
0.35 ± 0.005
9p
0.47 ± 0.07
9q
0.26 ± 0.03
1 The data are presented as mean ± s.d. (n = 3). the sulfonamide to the C4 position of alizarin by substituting the hydroxyl,
31.3 ± 1.9
n.d. n.d. 9h
6 of 19
Table 2. Cont. 9f
0.54 ± 0.07
9g
0.79 ± 0.04
9h
0.89 ± 0.13
9i
0.27 ± 0.04
9j
0.28 ± 0.07
9k
0.89 ± 0.33
9l
0.90 ± 0.26
9m
0.26 ± 0.02
9n
0.20 ± 0.04
9o
0.35 ± 0.005
9p
0.47 ± 0.07
9q
0.26 ± 0.03
1 The data are presented as mean ± s.d. (n = 3). Table 5 Data collection and refinement statistic
2.4. Inhibition Activity of Selected Compounds on Cancer Cell Proliferation the sulfonamide to the C4 position of alizarin by substituting the hydroxyl,
45.7 ± 2.7
n.d. n.d. 9i
6 of 19
Table 2. Cont. 9f
0.54 ± 0.07
9g
0.79 ± 0.04
9h
0.89 ± 0.13
9i
0.27 ± 0.04
9j
0.28 ± 0.07
9k
0.89 ± 0.33
9l
0.90 ± 0.26
9m
0.26 ± 0.02
9n
0.20 ± 0.04
9o
0.35 ± 0.005
9p
0.47 ± 0.07
9q
0.26 ± 0.03
1 The data are presented as mean ± s.d. (n = 3). the sulfonamide to the C4 position of alizarin by substituting the hydroxyl,
6.9 ± 1.2
12.7 ± 2.7
13.8 ± 1.0
9j
6 of 19
Table 2. Cont. 9f
0.54 ± 0.07
9g
0.79 ± 0.04
9h
0.89 ± 0.13
9i
0.27 ± 0.04
9j
0.28 ± 0.07
9k
0.89 ± 0.33
9l
0.90 ± 0.26
9m
0.26 ± 0.02
9n
0.20 ± 0.04
9o
0.35 ± 0.005
9p
0.47 ± 0.07
9q
0.26 ± 0.03
1 The data are presented as mean ± s.d. (n = 3). the sulfonamide to the C4 position of alizarin by substituting the hydroxyl,
23.7 ± 1.1
n.d. n.d. 9k
6 of 19
Table 2. Cont. 9f
0.54 ± 0.07
9g
0.79 ± 0.04
9h
0.89 ± 0.13
9i
0.27 ± 0.04
9j
0.28 ± 0.07
9k
0.89 ± 0.33
9l
0.90 ± 0.26
9m
0.26 ± 0.02
9n
0.20 ± 0.04
9o
0.35 ± 0.005
9p
0.47 ± 0.07
9q
0.26 ± 0.03
1 The data are presented as mean ± s.d. (n = 3). the sulfonamide to the C4 position of alizarin by substituting the hydroxyl
34.6 ± 2.4
n.d. n.d. 9l
6 of 19
Table 2. Cont. 9f
0.54 ± 0.07
9g
0.79 ± 0.04
9h
0.89 ± 0.13
9i
0.27 ± 0.04
9j
0.28 ± 0.07
9k
0.89 ± 0.33
9l
0.90 ± 0.26
9m
0.26 ± 0.02
9n
0.20 ± 0.04
9o
0.35 ± 0.005
9p
0.47 ± 0.07
9q
0.26 ± 0.03
1 The data are presented as mean ± s.d. (n = 3). the sulfonamide to the C4 position of alizarin by substituting the hydroxyl
6.2 ± 1.2
>50
>50
9m
6 of 19
Table 2. Cont. Table 5 Data collection and refinement statistic
2.4. Inhibition Activity of Selected Compounds on Cancer Cell Proliferation 9f
0.54 ± 0.07
9g
0.79 ± 0.04
9h
0.89 ± 0.13
9i
0.27 ± 0.04
9j
0.28 ± 0.07
9k
0.89 ± 0.33
9l
0.90 ± 0.26
9m
0.26 ± 0.02
9n
0.20 ± 0.04
9o
0.35 ± 0.005
9p
0.47 ± 0.07
9q
0.26 ± 0.03
1 The data are presented as mean ± s.d. (n = 3). th
lf
id
t
th
C4
iti
f
li
i
b
b tit ti
th
h d
l
28.1 ± 1.9
n.d. n.d. 9n
6 of 19
Table 2. Cont. 9f
0.54 ± 0.07
9g
0.79 ± 0.04
9h
0.89 ± 0.13
9i
0.27 ± 0.04
9j
0.28 ± 0.07
9k
0.89 ± 0.33
9l
0.90 ± 0.26
9m
0.26 ± 0.02
9n
0.20 ± 0.04
9o
0.35 ± 0.005
9p
0.47 ± 0.07
9q
0.26 ± 0.03
1 The data are presented as mean ± s.d. (n = 3). 50 ± 0.6
n.d. n.d. 9o
6 of 19
Table 2. Cont. 9f
0.54 ± 0.07
9g
0.79 ± 0.04
9h
0.89 ± 0.13
9i
0.27 ± 0.04
9j
0.28 ± 0.07
9k
0.89 ± 0.33
9l
0.90 ± 0.26
9m
0.26 ± 0.02
9n
0.20 ± 0.04
9o
0.35 ± 0.005
9p
0.47 ± 0.07
9q
0.26 ± 0.03
1 The data are presented as mean ± s.d. (n = 3). 24.5 ± 0.2
n.d. n.d. 9p
6 of 19
Table 2. Cont. 9f
0.54 ± 0.07
9g
0.79 ± 0.04
9h
0.89 ± 0.13
9i
0.27 ± 0.04
9j
0.28 ± 0.07
9k
0.89 ± 0.33
9l
0.90 ± 0.26
9m
0.26 ± 0.02
9n
0.20 ± 0.04
9o
0.35 ± 0.005
9p
0.47 ± 0.07
9q
0.26 ± 0.03
1 The data are presented as mean ± s.d. (n = 3). 17.3 ± 3.9
10.3 ± 1.4
13.4 ± 1.8
9q
6 of 19
Table 2. Cont. 9f
0.54 ± 0.07
9g
0.79 ± 0.04
9h
0.89 ± 0.13
9i
0.27 ± 0.04
9j
0.28 ± 0.07
9k
0.89 ± 0.33
9l
0.90 ± 0.26
9m
0.26 ± 0.02
9n
0.20 ± 0.04
9o
0.35 ± 0.005
9p
0.47 ± 0.07
9q
0.26 ± 0.03
1 The data are presented as mean ± s.d. (n = 3). 26.4 ± 3.8
n.d. n.d. 1 The data are presented as mean ± s.d. (n = 3) n.d. = not determined. 0.90 ± 0.26
0.2 ± 1.7%
0.26 ± 0.02
−0.1 ± 2.5%
%
0.90 ± 0.26
0.2 ± 1.7%
0.26 ± 0.02
−0.1 ± 2.5%
3.2. Synthesis of diethyl 2,2′-((9,10-dioxo-9,10-dihydroanthracene-1,2-diyl) bis(oxy)) diacetate (6a) n
0.20 ± 0.04
6.1 ± 0.8%
o
0.35 ± 0.005
7.7 ± 0.2%
p
0.47 ± 0.07
1.9 ± 0.1%
q
0.26 ± 0.03
−2.3 ± 2.4%
1 The data are presented as mean ±s.d. (n = 2). Mode of Compound 9i with PGAM1
her understand the molecular mechanism of the anthraquinone derivatives interacting
1, we determined the X-ray structure of PGAM1 in complex with compound 9i at
1.98 Å (Table 5). Compound 9i occupied a novel allosteric site adjacent to substrate
with nice electron density (Figure 3A and Figure 3B). The allosteric pocket was
by the residues of F22, R90, K100, R116 and R191. In detail, the anthraquinone scaffold
mide of compound 9i interacted with the main chain carbonyl of K100 through water
n
0.20 ± 0.04
6.1 ± 0.8%
o
0.35 ± 0.005
7.7 ± 0.2%
p
0.47 ± 0.07
1.9 ± 0.1%
q
0.26 ± 0.03
−2.3 ± 2.4%
1 The data are presented as mean ±s.d. (n = 2). Mode of Compound 9i with PGAM1
her understand the molecular mechanism of the anthraquinone derivatives interacting
1, we determined the X-ray structure of PGAM1 in complex with compound 9i at
1.98 Å (Table 5). Compound 9i occupied a novel allosteric site adjacent to substrate
with nice electron density (Figure 3A and Figure 3B). The allosteric pocket was
by the residues of F22, R90, K100, R116 and R191. In detail, the anthraquinone scaffold
mide of compound 9i interacted with the main chain carbonyl of K100 through water
1,2-Dihydroxyanthracene-9,10-dione (1.2 g, 5 mmol), ethyl 2-bromoacetate (2 g, 12 mmol) and
K2CO3 (1.66 g, 12 mmol) were added to N,N-dimethylformamide (DMF, 50 mL) and the mixture was
stirred at 80 ◦C. The mixture was cooled to room temperature and poured into 10% aqueous HCl
(350 mL). Then the suspension was extracted with ethyl acetate and the organic phase was washed with
brine, dried over anhydrous Na2SO4 and filtered. The residue was purified by silica chromatography
after removal of ethyl acetate to afford 6a as a yellow solid (0.89 g, 43%). 1H-NMR (400 MHz, DMSO-d6)
δ 8.16–8.10 (m, 2H), 8.01 (d, J = 8.8 Hz, 1H), 7.93–7.84 (m, 2H), 7.53 (d, J = 8.8 Hz, 1H), 5.07 (s, 2H), 4.74
(s, 2H), 4.20 (qd, J = 4.0, 7.2 Hz, 4H), 1.23 (td, J = 2.4, 7.2 Hz, 6H). Table 5 Data collection and refinement statistic
2.4. Inhibition Activity of Selected Compounds on Cancer Cell Proliferation 1299 IC50 (μM)
A549 IC50 (μM)
PC9 IC50 (μM) 1
H1299 IC50 (µM)
A549 IC50 (µM)
PC9 IC50 (µM) 1
O
GAM1 IC50 (μM) 1
O
GAM1 IC50 (μM) 1
O
GAM1 IC50 (μM) 1
O
GAM1 IC (μM) 1
O
GAM1 IC ( M) 1 99999
9k 9999999 to the C4 position of alizarin by substituting the hydrox
to the C4 position of alizarin by substituting the hydrox
to the C4 position of alizarin by substituting the hydrox
to the C4 position of alizarin by substituting the hydrox
to the C4 position of alizarin by substituting the hydrox
to the C4 position of alizarin by substituting the hydrox
e to the C4 position of alizarin by substituting the hydrox
p
to the C4 position of alizarin by substituting the hydrox
p
(
)
t
th
C4
iti
f
li
i
b
b tit ti
th
h d
are presented as mean ± s.d. (n 3). are presented as mean ± s.d. (n = 3). are presented as mean ± s.d. (n = 3). 1 The data are presented as mean ± s.d. (n = 3) n.d. = not determined. 11 of 20 Molecules 2019, 24, 845
1 Table 7. Inhibition activity of compounds 14a–g on cancer cell proliferation. Table 7. Inhibition activity of compounds 14a g on cancer cell proliferation. Table 7. Inhibition activity of compounds 14a–g on cancer cell proliferation. Table 7. Inhibition activity of compounds 14a g on cancer cell proliferation. Table 7. Inhibition activity of compounds 14a–g on cancer cell proliferation. Table 7. Inhibition activity of compounds 14a g on cancer cell proliferation. PGAM1 IC50 (μM) 1
>10 μM
>10 μM
PGAM1 IC50 (μM) 1
>10 μM
>10 μM mpound
R5
R6
H1299 IC50 (μM)
A549 IC50 (μM)
PC9 IC50 (μM) 1
14c
CH3
H
26.9 ± 2.1
n.d. n.d. 14d
CH3
CH3
10.3 ± 0.8
9.9 ± 0.2
49.1 ± 1.9
14f
H
>100
n.d. n.d. 14g
H
28.5 ± 0.6
n.d. n.d. 1 The data are presented as mean ±s.d. (n = 3) n.d. = not determined. Compound
R5
R6
H1299 IC50 (µM)
A549 IC50 (µM)
PC9 IC50 (µM) 1
14c
CH3
H
26.9 ± 2.1
n.d. n.d. Table 5 Data collection and refinement statistic
2.4. Inhibition Activity of Selected Compounds on Cancer Cell Proliferation 14d
CH3
CH3
10.3 ± 0.8
9.9 ± 0.2
49.1 ± 1.9
14f
14c
CH3
H
2.92 ± 0.11
14d
CH3
CH3
7.01 ± 0.92
14e
-CH2-
-CH2-
>10 μM
14f
H
2.0 ± 0.2
14g
H
>10 μM
1 The data are presented as mean ±s.d. (n = 3). T bl 4 th
d di
l
d l
t
i hibiti
th th
d
t
H
>100
n.d. n.d. 14g
14c
CH3
H
2.92 ± 0.11
14d
CH3
CH3
7.01 ± 0.92
14e
-CH2-
-CH2-
>10 μM
14f
H
2.0 ± 0.2
14g
H
>10 μM
1 The data are presented as mean ±s.d. (n = 3). T bl 4 th
d di
l
d l
t
i hibiti
th th
d
t
H
28.5 ± 0.6
n.d. n.d. 1 The data are presented as mean ± s.d. (n = 3) n.d. = not determined. he data are presented as mean ±s.d. (n = 3) n.d. = not determined. a are presented as mean ±s.d. (n 3). a are presented as mean ±s.d. (n = 3). 1 The data are presented as mean ± s.d. (n = 3) n.d. = not determined. a
e
e i
i i io
i
e
ound
PGAM1 IC50 (μM)
I
Table 4. The inhibition in the
ound
PGAM1 IC50 (μM)
I
3.1. General Procedures (μ
)
y
y (
μ
)
a
0.54 ± 0.04
8.0 ± 5.0%
b
0.47 ± 0.08
−2.9 ± 0.3%
c
0.90 ± 0.07
3.7 ± 1.8%
e
0.61 ± 0.12
6.7 ± 0.2%
f
0.54 ± 0.07
5.7 ± 0.7%
g
0.79 ± 0.04
10.2 ± 3.0%
h
0.89 ± 0.13
0.06 ± 0.4%
0.27 ± 0.04
−6.4 ± 5.0%
0.28 ± 0.07
−2.0 ± 5.1%
k
0.89 ± 0.33
0.5 ± 3.2%
(μ
)
y
y (
μ
)
a
0.54 ± 0.04
8.0 ± 5.0%
b
0.47 ± 0.08
−2.9 ± 0.3%
c
0.90 ± 0.07
3.7 ± 1.8%
e
0.61 ± 0.12
6.7 ± 0.2%
f
0.54 ± 0.07
5.7 ± 0.7%
g
0.79 ± 0.04
10.2 ± 3.0%
h
0.89 ± 0.13
0.06 ± 0.4%
0.27 ± 0.04
−6.4 ± 5.0%
0.28 ± 0.07
−2.0 ± 5.1%
k
0.89 ± 0.33
0.5 ± 3.2%
All reagents were purchased commercially. 1H-NMR and 13C-NMR spectra were recorded
on Bruker AC400 and Bruker AC600 NMR spectrometers, respectively (Billerica, MA, USA). Low-resolution mass spectra were recorded on a 6120 Quadrupole mass spectrometer (Agilent, Santa
Clara, CA, USA) equipped with electrospray ionization (ESI). High-resolution mass spectra were
determined on triple TOF 5600+ MS/MS system (AB Sciex, Concord, ON, Canada) in negative ESI
mode. The purity of target compounds was determined by high-performance liquid chromatography
(Agilent, Santa Clara, CA, USA, DIKMA Diamonsil Plus C18, 250 × 4.6 mm, 5 µm, 25 ◦C, UV 290 nM). All the biologically tested compounds achieved ≥95% purity. l
0.90 ± 0.26
0.2 ± 1.7%
m
0.26 ± 0.02
−0.1 ± 2.5%
0 20
0 04
6 1
0 8%
l
0.90 ± 0.26
0.2 ± 1.7%
m
0.26 ± 0.02
−0.1 ± 2.5%
%
3.2. Synthesis of diethyl 2,2′-((9,10-dioxo-9,10-dihydroanthracene-1,2-diyl) bis(oxy)) diacetate (6a) l
0.90 ± 0.26
0.2 ± 1.7%
m
0.26 ± 0.02
−0.1 ± 2.5%
%
l
0.90 ± 0.26
0.2 ± 1.7%
m
0.26 ± 0.02
−0.1 ± 2.5%
3.2. Synthesis of diethyl 2,2′-((9,10-dioxo-9,10-dihydroanthracene-1,2-diyl) bis(oxy)) diacetate (6a) 0.90 ± 0.26
0.2 ± 1.7%
0.26 ± 0.02
−0.1 ± 2.5%
%
0.90 ± 0.26
0.2 ± 1.7%
0.26 ± 0.02
−0.1 ± 2.5%
3.2. Synthesis of diethyl 2,2′-((9,10-dioxo-9,10-dihydroanthracene-1,2-diyl) bis(oxy)) diacetate (6a) 13C-NMR (151 MHz, DMSO) δ 181.69,
181.42, 168.27, 167.97, 156.49, 146.23, 134.58, 134.32, 133.94, 132.26, 127.15, 126.94, 126.67, 126.18, 124.64,
118.20, 68.72, 65.22, 60.99, 60.44, 14.07, 13.98. MS (ESI) (m/z): 413.1 (M −H)−. HRMS (ESI) calcd. for
C22H20O8 [M −H]−: 413.1231; found: 413.1239. Purity 95.1% by HPLC. ure 3C). In addition, a hydrophobic interaction was observed between F22 and
stituted phenyl ring of compound 9i (Figure 3C). Compound 9i also engaged in a
ure 3C). In addition, a hydrophobic interaction was observed between F22 and
stituted phenyl ring of compound 9i (Figure 3C). Compound 9i also engaged in a
3.3. Synthesis of 2,2′-((9,10-dioxo-9,10-dihydroanthracene-1,2-diyl) bis(oxy)) diacetic acid (6b) 3.5. General Procedure for the Synthesis of Compounds 9a–q 3.5. General Procedure for the Synthesis of Compounds 9a–q Step 1. Nitric acid (1.5 mL, 33.33 mmol, Sinopharm Chemical Reagent Co., Ltd, Shanghai, China)
was added to the suspension of alizarin (5 g, 20.8 mmol) in acetic acid (350 mL) at 50 ◦C. A yellow
solid (4.15 g, 70%) of crude product 6 was obtained by filtration [40]. Step 2. Sn (10.5 g, 341 mmol, Sinopharm Chemical Reagent Co., Ltd, Shanghai, China),
SnCl2·2H2O (12.5 g, 55.4 mmol) and concentrated HCl (50.4 mL, 604.8 mmol) were added to the
suspension of crude product 6 (1.75 g, 6.14 mmol) in ethanol (350 mL, Sinopharm) and the mixture was
stirred at room temperature overnight. Then the mixture was poured into water (1 L) and a red solid
precipitated. A black solid of crude product 7 (1.41 g, 90%) was obtained after filtration and being
dried under vacuum [40]. Step 3. To the solution of crude product 7 (255 mg, 1 mmol) in dry pyridine (5 mL, Sinopharm),
corresponding sulfonyl chloride (1.5 mmol, Energy Chemical, Shanghai, China) was added and the
mixture was stirred at room temperature. Then it was added to 10% aqueous HCl (50 mL, Sinopharm)
and the suspension was extracted with ethyl acetate (Sinopharm). The organic phase was washed with
brine, dried over anhydrous Na2SO4 and filtered. The residue was purified by silica chromatography
after removal of ethyl acetate to give compounds 9a–q. N-(3,4-Dihydroxy-9,10-dioxo-9,10-dihydroanthracen-2-yl)-4-(trifluoromethyl) benzenesulfonamide (9a). Yellow solid, 25% yield. 1H-NMR (400 MHz, DMSO-d6) δ 12.60 (brs, 1H), 10.90 (brs, 1H), 10.67 (brs,
1H), 8.22–8.12 (m, 2H), 8.09 (d, J = 8.4 Hz, 2H), 8.01 (d, J = 8.4 Hz, 2H), 7.95–7.86 (m, 2H), 7.73 (s, 1H). 13C-NMR (151 MHz, DMSO) δ 187.78, 180.56, 150.37, 144.20, 143.23, 135.02, 134.22, 133.28, 132.79,
132.68(q, J = 31.7Hz), 130.34, 127.60 (2C), 126.77, 126.61, 126.59, 126.39, 123.71, 123.38(q, J = 273.3 Hz),
113.49, 113.35. MS (ESI) (m/z): 462.0 (M −H)−. HRMS (ESI) calcd. for C21H12F3NO6S [M −H]−:
462.0265; found: 462.0284. Purity 99.8% by HPLC. N-(3,4-Dihydroxy-9,10-dioxo-9,10-dihydroanthracen-2-yl)-4-nitrobenzenesulfonamide (9b). Orange
solid, 50% yield. 1H-NMR (400 MHz, DMSO-d6) δ 12.61 (brs, 1H), 10.98 (brs, 1H), 10.79 (brs, 1H), 8.41
(d, J = 8.0 Hz, 2H), 8.25–8.06 (m, 4H), 7.97–7.86 (m, 2H), 7.73 (s, 1H). 13C-NMR (151 MHz, DMSO) δ
187.79, 180.53, 150.41, 149.90, 145.71, 143.61, 135.05, 134.24, 133.27, 132.79, 130.11, 128.22(2C), 126.77,
126.41, 124.65(2C), 123.70, 114.04, 113.50. MS (ESI) (m/z): 439.0 (M −H)−. HRMS (ESI) calcd. C). In addition, a hydrophobic interaction was observed between F22 and
d phenyl ring of compound 9i (Figure 3C). Compound 9i also engaged in a
C). In addition, a hydrophobic interaction was observed between F22 and
d phenyl ring of compound 9i (Figure 3C). Compound 9i also engaged in a
Synthesis of 2,2′-((9,10-dioxo-9,10-dihydroanthracene-1,2-diyl) bis(oxy)) diacetic acid (6b) Compound 6a (206 mg, 0.5 mmol) and 0.5M aqueous LiOH (8 mL) were added into methanol
(8 mL) and the mixture was stirred at 30 ◦C. The mixture was added dropwise to 10% aqueous HCl
and acidified to pH 1–2. A yellow solid (169 mg, 95%) of 6b was obtained by filtration. 1H-NMR
(400 MHz, DMSO-d6) δ 12.76 (brs, 2H), 8.14 (d, J = 7.6 Hz, 2H), 8.01 (d, J = 8.4 Hz, 1H), 7.93–7.85 (m,
2H), 7.51 (d, J = 8.8 Hz, 1H), 4.98 (s, 2H), 4.67 (s, 2H). 13C-NMR (151 MHz, DMSO) δ 181.95, 181.45,
169.75, 169.45, 156.76, 146.32, 134.62, 134.31, 133.98, 132.34, 126.97, 126.82, 126.71, 126.20, 124.61, 118.12, Molecules 2019, 24, 845
12 of 20
68.62, 65.05. MS (ESI) (m/z): 355.0 (M −H)−. HRMS (ESI) calcd. for C18H12O8 [M −H]−: 355.0459;
found: 355 0462 Purity 95 1% by HPLC 12 of 20 Molecules 2019, 24, 845 68.62, 65.05. MS (ESI) (m/z): 355.0 (M −H)−. HRMS (ESI) calcd. for C18H12O8 [M −H]−: 355.0459;
found: 355.0462. Purity 95.1% by HPLC. 3.4. Synthesis of 2-((1-hydroxy-9,10-dioxo-9,10-dihydroanthracen-2-yl) oxy)-N,N-dimethylacetamide (6e) Compound 6d (149 mg, 0.5 mmol), dimethylamine hydrochloride (31 mg, 0.68 mmol),
N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDCI, 120 mg,
0.6 mmol),
1-hydroxybenzotriazole (HOBt, 80 mg, 0.6 mmol) and diisopropylethylamine (DIPEA, 175 µL, 1 mmol)
were added to dry DMF (10 mL) and the mixture was stirred at room temperature. The mixture was
added dropwise to H2O (140 mL). An orange solid (114 mg, 70%) of 6e was obtained by filtration. 1H-NMR (400 MHz, DMSO-d6) δ 12.74 (s, 1H), 8.28–8.11 (m, 2H), 7.99–7.87 (m, 2H), 7.68 (d, J = 8.4 Hz,
1H), 7.31 (d, J = 8.8 Hz, 1H), 5.09 (s, 2H), 3.02 (s, 3H), 2.87 (s, 3H). 13C-NMR (151 MHz, DMSO) δ 188.67,
180.76, 166.18, 152.60, 151.79, 135.20, 134.26, 133.48, 132.95, 126.81, 126.60, 124.89, 120.20, 118.29, 115.94,
66.12, 35.46, 35.01. MS (ESI) (m/z): 324.0 (M −H)−. HRMS (ESI) calcd. for C18H15NO5 [M −H]−:
324.0877; found: 324.0876. Purity 96.4% by HPLC. 3.5. General Procedure for the Synthesis of Compounds 9a–q for
C20H12N2O8S [M −H]−: 439.0242; found: 439.0228. Purity 98.4% by HPLC. y
y
N-(3,4-Dihydroxy-9,10-dioxo-9,10-dihydroanthracen-2-yl)-4-(trifluoromethoxy) benzenesulfonamide (9c). Yellow solid, 41% yield. 1H-NMR (400 MHz, DMSO-d6) δ 12.61 (brs, 1H), 10.94 (brs, 1H), 10.51 (brs,
1H), 8.22–8.12 (m, 2H), 8.05–7.99 (m, 2H), 7.95–7.85 (m, 2H), 7.74 (s, 1H), 7.61 (dd, J = 1.2, 9.2 Hz, 2H). 13C-NMR (151 MHz, DMSO) δ 187.78, 180.60, 151.20, 150.35, 142.96, 139.19, 135.02, 134.23, 133.30,
132.82, 130.63, 129.29, 126.78, 126.40, 123.74, 121.47,119.80 (q, J = 259.7 Hz), 113.22, 113.12. MS (ESI) Molecules 2019, 24, 845 13 of 20 13 of 20 (m/z): 478.0 (M −H)−. HRMS (ESI) calcd. for C21H12F3NO7S [M −H]−: 478.0214; found: 478.0207. Purity 96.8% by HPLC. (m/z): 478.0 (M −H)−. HRMS (ESI) calcd. for C21H12F3NO7S [M −H]−: 478.0214; found: 478.0207. Purity 96.8% by HPLC. N-(3,4-Dihydroxy-9,10-dioxo-9,10-dihydroanthracen-2-yl)-2-(trifluoromethoxy) benzenesulfonamide (9d). Yellow solid, 40% yield. 1H-NMR (400 MHz, DMSO-d6) δ 12.62 (brs, 1H), 11.00 (brs, 1H), 10.37 (brs,
1H), 8.24–8.09 (m, 2H), 8.00 (dd, J = 2.0, 8.0 Hz, 1H), 7.96–7.86 (m, 2H), 7.83–7.75 (m, 1H), 7.71 (s,
1H), 7.63–7.51 (m, 2H). 13C-NMR (151 MHz, DMSO) δ 187.81, 180.54, 150.37, 145.18, 143.20, 135.55,
135.05, 134.25, 133.28, 132.82, 132.34, 130.65, 130.41, 127.45, 126.80, 126.43, 123.61, 120.97, 119.81 (q,
J = 259.7 Hz), 113.72, 113.26. MS (ESI) (m/z): 477.8 (M −H)−. HRMS (ESI) calcd. for C21H12F3NO7S
[M −H]−: 478.0214; found: 478.0225. Purity 95.2% by HPLC. y
y
4-Cyano-N-(3,4-dihydroxy-9,10-dioxo-9,10-dihydroanthracen-2-yl) benzenesulfonamide (9e). Red solid,
60% yield. 1H-NMR (400 MHz, DMSO-d6) δ 12.60 (brs, 1H), 10.77 (brs, 2H), 8.22–8.12 (m, 2H), 8.09 (d,
J = 8.0 Hz, 2H), 8.02 (d, J = 8.4 Hz, 2H), 7.96–7.87 (m, 2H), 7.70 (s, 1H). 13C-NMR (151 MHz, DMSO) δ
187.81, 180.57, 150.39, 144.34, 143.46, 135.05, 134.25, 133.49, 133.29, 132.81, 130.22, 127.34, 126.81, 126.75,
126.44, 123.71, 117.60, 115.48, 113.78(2C), 113.44. MS (ESI) (m/z): 419.0 (M −H)−. HRMS (ESI) calcd. for C21H12N2O6S [M −H]−: 419.0343; found: 419.0348. Purity 95.1% by HPLC. y
y
N-(3,4-Dihydroxy-9,10-dioxo-9,10-dihydroanthracen-2-yl)-4-fluorobenzenesulfonamide (9f). Yellow
solid, 62% yield. 1H-NMR (400 MHz, DMSO-d6) δ 12.58 (brs, 1H), 10.84 (brs, 1H), 10.43 (brs, 1H),
8.19–8.11 (m, 2H), 8.01–7.84 (m, 4H), 7.73 (s, 1H), 7.46 (t, J = 8.8 Hz, 2H). 13C-NMR (151 MHz, DMSO)
δ 187.75, 180.59, 164.47 (d, J = 250.7 Hz), 150.32, 142.76, 136.58, 134.99, 134.21, 133.28, 132.81, 130.82,
129.75 (d, J = 9.1 Hz,2C), 126.76, 126.38, 123.72, 116.57 (d, J = 22.7 Hz, 2C), 113.10, 112.89. MS (ESI)
(m/z): 412.0 (M −H)−. HRMS (ESI) calcd. 3.5. General Procedure for the Synthesis of Compounds 9a–q for C20H12FNO6S [M −H]−: 412.0297; found: 412.0305. Purity 98.7% by HPLC. N-(3,4-Dihydroxy-9,10-dioxo-9,10-dihydroanthracen-2-yl)-2-fluorobenzenesulfonamide (9g). Yellow
solid, 42% yield. 1H-NMR (400 MHz, DMSO-d6) δ 12.61 (brs, 1H), 10.95 (brs, 1H), 10.49 (brs, 1H),
8.21–8.09 (m, 2H), 7.95–7.80 (m, 3H), 7.77–7.67 (m, 2H), 7.46 (t, J = 9.4 Hz, 1H), 7.38 (t, J = 7.6 Hz, 1H). 13C-NMR (151 MHz, DMSO) δ 187.80, 180.56, 158.34 (d, J = 256.7Hz), 150.40, 143.42, 136.02, 135.01,
134.21, 133.28, 132.81, 130.39, 129.87, 127.94 (d, J = 12.1 Hz), 126.77, 126.41, 124.90, 123.57, 117.44 (d,
J = 21.1 Hz), 113.88, 113.32. MS (ESI) (m/z): 412.0 (M −H)−. HRMS (ESI) calcd. for C20H12FNO6S
[M −H]−: 412.0297; found: 412.0300. Purity 96.1% by HPLC. N-(3,4-Dihydroxy-9,10-dioxo-9,10-dihydroanthracen-2-yl)-2,3-difluorobenzenesulfonamide (9h). Red
solid, 52% yield. 1H-NMR (400 MHz, DMSO-d6) δ 12.60 (brs, 1H), 10.83 (brs, 2H), 8.23–8.11 (m, 2H),
7.96–7.86 (m, 2H), 7.79 (q, J = 8.6 Hz, 1H), 7.69 (s, 1H), 7.62 (t, J = 7.0 Hz, 1H), 7.44–7.34 (m, 1H). 13C-NMR (151 MHz, DMSO) δ 187.86, 180.52, 150.46, 149.96 (dd, J = 249.9 Hz, J=12.1 Hz), 146.64 (dd,
J = 256.7 Hz, J = 13.6 Hz) 144.45, 135.05, 134.22, 133.29, 132.81, 130.25 (d, J = 10.6 Hz), 129.75, 126.78 (d,
J = 10.6 Hz), 126.43 d, J = 7.6 Hz), 125.26, 124.95, 123.51, 122.86 (d, J = 16.6 Hz), 115.28 (d, J = 6.0 Hz),
113.72. MS (ESI) (m/z): 430.0 (M −H)−. HRMS (ESI) calcd. for C20H11FNO6S [M −H]−: 430.0202;
found: 430.0206. Purity 95.4% by HPLC. 4-Chloro-N-(3,4-dihydroxy-9,10-dioxo-9,10-dihydroanthracen-2-yl) benzenesulfonamide (9i). Yellow
solid, 56% yield. 1H-NMR (400 MHz, DMSO-d6) δ 12.61 (brs, 1H), 10.94 (brs, 1H), 10.47 (brs, 1H),
8.22–8.11 (m, 2H), 7.96–7.83 (m, 4H), 7.78–7.63 (m, 3H). 13C-NMR (151 MHz, DMSO) δ 187.76, 180.58,
150.34, 142.90, 139.07, 138.04, 135.00, 134.22, 133.28, 132.80, 130.63, 129.50(2C), 128.57(2C), 126.77,
126.39, 123.72, 113.18, 113.08. MS (ESI) (m/z): 427.8 (M −H)−. HRMS (ESI) calcd. for C20H12ClNO6S
[M −H]−: 428.0001; found: 428.0005. Purity 99.4% by HPLC. 2,4-Dichloro-N-(3,4-dihydroxy-9,10-dioxo-9,10-dihydroanthracen-2-yl) benzenesulfonamide (9j). Orange
solid, 60% yield. 1H-NMR (400 MHz, DMSO-d6) δ 12.62 (brs, 1H), 11.00 (brs, 1H), 10.56 (brs, 1H),
8.20–8.08 (m, 2H), 8.01 (d, J = 8.8 Hz, 1H), 7.95–7.84 (m, 3H), 7.68–7.63 (m, 2H). 13C-NMR (151 MHz,
DMSO) δ 187.76, 180.50, 150.44, 143.33, 138.62, 136.55, 134.99, 134.20, 133.24, 132.79, 132.25, 132.12,
131.49, 130.24, 127.85, 126.75, 126.38, 123.63, 113.52, 113.36. MS (ESI) (m/z): 461.8 (M −H)−. HRMS
(ESI) calcd. for C20H11Cl2NO6S [M −H]−: 461.9611; found: 461.9614. Purity 99.2% by HPLC. 3.5. General Procedure for the Synthesis of Compounds 9a–q Molecules 2019, 24, 845 14 of 20 14 of 20 2,6-Dichloro-N-(3,4-dihydroxy-9,10-dioxo-9,10-dihydroanthracen-2-yl) benzenesulfonamide (9k). Red
solid, 12% yield. 1H-NMR (400 MHz, DMSO-d6) δ 12.62 (brs, 1H), 11.00 (brs, 1H), 10.56 (brs, 1H),
8.20–8.09 (m, 2H), 7.94–7.83 (m, 2H), 7.72–7.52 (m, 4H). 13C-NMR (151 MHz, DMSO) δ 187.71, 180.43,
150.40, 142.57, 134.95, 134.80, 134.29, 134.17, 134.13 (2C), 133.22, 132.78, 131.85(2C), 130.41, 126.75,
126.36, 123.79, 113.20, 112.18. MS (ESI) (m/z): 461.8 (M −H)−. HRMS (ESI) calcd. for C20H11Cl2NO6S
[M −H]−: 461.9611; found: 461.9601. Purity 95.7% by HPLC. y
y
2,4,6-Trichloro-N-(3,4-dihydroxy-9,10-dioxo-9,10-dihydroanthracen-2-yl)
benzenesulfonamide
(9l). Yellow solid, 10% yield. 1H-NMR (400 MHz, DMSO-d6) δ 12.62 (brs, 1H), 10.96 (brs, 1H), 10.73 (brs,
1H), 8.21–8.11 (m, 2H), 7.96–7.86 (m, 4H), 7.66 (s, 1H). 13C-NMR (151 MHz, DMSO) δ 187.77, 180.49,
150.48, 143.28, 137.64, 135.25 (2C), 135.00, 134.43, 134.22, 133.27, 132.83, 131.20 (3C), 126.79, 126.41,
123.78, 113.43, 113.21. MS (ESI) (m/z): 495.8 (M −H)−. HRMS (ESI) calcd. for C20H10Cl3NO6S
[M −H]−: 495.9222; found: 495.9220. Purity 99.4% by HPLC. y
y
4-Bromo-N-(3,4-dihydroxy-9,10-dioxo-9,10-dihydroanthracen-2-yl) benzenesulfonamide (9m). Yellow
solid, 65% yield. 1H-NMR (400 MHz, DMSO-d6) δ 12.60 (brs, 1H), 10.88 (brs, 1H), 10.47 (brs, 1H),
8.22–8.11 (m, 2H), 7.95–7.78 (m, 6H), 7.73 (s, 1H). 13C-NMR (151 MHz, DMSO) δ 187.77, 180.59, 150.33,
142.88, 139.50, 135.01, 134.23, 133.29, 132.81, 132.44 (2C), 130.65, 128.64 (2C), 127.08, 126.78, 126.39,
123.73, 113.18, 113.05. MS (ESI) (m/z): 471.8 (M −H)−. HRMS (ESI) calcd. for C20H12BrNO6S
[M −H]−: 471.9496; found: 471.9495. Purity 96.0% by HPLC. 2-Bromo-N-(3,4-dihydroxy-9,10-dioxo-9,10-dihydroanthracen-2-yl) benzenesulfonamide (9n). Yellow
solid, 46% yield. 1H-NMR (400 MHz, DMSO-d6) δ 12.63 (brs, 1H), 11.10 (brs, 1H), 10.24 (brs, 1H),
8.20–8.05 (m, 3H), 7.93–7.84 (m, 3H), 7.67–7.53 (m, 3H). 13C-NMR (151 MHz, DMSO) δ 187.72, 180.55,
150.37, 142.31, 138.96, 135.62, 134.97, 134.74, 134.21, 133.23, 132.80, 130.93, 130.76, 128.28, 126.75, 126.38,
123.75, 119.31, 113.07, 112.14. MS (ESI) (m/z): 471.8 (M −H)−. HRMS (ESI) calcd. for C20H12BrNO6S
[M −H]−: 417.9496; found: 417.9498. Purity 97.5% by HPLC. 3-Bromo-N-(3,4-dihydroxy-9,10-dioxo-9,10-dihydroanthracen-2-yl) benzenesulfonamide (9o). Yellow
solid, 54% yield. 1H-NMR (400 MHz, DMSO-d6) δ 12.60 (brs, 1H), 10.99 (brs, 1H), 10.53 (brs, 1H),
8.21–8.11 (m, 2H), 8.10 (s, 1H), 7.94–7.83 (m, 4H), 7.71 (s, 1H), 7.57 (t, J = 8.0 Hz, 1H). 13C-NMR (151 MHz,
DMSO) δ 187.75, 180.55, 150.33, 142.90, 142.23, 136.02, 134.98, 134.19, 133.27, 132.79, 131.56, 130.49,
129.08, 126.76, 126.37, 125.61, 123.71, 122.16, 113.22, 112.97. MS (ESI) (m/z): 473.8 (M −H)−.HRMS
(ESI) calcd. for C20H12BrNO6S [M −H]−: 417.9496; found: 417.9500. Purity 96.1% by HPLC. 3.5. General Procedure for the Synthesis of Compounds 9a–q (
)
y
y
4-Iodo-N-(3,4-dihydroxy-9,10-dioxo-9,10-dihydroanthracen-2-yl) benzenesulfonamide (9p). Yellow solid,
70% yield. 1H-NMR (400 MHz, DMSO-d6) δ 12.60 (brs, 1H), 10.87 (brs, 1H), 10.44 (brs, 1H), 8.23–8.10
(m, 2H), 7.99 (d, J = 8.0 Hz, 2H), 7.96–7.85 (m, 2H), 7.73 (s, 1H), 7.64 (d, J = 8.0 Hz, 2H). 13C-NMR
(151 MHz, DMSO) δ 187.75, 180.61, 150.31, 142.73, 139.85, 138.23 (2C), 135.00, 134.22, 133.29, 132.82,
130.76, 128.27 (2C), 126.78, 126.39, 123.74, 113.12, 112.85, 101.56. MS (ESI) (m/z): 519.8 (M −H)−. HRMS (ESI) calcd. for C20H12INO6S [M −H]−: 519.9357; found: 519.9334. Purity 99.3% by HPLC. 2-Iodo-N-(3,4-dihydroxy-9,10-dioxo-9,10-dihydroanthracen-2-yl) benzenesulfonamide (9q). Yellow solid,
48% yield.1H-NMR (400 MHz, DMSO-d6) δ 12.64 (brs, 1H), 11.18 (brs, 1H), 10.02 (brs, 1H), 8.19–8.06 (m,
4H), 7.93–7.85 (m, 2H), 7.65 (s, 1H), 7.61 (td, J = 1.2, 8.0 Hz, 1H), 7.33 (td, J = 1.6, 8.0 Hz, 1H). 13C-NMR
(151 MHz, DMSO) δ 187.72, 180.58, 150.30, 142.77, 142.13, 141.83, 134.97, 134.22, 134.21, 133.24, 132.81,
131.01, 130.16, 128.64, 126.76, 126.38, 123.83, 112.96, 111.59, 93.30. MS (ESI) (m/z): 519.8 (M −H)−. HRMS (ESI) calcd. for C20H12INO6S [M −H]−: 519.9357; found: 519.9356. Purity 96.3% by HPLC. 4-Iodo-N-(3,4-dihydroxy-9,10-dioxo-9,10-dihydroanthracen-2-yl) benzenesulfonamide (9p). Yellow solid,
70% yield. 1H-NMR (400 MHz, DMSO-d6) δ 12.60 (brs, 1H), 10.87 (brs, 1H), 10.44 (brs, 1H), 8.23–8.10
(m, 2H), 7.99 (d, J = 8.0 Hz, 2H), 7.96–7.85 (m, 2H), 7.73 (s, 1H), 7.64 (d, J = 8.0 Hz, 2H). 13C-NMR
(151 MHz, DMSO) δ 187.75, 180.61, 150.31, 142.73, 139.85, 138.23 (2C), 135.00, 134.22, 133.29, 132.82,
130.76, 128.27 (2C), 126.78, 126.39, 123.74, 113.12, 112.85, 101.56. MS (ESI) (m/z): 519.8 (M −H)−. HRMS (ESI) calcd. for C20H12INO6S [M −H]−: 519.9357; found: 519.9334. Purity 99.3% by HPLC. 3.6. Synthesis of ethyl 2-((4-((4-chlorophenyl) sulfonamido)-1-hydroxy-9,10-dioxo-9,10-dihydroanthracen-2-yl)
oxy) acetate (12) 3.6. Synthesis of ethyl 2-((4-((4-chlorophenyl) sulfonamido)-1-hydroxy-9,10-dioxo-9,10-dihydroanthracen-2-yl)
oxy) acetate (12) 3.6. Synthesis of ethyl 2-((4-((4-chlorophenyl) sulfonamido)-1-hydroxy-9,10-dioxo-9,10-dihydroanthracen-2-yl)
oxy) acetate (12) Step 1. Compound 10 was synthesized according to the same route of compound 7 in which
compound 5 was replaced by compound 6c. Yellow solid, 73% yield. Step 1. Compound 10 was synthesized according to the same route of compound 7 in which
compound 5 was replaced by compound 6c. Yellow solid, 73% yield. Step 2. Compound 11 was synthesized according to the same route of compound 8 in which
compound 7 was replaced by compound 10. Purple solid, 93% yield. Step 2. Step 2. Compound 11 was synthesized according to the same route of compound 8 in which
compound 7 was replaced by compound 10. Purple solid, 93% yield. Step 1. Compound 10 was synthesized according to the same route of compound 7 in which
compound 5 was replaced by compound 6c. Yellow solid, 73% yield. 3.5. General Procedure for the Synthesis of Compounds 9a–q for C22H14ClNO8S [M −H]−: 486.0056; found: 486.0061. Purity 98.0% by HPLC. 3.8. Synthesis of ethyl 2-((3-((4-chlorophenyl) sulfonamido)-1-hydroxy-9,10-dioxo-9,10-dihydroanthracen-2-yl)
oxy) acetate (14a) 3.8. Synthesis of ethyl 2-((3-((4-chlorophenyl) sulfonamido)-1-hydroxy-9,10-dioxo-9,10-dihydroanthracen-2-yl)
oxy) acetate (14a) Compound 14a was synthesized according to the same route of 6c in which compound 5 was
replaced by compound 9i. Yellow solid, 24% yield. 1H-NMR (400 MHz, DMSO-d6) δ 12.74 (s, 1H),
10.59 (s, 1H), 8.22–8.09 (m, 2H), 8.01–7.87 (m, 4H), 7.83–7.68 (m, 3H), 4.85 (s, 2H), 4.19 (q, J = 7.2 Hz,
2H), 1.22 (t, J = 7.2 Hz, 3H). 13C-NMR (151 MHz, DMSO) δ 187.53, 180.83, 169.46, 154.03, 139.72, 138.49,
138.21, 136.81, 135.08, 134.58, 132.87, 132.72, 129.71(2C), 128.70(2C), 128.46, 126.88, 126.45, 113.29, 109.22,
68.53, 60.98, 13.98. MS (ESI) (m/z): 514.0 (M −H)−. HRMS (ESI) calcd. for C24H18ClNO8S [M −H]−:
514.0369; found: 514.0363. Purity 95.6% by HPLC. 3.9. Synthesis of 2-((3-((4-chlorophenyl) sulfonamido)-1-hydroxy-9,10-dioxo-9,10-dihydroanthracen-2-yl) oxy)
acetic acid (14b) 3.9. Synthesis of 2-((3-((4-chlorophenyl) sulfonamido)-1-hydroxy-9,10-dioxo-9,10-dihydroanthracen-2-yl) oxy)
acetic acid (14b) .9. Synthesis of 2-((3-((4-chlorophenyl) sulfonamido)-1-hydroxy-9,10-dioxo-9,10-dihydroanthracen-2-yl)
cetic acid (14b) Compound 14b was synthesized according to the same route of 6b in which compound 6a was
replaced by compound 14a. Yellow solid, 95% yield. 1H-NMR (400 MHz, DMSO-d6) δ 12.72 (s, 1H),
8.19–8.10 (m, 2H), 7.97–7.87 (m, 4H), 7.75 (s, 1H), 7.71 (d, J = 8.4 Hz, 2H), 4.80 (s, 2H). 13C-NMR
(151 MHz, DMSO) δ 187.49, 180.86, 171.92, 154.23, 139.93, 138.53, 138.14, 137.00, 135.06, 134.59, 132.86,
132.74, 129.76 (2C), 128.67 (2C), 128.58, 126.89, 126.45, 113.28, 108.86, 68.92. MS (ESI) (m/z): 486.0
(M −H)−. HRMS (ESI) calcd. for C22H14ClNO8S [M −H]−: 486.0056; found: 486.0049. Purity 99.0%
by HPLC. 3.10. Synthesis of 4-chloro-N-(4-hydroxy-3-methoxy-9,10-dioxo-9,10-dihydroanthracen-2-yl) benzene-
sulfonamide (14c) Compound 9i (215 mg, 0.5 mmol), K2CO3 (70 mg, 0.5 mmol) and iodomethane (70 mg, 12 mmol)
were added to DMF (2.5 mL) and mixture was stirred at room temperature. The mixture was added to
10% aqueous HCl (20 mL). The suspension was extracted with ethyl acetate and the organic phase
was washed with brine, dried over anhydrous Na2SO4 and filtered. The residue was purified by silica
chromatography after removal of ethyl acetate to afford 14c as a yellow solid (115 mg, 52%). 1H-NMR
(400 MHz, DMSO-d6) δ 12.68 (brs, 1H), 11.02 (brs, 1H), 8.28–8.14 (m, 2H), 7.99–7.89 (m, 2H), 7.70 (q,
J = 8.5 Hz, 4H), 7.45 (s, 1H), 3.20 (s, 3H). 3.5. General Procedure for the Synthesis of Compounds 9a–q Compound 11 was synthesized according to the same route of compound 8 in which
compound 7 was replaced by compound 10. Purple solid, 93% yield. 15 of 20 Molecules 2019, 24, 845 15 of 20 Step 3. Compound 12 was synthesized according to the same route of 9i in which compound 8
was replaced by compound 11. Red solid, 6% yield. 1H-NMR (400 MHz, DMSO-d6) δ 13.37 (s, 1H),
12.44 (s, 1H), 8.20 (d, J = 7.2 Hz, 2H), 8.00–7.83 (m, 4H), 7.64 (d, J = 8.0 Hz, 2H), 7.32 (s, 1H), 5.09 (s, 2H),
4.25 (q, J = 7.2 Hz, 2H), 1.27 (t, J = 7.2 Hz, 3H).13C-NMR (151 MHz, DMSO) δ 187.87, 184.15, 167.48,
153.41, 150.30, 138.86, 137.31, 135.41, 135.23, 134.74, 133.52, 132.12, 129.90 (2C), 128.71 (2C), 127.06,
126.50, 114.75, 109.81, 107.99, 65.44, 61.24, 14.04. MS (ESI) (m/z): 514.2 (M −H)−. HRMS (ESI) calcd. for C24H18ClNO8S [M −H]−: 514.0369; found: 514.0364. Purity 96.0% by HPLC. Step 3. Compound 12 was synthesized according to the same route of 9i in which compound 8
was replaced by compound 11. Red solid, 6% yield. 1H-NMR (400 MHz, DMSO-d6) δ 13.37 (s, 1H),
12.44 (s, 1H), 8.20 (d, J = 7.2 Hz, 2H), 8.00–7.83 (m, 4H), 7.64 (d, J = 8.0 Hz, 2H), 7.32 (s, 1H), 5.09 (s, 2H),
4.25 (q, J = 7.2 Hz, 2H), 1.27 (t, J = 7.2 Hz, 3H).13C-NMR (151 MHz, DMSO) δ 187.87, 184.15, 167.48,
153.41, 150.30, 138.86, 137.31, 135.41, 135.23, 134.74, 133.52, 132.12, 129.90 (2C), 128.71 (2C), 127.06,
126.50, 114.75, 109.81, 107.99, 65.44, 61.24, 14.04. MS (ESI) (m/z): 514.2 (M −H)−. HRMS (ESI) calcd. for C24H18ClNO8S [M −H]−: 514.0369; found: 514.0364. Purity 96.0% by HPLC. 3.7. Synthesis of 2-((4-((4-chlorophenyl) sulfonamido)-1-hydroxy-9,10-dioxo-9,10-dihydroanthracen-2-yl) oxy)
acetic acid (13) 3.7. Synthesis of 2-((4-((4-chlorophenyl) sulfonamido)-1-hydroxy-9,10-dioxo-9,10-dihydroanthracen-2-yl) oxy)
acetic acid (13) Synthesis of 2-((4-((4-chlorophenyl) sulfonamido)-1-hydroxy-9,10-dioxo-9,10-dihydroanthracen-2-yl) oxy
c acid (13) Compound 13 was synthesized according to the same route of 6b in which compound 6a was
replaced by compound 12. Red solid, 95% yield. 1H-NMR (400 MHz, DMSO-d6) δ 13.36 (s, 1H), 12.53
(s, 1H), 8.23–8.13 (m, 2H), 7.98–7.84 (m, 4H), 7.64 (d, J = 8.4 Hz, 2H), 7.35 (s, 1H), 4.99 (s, 2H). 13C-NMR
(151 MHz, Pyr) δ 188.75, 185.01, 171.15, 155.76, 152.45, 140.30, 139.12, 137.34, 134.82, 133.21, 130.44 (2C),
129.85 (2C), 127.85, 127.27, 121.12, 119.10, 115.52, 110.23, 108.94, 67.24. MS (ESI) (m/z): 485.8 (M −H)−. HRMS (ESI) calcd. 3.5. General Procedure for the Synthesis of Compounds 9a–q 13C-NMR (151 MHz, DMSO) δ 188.19, 180.25, 151.50, 150.13,
138.19, 137.11, 135.30, 134.32, 133.37, 132.82, 132.10, 129.46 (2C), 129.16 (2C), 126.88, 126.62, 122.74,
121.75, 115.43, 37.30. MS (ESI) (m/z): 442.0 (M −H)−. HRMS (ESI) calcd. for C21H14ClNO6S [M −
H]−: 442.0158; found: 442.0162. Purity 100% by HPLC. Purity 97.0% by HPLC. Molecules 2019, 24, 845 16 of 20 16 of 20 3.11. Synthesis of 4-chloro-N-(4-hydroxy-3-methoxy-9,10-dioxo-9,10-dihydroanthracen-2-yl)-N-methyl-
benzenesulfonamide (14d) 3.11. Synthesis of 4-chloro-N-(4-hydroxy-3-methoxy-9,10-dioxo-9,10-dihydroanthracen-2-yl)-N-methyl-
benzenesulfonamide (14d) Compound 14d was synthesized according to the same route of 14c in which room temperature
was raised to 40 ◦C. Orange solid, 50% yield. 1H-NMR (400 MHz, DMSO-d6) δ 12.79 (s, 1H), 8.28–8.13
(m, 2H), 8.00–7.92 (m, 2H), 7.76 (q, J = 8.4 Hz,4H), 7.40 (s, 1H), 3.80 (s, 3H), 3.23 (s, 3H). 13C-NMR
(151 MHz, DMSO) δ 188.22, 180.56, 155.48, 150.66, 138.57, 138.37, 136.79, 135.40, 134.67, 132.96, 132.83,
129.62 (2C), 129.23 (2C), 127.10, 126.96, 126.72, 119.91, 116.60, 60.65, 38.12. MS (ESI) (m/z): 456.0
(M −H)−. HRMS (ESI) calcd. for C22H16ClNO6S [M −H]−: 456.0314; found: 456.0305. Purity 100%
by HPLC. 3.12. Synthesis of 4-((4-chlorophenyl) sulfonyl)-12-hydroxy-3,4-dihydro-2H-anthra[2,3-b][1,4]-oxazine-
6,11-dione (14e) 3.12. Synthesis of 4-((4-chlorophenyl) sulfonyl)-12-hydroxy-3,4-dihydro-2H-anthra[2,3-b][1,4]-oxazine-
6,11-dione (14e) Compound 14e was synthesized according to the same route of 14c in which iodomethane was
replaced by 1,2-dibromoethane. Orange solid, 32% yield. 1H-NMR (400 MHz, DMSO-d6) δ 12.63 (s,
1H), 8.18 (t, J = 8.2 Hz, 2H), 8.11 (s, 1H), 7.98–7.88 (m, 2H), 7.84 (d, J = 8.4 Hz, 2H), 7.72 (d, J = 8.4 Hz,
2H), 4.06 (dd, J = 4.4, 16.8 Hz, 4H). 13C-NMR (151 MHz, DMSO) δ 187.52, 180.70, 151.78, 140.17, 139.33,
136.21, 135.07, 134.43, 133.27, 132.79, 130.21(2C), 129.30, 129.04(2C), 126.92, 126.55, 124.47, 113.41, 112.82,
64.03, 43.77. MS (ESI) (m/z): 456.0 (M + H)+. HRMS (ESI) calcd. for C22H14ClNO6S [M + H]+: 456.0303;
found: 456.0307. Purity 100% by HPLC. 3.13. Synthesis of 4-chloro-N-(3-(2-hydrazinyl-2-oxoethoxy)-4-hydroxy-9,10-dioxo-9,10-dihydroanthracen-2-yl)
benzenesulfonamide (14f) 3.13. Synthesis of 4-chloro-N-(3-(2-hydrazinyl-2-oxoethoxy)-4-hydroxy-9,10-dioxo-9,10-dihydroanthracen-2-yl)
benzenesulfonamide (14f) Compound 14a (20 mg, 0.04 mmol) and hydrazine hydrate (85%, 5 µL, 0.16 mmol) were added to
ethanol (1.5 mL) and mixture was stirred at 76 ◦C. A yellow solid (14 mg, 72%) of 14f was obtained by
filtration. 1H-NMR (400 MHz, DMSO-d6) δ 12.95 (brs, 1H), 8.43–7.36 (m, 9H), 4.53 (s, 2H). 13C-NMR
(151 MHz, DMSO) δ 187.43, 180.96, 168.15, 154.79, 140.49, 138.43, 135.10, 134.98, 134.67, 132.89, 132.81,
129.78 (2C), 129.08, 128.64 (2C), 128.51, 126.95, 126.49, 113.41, 109.56, 70.47. MS (ESI) (m/z): 500.0
(M −H)−. HRMS (ESI) calcd. 3.14. Synthesis of 3-((4-chlorophenyl) sulfonamido)-1-hydroxy-9,10-dioxo-9,10-dihydroanthracen-2-yl
methanesulfonate (14g) Compound 9i (107 mg, 0.25 mmol) and DIPEA (50 µL, 0.3mmol) were added to pyridine (1.5 mL),
and then methanesulfonyl chloride (23 µL,0.3 mmol) was added to the mixture. After being stirred
at room temperature for 4 h, the mixture was added to 10% aqueous HCl (20 mL). The suspension
was extracted with ethyl acetate and the organic phase was washed with brine, dried over anhydrous
Na2SO4 and filtered. The residue was purified by silica chromatography after removal of ethyl acetate
to afford 14 g as a yellow solid (83 mg, 65%). 1H-NMR (400 MHz, DMSO-d6) δ 12.81 (s, 1H), 8.23–8.11
(m, 2H), 8.00–7.90 (m, 4H), 7.72 (d, J = 8.4 Hz, 2H), 7.64 (s, 1H), 3.61 (s, 3H). 13C-NMR (151 MHz,
DMSO) δ 186.86, 180.93, 155.49, 138.86, 138.32, 135.07, 134.76, 132.83 (2C), 131.81, 131.31, 129.70 (3C),
128.79, (3C) 126.98, 126.50, 109.55, 40.45. MS (ESI) (m/z): 505.8 (M −H)−. HRMS (ESI) calcd. for
C21H14ClNO8S2 [M −H]−: 505.9777; found: 505.9791. Purity 99.5% by HPLC. 3.5. General Procedure for the Synthesis of Compounds 9a–q for C22H16ClN3O7S [M −H]−: 500.0325; found: 500.0340. Purity 95.1%
by HPLC. 3.14. Synthesis of 3-((4-chlorophenyl) sulfonamido)-1-hydroxy-9,10-dioxo-9,10-dihydroanthracen-2-yl
methanesulfonate (14g) 3.16. Constructions of Mutations of PGAM1 The wild-type plasmid was a gift from Department of Chemistry and Institute for Biophysical
Dynamics, University of Chicago (Chicago, IL, USA). To obtain PGAM1 F22A/R116H/R191H mutant
construct, the quick change kit (#KOD-401, TOYOBO, Osaka, Japan) was used to make site-specific
mutagenesis, which confirmed by standard DNA sequencing methods. The primers used for the
mutation were listed below: 3.15. PGAM1 Enzymatic Assay Firstly, 1 µL inhibitor in dimethyl sulfoxide (DMSO) incubated with 49 µL 4.6 nM recombinant
PGAM1, then 50 µL enzyme mix containing 3 units/mL enolase (Sigma-Aldrich, Saint Louis, MO,
USA), 3 units/mL recombinant pyruvate kinase M2 (Sigma-Aldrich), 0.6 units/mL recombinant
lactatdehydrogenase (LDH, Sigma-Aldrich), 100 mM Tris-HCl, 100 mM KCl, 5 mM MgCl2, 1 mM
adenosine diphosphate (ADP), 0.2 mM the reduced form of nicotinamide adenine dinucleotide (NADH)
and 4 mM 3PG was added. PGAM1 activity was measured as the decrease in OD at 340 nm. In addition, 17 of 20 17 of 20 Molecules 2019, 24, 845 we performed a counter screening in which 4mM 2PG was added to 50 µL reaction mix containing the
indicated inhibitor, 3 units/mL enolase (Sigma-Aldrich), 3 units/mL recombinant pyruvate kinase M2
(Sigma-Aldrich), 0.6 units/mL recombinant LDH (Sigma-Aldrich), 100 mM Tris-HCl, 100 mM KCl,
5 mM MgCl2, 1 mM ADP and 0.2 mM NADH. The other three enzyme activity was measured as the
decrease in OD at 340 nm [17]. 3.17. Protein Purification, Crystallization, Data Collection and Structure Determination The C-terminal His6-tagged PGAM1 was expressed and affinity purified following the reported
protocols [22]. The crystals of PGAM1 were obtained using the hanging drop vapor-diffusion method
at 16 ◦C in a crystallization buffer containing of 8% (w/v) PEG3350 and 100 mM MES 6.0. To obtain
the co-crystal of PGAM1 with compound 9i, the crystals of PGAM1 were soaked in stock solution
containing 500 µM 9i for 2 h. Crystals were then cryo-protected by brief soaking in a mixture solution
of crystallization buffer:glycerol (76:24) and quickly cooled in liquid nitrogen. Diffraction data were
collected at beamline BL17U1, BL18U1 and BL19U1 in the Shanghai Synchrotron Radiation Facility
(SSRF, Shanghai, China) using an X-ray beam of wavelength 0.97851 Å. The data was handled with
HKL3000 [41] and the structure was determined by using molecular replacement in CCP4 [42] via an
initial model of PGAM1 derived from PDB entry 4GPZ [22]. The model was then refined to 1.98 Å
resolution using Phenix. The ligand restraints were generated using eLBOW in Phenix and manual
rebuilding of the model was completed using the molecular graphics program COOT [43] according
to the electronic density. All the graphs were plotted by Pymol (DeLano Scientific LLC, San Carlos,
CA, USA). 3.18. Cell Viability Assays The H1299 cell line was obtained from Guangzhou Jenniobio Biotechnology Co., Ltd. (Guangzhou,
China), PC9 and A549 cells were gifted from Deng Jiong’s lab in the School of Medicine, Shanghai Jiao
Tong University, Shanghai, China). H1299, PC9 cells were cultured in RPMI-1640 medium and A549 cells
were cultured in F-12K medium containing 10% fetal bovine serum (FBS), 100 units/mL of Penicillin and
100 µg/mL Streptomycin. In cell viability assays, 2000 H1299 cells, A549 cells or 1000 PC9 cells per well
were seeded in 96-well plate. After attachment for 24 h, the cells were treated with indicated inhibitor for
72 h. After incubation with 0.5 mg/mL methylthiazolyldiphenyl-tetrazolium bromide (MTT) for 4 h, cell
viability was measured as the OD at 570 nm. PGAM1 F22A Forward: GAACCTGGAGAACCGCGCCAGCGGCTGGTACGAC
PGAM1 F22A Reverse: GTCGTACCAGCCGCTGGCGCGGTTCTCCAGGTTC
PGAM1 K116H Forward: CAGGTGAAGATCTGGCACCGCTCCTATGATGTCC
PGAM1 K116H Reverse: GGACATCATAGGAGCGGTGCCAGATCTTCATCTG
PGAM1 K119H Forward: CATGGCAACAGCCTCCACGGCATTGTCAAGCAT
PGAM1 K119H Reverse: ATGCTTGACAATGCCGTGGAGGCTGTTGCCATG 3.17. Protein Purification, Crystallization, Data Collection and Structure Determination 4. Conclusions In summary, we designed and synthesized 31 anthraquinone derivatives as PGAM1 inhibitors. The SAR study focused on the effect of 1,2-substituents, 3-substituents, 2,4-substituents, and
2,3-substituents on the anthraquinone core scaffold activity against PGAM1. Among them, 3- 18 of 20 18 of 20 Molecules 2019, 24, 845 sulfonamide substituents but not 4-substituents on the anthraquinone scaffold were essential for
maintaining potency, which was further revealed by the X-ray structure of PGAM1 complexed with
compound 9i with IC50 value of 0.27 µM. In addition, modifications of the hydroxyl of alizarin
disrupted the π-cation interaction of PGAM1 and the anthraquinone derivatives which led to the loss
of potency. Taken as a whole, the SAR study of anthraquinone derivatives against PGAM1 provided
useful information for further discovery of PGAM1 inhibitors. Supplementary Materials: The following are available online, Figure S1: The activity of PGAM1 mutant. Author Contributions: conceptualization, K.H., D.Y. and L.Z.; chemistry, K.H. and H.L.; biological assay, K.H. and L.J.; structure determination, L.J. and L.Z.; writing—original draft preparation, K.H.; writing—review and
editing, K.H., D.Y. and L.Z. Author Contributions: conceptualization, K.H., D.Y. and L.Z.; chemistry, K.H. and H.L.; biological assay, K.H. and L.J.; structure determination, L.J. and L.Z.; writing—original draft preparation, K.H.; writing—review and
editing, K.H., D.Y. and L.Z. Funding: This research was funded by the Chinese National Natural Science Foundation (grant no. 21472026,
21877014), the Shanghai Municipal Committee of Science and Technology(14XD1400300), the program for Shanghai
Rising Star (15QA1400300), and the open grant of the State Key Laboratory of Bio-organic and Natural Products
Chemistry, CAS. Funding: This research was funded by the Chinese National Natural Science Foundation (grant no. 21472026,
21877014), the Shanghai Municipal Committee of Science and Technology(14XD1400300), the program for Shanghai
Rising Star (15QA1400300), and the open grant of the State Key Laboratory of Bio-organic and Natural Products
Chemistry, CAS. Acknowledgments: We thank Deng Jiong’s lab in the School of Medicine, Shanghai Jiao Tong University for
providing the A549 and PC9 cells. Acknowledgments: We thank Deng Jiong’s lab in the School of Medicine, Shanghai Jiao Tong University for
providing the A549 and PC9 cells. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. References Chaneton, B.; Gottlieb, E. PGAMgnam style: A glycolytic switch controls biosynthesis. Cancer Cell 2012, 22,
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article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).
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Colonos ou reassentados: disputas políticas e identitárias em torno da construção da prainha do reassentamento São Francisco de Assis (1995-2009)
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COLONOS OU REASSENTADOS: DISPUTAS POLÍTICAS E IDENTITÁRIAS
EM TORNO DA CONSTRUÇÃO DA PRAINHA DO REASSENTAMENTO
SÃO FRANCISCO DE ASSIS (1995-2009) Jorge Pagliarini Junior* Jorge Pagliarini Junior* Resumo: Esta pesquisa pretende problematizar a construção de território pelos moradores
do Reassentamento São Francisco de Assis, em Cascavel/PR, reassentamento rural surgido
após movimento de luta de moradores do Sudoeste do Estado do Paraná com o Estado,
quando da construção da Hidrelétrica de Salto Caxias. Assim, a partir do estudo das
memórias dos moradores envolvidos com esse processo de luta e de outros que adquiriram
propriedades no reassentamento nos dez anos passados de sua construção, destacamos
disputas presentes nos viveres dentro do reassentamento e também nas cidades vizinhas a
ele, lugares que também fazem parte do território a ser reconstruído. Palavras-chave: Reassentamento São Francisco de Assis (Cascavel/PR), construção de
território. COLONISTS OR RELOCATED PEOPLE: IDENTITY AND POLITICAL DISPUTES
SURROUNDING THE CONSTRUCTION OF THE "SMALL BEACH" OF SÃO FRANCISCO DE
ASSIS' RESETTLEMENT (1995-2009) Abstract: This research intends to discuss about the territory construction for the
inhabitants of the São Francisco de Assis Resettlement (Cascavel/PR), an agricultural
resettlement created after a strike between a Paraná southwestern movements with the
Paraná State, when it had been constructing the Salto Caxias Hydroelectric. Therefore,
taking as base the memories study of the involved inhabitants in the strike and from those
who purchase properties in the re-settlement ten years after its construction, it is noticed
current disputes in the life inside the re-settlement as well as in the cities nearby. Those are
places that also are part of the territory to be reconstructed. Keywords: The São Francisco de Assis Resettlement (Cascavel/PR), the construction of the
territory. Introdução 1
O objetivo deste trabalho é o de problematizar a construção de
territórios pelos moradores do reassentamento São Francisco de Assis,
Cascavel, Paraná, moradores esses que, a partir de um processo de migração
forçada, são designados e passam a se designar a si mesmos como
2
reassentados . Para tanto, apresentaremos a materialidade do reassentamento
e suas práticas atuais, seja a partir das políticas de implantação do turismo rural
e do agronegócio, seja por práticas cotidianas como as relações de vizinhança,
religiosidade e lazer, com o jogo de futebol. Essas práticas demonstram a
existência de disputas econômicas e de apropriações culturais dos moradores
locais ao reconstruírem seus territórios. Revista NUPEM, Campo Mourão, v. 3, n. 4, jan./jul. 2011 Revista NUPEM, Campo Mourão, v. 3, n. 4, jan./jul. 2011 Revista NUPEM, Campo Mourão, v. 3, n. 4, jan./jul. 2011
115 115 Colonos ou reassentados: disputas políticas e identitárias em torno da construção da prainha do reassentamento Essas disputas, como veremos, se dão ainda ao nível da memória
(POLLAK, 1989), pois é significativo muitos dos entrevistados se dizerem
próximos ao passado de luta no qual fora conquistado o reassentamento. Isso
daria autoridade para se posicionarem diante da administração atual. Da
mesma maneira, perceberemos como fora significativo enfatizar o passado de
agricultor, referenciado quando se falava da trajetória anterior ao processo de
migração forçado pela barragem de Salto Caxias, identidade assumida
inclusive enquanto uma estratégia de inserção perante os moradores já
estabelecidos na região. Assim, enfatizaremos que essa conquista por territórios está permeada
por disputas identitárias. Elas foram apresentadas a partir daqueles que se
dizem empreendedores rurais e assim se destacariam nas atividades
cotidianas; a partir da reivindicação de quem justifica a terra recebida por
indenização pelo fato de que sempre esteve ligado ao trabalho com a terra; ou
ainda, a partir da ligação dos sujeitos com o passado de luta contra o Estado
para que fosse criado o reassentamento. Para apreendermos parte desse
cotidiano seguiremos com as possibilidades teórico-metodológicas da Historia
Oral, em específico numa linha próxima à problematizada por Portelli
(PORTELLI, 1997). Surgido nos anos de 1990, após movimento de lutas de agricultores que
foram deslocados de suas terras no Sudoeste paranaense quando da
construção da barragem de Salto Caxias, o reassentamento é formado também
3
por agricultores que compraram terras na localidade . Introdução Nas narrativas dos
entrevistados e nos documentos estudados, produzidos pela Comissão
Regional dos Atingidos por Barragem do Rio Iguaçu (CRABI), instituição que
liderou os embates dos moradores até a chagada no reassentamento e nos
primeiros anos de seu funcionamento, percebemos um forte discurso em torno
da construção e da continuidade de uma união entre os moradores. Todavia,
agora, com a segurança dos títulos das propriedades pelos moradores, essa
união cede lugar, nas narrativas, às disputas e aos posicionamentos diversos
em relação à administração local, e se propaga nas práticas culturais e sociais
dos reassentados e visitantes do reassentamento. Assim, portanto, a construção do reassentamento reflete tanto as
apropriações de costumes, como relações de vizinhança e a tradições
atribuídas ao homem da terra, práticas trazidas já no processo de migração,
afinal, os reassentados eram na maioria dos casos agricultores, quanto disputas
políticas atuais permeadas pela cobrança por certo empreendedorismo por
parte dos moradores dali. O resultado desses embates será discutido a partir do estudo de
narrativas e envolve questões relativas à polêmica sobre a placa localizada na
entrada do reassentamento, a qual nomeia o local como Colônia São Francisco
de Assis, substituindo outra na qual se lia Reassentamento São Francisco de
Assis. Além disso, o estudo também envolve as práticas com as quais ainda se Revista NUPEM, Campo Mourão, v. 3, n. 4, jan./jul. 2011 116 116 Colonos ou reassentados: disputas políticas e identitárias em torno da construção da prainha do reassentamento mantém certa união, que é o caso da ida à igreja e do jogo de futebol. Ainda cabe destacar que este artigo é um recorte de uma pesquisa
concluída em mestrado em 2009, e iniciada ainda com trabalho de monografia
há aproximadamente quatro anos. Isto, de certa forma, contribuiu para que
pudéssemos perceber disputas e configurações internas do processo de
reassentamento. Da mesma forma, ser morador da cidade de Corbélia e
vivenciar a construção desses territórios dos reassentados somou-se aos
resultados alcançados (a respeito das relações campo-cidade e do
questionamento de uma visão dicotômica para essas categorias históricas a
partir da literatura, ver: WILLIAMS, 1998). Existe uma “Colônia” São Francisco de Assis? A sede do reassentamento localiza-se aproximadamente a dois
quilômetros do trevo de acesso à localidade. Tem destaque, no trevo, a placa
que nomeia a localidade, na qual se lê: Colônia São Francisco de Assis,
4
desenvolvendo o turismo rural . Uma estrada pavimentada com paralelepípedos
liga esses espaços. A sede encontra-se na comunidade Alto Alegre e é utilizada
por moradores de todo o reassentamento. Ali estão localizados o Colégio
Estadual de Ensino Fundamental São Francisco de Assis, o posto de saúde, a
igreja católica com salão de festa, a igreja evangélica, o campo de futebol e uma
área de lazer, composta pelo lago, conhecido por Prainha, e, ao lado, o jóquei
clube ou a hípica. Em frente ao lago foi construída uma lanchonete, local
utilizado principalmente por visitantes das cidades vizinhas. Já ao lado da igreja
há um bar, frequentado pelos moradores do reassentamento. A Copel procurou manter as relações de vizinhança na distribuição
espacial das famílias no reassentamento. Essa divisão pode ser percebida,
inclusive, nos jogos de futebol que ocorrem no campo ao lado do colégio e do
5
lago, onde as comunidades jogam entre si ou se juntam para desafiar outras
equipes que representam localidades das regiões Oeste e Sudoeste do Estado. Todos os entrevistados no reassentamento ressaltaram a boa estrutura
do local: a construção do colégio, ocorrida logo no segundo ano do
reassentamento; o posto de saúde entregue no ano seguinte; os bailes que
organizam no salão de festas da igreja católica; a infraestrutura da igreja
evangélica; as disputas de futebol de campo; e as quadras de futsal. Tal
infraestrutura não existe somente na sede, pois nas comunidades foram
também construídas capelas da igreja católica, campo de futebol e salão de
festas. Para estudarmos a operacionalidade desse lugar, entrevistamos também
moradores da cidade de Corbélia, cidade vizinha ao reassentamento. Diariamente os reassentados buscam a rede de comércio dessa cidade. Por sua
vez, parte significativa dos visitantes da “Prainha” é oriunda dos municípios de
Corbélia e de Cascavel. Esses visitantes enfatizaram a existência de uma boa
infraestrutura. Não foram raras as vezes em que o São Francisco de Assis foi Revista NUPEM, Campo Mourão, v. 3, n. 4, jan./jul. 2011 117 117 Colonos ou reassentados: disputas políticas e identitárias em torno da construção da prainha do reassentamento destacado por todos esses entrevistados enquanto um exemplo de
organização. Existe uma “Colônia” São Francisco de Assis? Para alguns, trata-se de exemplo de eficiência prestado pela
organização do Estado; para outros, resultado da organização de moradores
diante das reivindicações perante Copel, ou de ambas, para a maioria dos que
se propõem a falar sobre o reassentamento. Essa ênfase no resultado positivo e na ausência de reclamações nos levou
a questionar se lidávamos com um silêncio dos entrevistados ou com uma
aceitação geral, pois pouco se ouvia sobre conflitos locais. igura 1. Sede do Reassentamento São Francisco de Assis. Hípica e Prainha
Fonte: Imagem de Google Earth (Acesso em: 18 fev. 2011). Figura 1. Sede do Reassentamento São Francisco de Assis. Hípica e Prainha Fonte: Imagem de Google Earth (Acesso em: 18 fev. 2011). Se as opiniões não divergem a respeito da “boa estrutura”, a
administração enfrentara disputas e divergências quanto ao uso da sede local,
da Prainha e da construção da hípica. Há de se considerar que os visitantes
geram rendas, consomem e dão lógica ao funcionamento do agronegócio e de
demais iniciativas que visam agregar valor à produção familiar. De maneira
geral, a problemática gira em torno da mudança do nome para Colônia São
Francisco de Assis e que envolve disputas de interesses, expectativas e de
memórias. A criação da colônia reflete como se encontra o dilema em torno do ideal
comunitário local. Percebemos as dificuldades dos moradores para se dizerem
membros atuantes de uma comunidade, pois se temos presentes disputas
internas, esse processo de construção do reassentamento necessita, muitas
vezes, de uma ideia de união e que justificaria o recebimento de terras. Como
6
bem destacou Bauman , a sensação de segurança presente na palavra
comunidade não dá conta de absorver as necessidades das sociedades
individualistas em que vivemos (BAUMAN, 2003). Outra problemática que norteia a questão é o próprio contato com o
outro. O morador da cidade foi destacado quando os reassentados e/ou Revista NUPEM, Campo Mourão, v. 3, n. 4, jan./jul. 2011 118 Colonos ou reassentados: disputas políticas e identitárias em torno da construção da prainha do reassentamento colonos falavam da importância das cidades para a própria vivência no
reassentamento. Todavia, o encontro agora se dá no reassentamento, nas
visitas à Prainha e à hípica. Existe uma “Colônia” São Francisco de Assis? 2011 119 119 Colonos ou reassentados: disputas políticas e identitárias em torno da construção da prainha do reassentamento em menor número, compram produtos artesanais produzidos ali, pagando R$
7
3,00 pela entrada . 8
Outros projetos, na opinião de Seu Zequinha , poderão agregar valor à
produção local e concretizar a implantação da colônia, favorecendo o
desenvolvimento local: É, turismo rural, daí o nome: Colônia São Francisco de Assis. Aqui ficou Colônia São Francisco de Assis. É um nome
turístico, né, e o povo, deu 90% aqui que aprovaram. A palavra colônia, que nem diz lá, uma colônia italiana, uma
colônia... Nós viemos de todo um processo de barragem,
formamos uma colônia aqui, né. E outra, aqui já tem, a Colônia Sapucaia, Colônia Esperança,
Colônia Barreto... Por que nós não vamos transformar a
Colônia São Francisco? Agora a Barateira ali, não sei se é Nossa Senhora dos
Navegantes, não tem nada de reassentamento. Agora nós
ali fica: reassentamento... Reassentamento é o processo. Agora é nome? Pergunta: Uma colônia e um reassentamento tem suas
diferenças? Zequinha: Sim, mas eu falei assim, um processo de
reassentamento, e nós vamos continuar falando, dessa luta,
de tudo, agora, nós não podemos... Até pro próprio
comércio ficou melhor, eles vão lá e falam: eu sou lá da
Colônia São Francisco, é diferente falar, sou lá do
reassentamento. Muita gente ainda fala dos sem terra. Entende? O pessoal aqui, quando saiu essa palavra ali, Colônia São
Francisco, muitos gostaram por causa disso. Tem gente que
fez questão de já fazer ficha com o nome Colônia São
Francisco. A gente tinha uma placona, coisa mais linda caiu
de lá, né, com o vento. Mas nós vamos tentar recuperar
(MEURER, José Rossi. Entrevista. A/A). As disputas em torno do nome reassentamento ou colônia são materiais e
simbólicas. As reticências e o emprego da palavra agora marcam as
divergências locais. O entrevistado reconhece o significado presente na
palavra reassentamento, a qual faz referência a um passado de luta, do
enfrentamento. Todavia, alerta para as confusões entre assentados com
reassentados e, por isso, defende a mudança para colônia. E mais
interpretações estão relacionadas à sua fala. O passado de agricultor ou colono, como nos lembra Hobsbawm,
quando evocado pode afirmar ou modificar o presente e futuro (Cf. HOBSBAWM, 1998, p. 22-35). Nesse caso, o passado comum de colono também
autorizaria a mudança do nome de reassentamento para colônia. Existe uma “Colônia” São Francisco de Assis? Assim, ao historicizar o processo de construção do reassentamento São
Francisco de Assis, seguimos com a preocupação de destacar lutas, inclusive
identitárias: disputas com o visitante ou com os que compram terras ali;
disputas pelas escolhas na aplicação de políticas agrárias e, ainda, pela própria
designação do grupo, ou seja, ser designado reassentado, colono, agricultor ou
mesmo empreendedor. Em outros termos, a questão que se coloca é a de se refletir o que
significaria, para os entrevistados, ser um reassentado ou ser um colono. Entretanto, antes de conclusões, uma indagação: Existe, de fato, uma “Colônia”
São Francisco de Assis? Quais os significados, concordâncias e discórdias no
momento atual? Nesse sentido, um morador do reassentamento procurou delimitar o
processo de construção do reassentamento da seguinte maneira: “Nós estamos
aqui, no São Francisco, não na colônia ou no reassentamento” (OLIVEIRA, Djair
Márcio. Entrevista – A/A). Nas palavras de seu Djair, a mudança de nome de
reassentamento para colônia não significaria uma mudança radical que
atingiria a estrutura e organização local, mesmo porque, em sua opinião, das
duas propostas, seja reassentamento ou colônia, sobressai a organização e
passado de grupo que originou e organiza o local. Todavia, os demais
entrevistados que falaram sobre a questão posicionaram-se criticamente. Foi o
caso do Seu Zequinha. Sua opinião representa o amálgama de relações que
norteiam a questão: É, penso eu, pra visitas, colônia seria uma boa coisa, mas não
podemos descartar o que foi essa Colônia São Francisco,
não descartar da história, da origem, você não pode
esquecer da origem, você nasceu, você tem uma
identidade, você tem uma origem, então porque da origem
(MORAIS, Adilson de. Entrevista. A/A). E esse posicionamento pode ser entendido se considerarmos o lugar
social ocupado pelo entrevistado no reassentamento. Definir o nome de um
lugar implica também isso. Então, seguindo os significados das políticas
internas do reassentamento, podemos destacar a placa instalada na entrada da
localidade, a qual informa: “Colônia São Francisco de Assis”. A Colônia São
Francisco de Assis oferece aos visitantes da região um espaço de lazer e de
compras de produtos beneficiados na localidade, como artesanato e
alimentos. O ecoturismo ainda está sendo implantado, mas, com a utilização da
“praia”, o local é procurado frequentemente por visitantes. E isso gera rendas,
pois são cerca de 800 visitantes por final de semana. Elas consomem bebidas e, Revista NUPEM, Campo Mourão, v. 3, n. 4, jan./jul. Existe uma “Colônia” São Francisco de Assis? Para o
entrevistado isso não implica o esquecimento do passado ligado ao Revista NUPEM, Campo Mourão, v. 3, n. 4, jan./jul. 2011 120 Colonos ou reassentados: disputas políticas e identitárias em torno da construção da prainha do reassentamento movimento dos atingidos por barragem. movimento dos atingidos por barragem. Ao se dizer representante dos moradores nas políticas de
desenvolvimento local, seu Zequinha defende a efetivação da colônia, pois
essa mudança andaria ao lado das novas necessidades locais (advindas do
turismo rural). Seguindo as palavras do entrevistado, a situação poderia ser lida
do seguinte modo: os colonos atingidos pelas políticas energéticas do Estado
foram indenizados com terras e continuaram com sua cultura, seu trabalho,
mas em outras terras, formando outra colônia. A placa na entrada aparece enquanto um símbolo para alguns
moradores, visto que há divergências quanto à sua legitimidade. Também é um
símbolo para os visitantes e para aqueles que passam por ela na rodovia que
liga Cascavel e Corbélia, mas, para os reassentados, a mudança no nome
remete, muitas vezes, à história do grupo. Figura 2. Placa presente no trevo de acesso ao reassentamento, nas margens
da rodovia BR 369, entre Cascavel e Corbélia Fonte: Arquivo do autor. Fonte: Arquivo do autor. Lidamos com disputas internas e o posicionamento de outros moradores
corroborando com a visão de seu Zequinha. É o caso de seu Alcideo e de sua
esposa, dona Ana. O casal adquiriu terras no reassentamento e hoje frequenta
as reuniões da associação de moradores local. Dizem, inclusive, que já são
reconhecidos pela Copel enquanto reassentados. Sobre a mudança de nome
para colônia, eles afirmam: Ana: Mas, é porque mudou, o nome foi mudado pra colônia
São Francisco. Pergunta: Mudaram né, me falaram, mas é... Essa mudança
foi pra melhor, para o senhor? Alcideo: Eu acho que foi pra melhor. Pergunta: Melhorou em qual sentido? Ana: Mas, é porque mudou, o nome foi mudado pra colônia
São Francisco. Pergunta: Mudaram né, me falaram, mas é... Essa mudança
foi pra melhor, para o senhor? Alcideo: Eu acho que foi pra melhor. Pergunta: Melhorou em qual sentido? Ana: Mas, é porque mudou, o nome foi mudado pra colônia
São Francisco. Pergunta: Mudaram né, me falaram, mas é... Essa mudança
foi pra melhor, para o senhor? Alcideo: Eu acho que foi pra melhor. Pergunta: Melhorou em qual sentido? Revista NUPEM, Campo Mourão, v. 3, n. 4, jan./jul. 2011 Revista NUPEM, Campo Mourão, v. 3, n. 4, jan./jul. 2011
121 121 Colonos ou reassentados: disputas políticas e identitárias em torno da construção da prainha do reassentamento Alcideo: Eu acho que você chegava no comércio e fala: você
mora aonde? Eu moro na Colônia São Francisco. movimento dos atingidos por barragem. Eles já não,
não confundem tanto as coisas... Ana: Que eles confundiam aqui muito os assentados com os
reassentados. Agora, que mudou o nome, em vez Colônia
São Francisco, então já não é assentado, já não vai dizer: é os
assentados, né. Pergunta: E pro pessoal que visita aqui, será que muda? Pergunta: E pro pessoal que visita aqui, será que muda? Alcideo: Muda, eu acho que muda. No meu ponto de vista
eu acho que muda, até pra quem vem de visita, é... Dizer
Colônia São Francisco você, fica melhor do que.... Se tivesse
a placa lá dizendo reassentamento São Francisco, bastante
gente ia ficar naquela, né...[...] É: reassentamento, o que é
reassentamento? Que quer dizer? (VOESE, Ana e VOESE,
Alcideo. Entrevista. A/A). Aqui, a referência ao comércio demonstra a preocupação em serem
reconhecidos como autônomos que têm crédito. Questionamo-nos, diante
desse depoimento, se não está posta uma vontade de tentar apagar esse
passado de discriminação. Essas divergentes formas de usos do espaço e do passado que formam o
reassentamento seguem nas memórias dos moradores. Na entrevista com seu
Agenor, um fragmento de nossa conversa resume essas divergências,
principalmente políticas, em torno da mudança do nome para colônia e já nos
indica caminhos para problematizar a questão: Pergunta: Quando eu cheguei lá estava escrito Colônia São
Francisco e eu me lembro que no outro trabalho, quando eu
conversava com um senhor, ele me falava: “olha, lê lá na
placa da entrada, Reassentamento São Francisco de Assis, (...)
as pessoas tem que saber o que é isso!” E agora, quando
voltei, esta lá: Colônia São Francisco. Resumindo, o que o
senhor acha de tudo isso, o nome não ajuda ou...? Agenor: Ele, ele atrapalha, esse nome atrapalha, sabe
porque, colônia, no meu ver, a colônia é coisa de 50, 60 anos
atrás. É aonde você adquiriu uma área de terra, através do Incra,
do governo federal, onde você... Uma colônia de terra, daí
ele titulava depois, porque essa aqui foi uma conquista, foi
uma luta e sem luta você não chega a lugar nenhum... A colônia é diferente, co-lo-ni-za-ção, é diferente, hoje não
existe isso mais. (...) A confusão, quem confunde é a cabeça
das pessoas, pessoas que não conhece a luta, não sabe da
luta, não sabe o porquê do reassentamento, eles
confundem, ele tá pensando que aquilo foi uma
colonização do governo. movimento dos atingidos por barragem. ç
g
Porque eu acho assim, no meu entender, que na verdade,
quem colocou aquilo ali são pessoas que não é da luta,
pessoas que compraram aquilo, não é pessoas que veio do
início. Revista NUPEM, Campo Mourão, v. 3, n. 4, jan./jul. 2011 Revista NUPEM, Campo Mourão, v. 3, n. 4, jan./jul. 2011
122 122 Colonos ou reassentados: disputas políticas e identitárias em torno da construção da prainha do reassentamento Na colônia... Pra quem tá comprando, isso é bom, porque a
colônia é diferente do reassentamento, pra quem tá
comprando, não tá comprando do reassentado ou do
assentado, tá comprando do colono (DANTAS, Agenor. Entrevista. A/A). Na colônia... Pra quem tá comprando, isso é bom, porque a
colônia é diferente do reassentamento, pra quem tá
comprando, não tá comprando do reassentado ou do
assentado, tá comprando do colono (DANTAS, Agenor. Entrevista. A/A). Questões políticas, como a luta, ainda estão na pauta e a fala de seu
Agenor, de certa forma, desautoriza a fala de seu Zequinha. Seu Agenor se
coloca entre aqueles que têm autoridade para falar em nome dos demais, uma
autoridade possivelmente legitimada pelo fato de ele ter participado da luta. Para expressar sua opinião, remete-se a uma outra acepção da palavra colônia:
significa, para ele, o resultado de um projeto de colonização pleiteado pelo
Estado. Algo do passado, que não condiz com os viveres e os desafios de hoje. Diferente de seu Zequinha (naquele momento da conversa vice-presidente do
Conselho Unificado), seu Agenor se diz afastado das reuniões locais. Ele é
crítico das propostas implantadas na localidade, em específico, a de mudança
de nome. Uma questão política e de identidade se coloca e a luta remete às
políticas estatais de distribuição de terras e à preocupação com a venda de
terras, pois, na denominação colônia, segundo seu Agenor, encontra-se uma
estratégia que facilitaria a negociação de terras: comprar áreas de
reassentamento é diferente de comprar de uma colônia. Outro ponto relevante, que complementa a interpretação da fala de seu
Agenor, é que ele esteve e está ligado à CRABI. Ou seja, atuou na implantação
das primeiras iniciativas que resultaram na constituição do São Francisco de
Assis. Ele diz que estava presente desde os momentos de enfrentamento e
posterior negociação com a Copel. Assim, reconhece no nome reassentamento
a necessidade de se destacar todo o processo de luta. movimento dos atingidos por barragem. Não bastaria apenas se
dizer um colono, pois necessitaria, sim, estar ligado ao passado de luta. Então, podemos resumir aqui as três leituras possíveis desse processo e
dos posicionamentos dos moradores em transformar o reassentamento numa
colônia. Na primeira, o entrevistado busca a defesa da mudança na origem
comum de agricultor e nas exigências do agronegócio. Uma segunda postura
reconhece o significado do fato de os reassentados terem no nome
Reassentamento São Francisco de Assis a demonstração da vitória contra o
Estado, nome que representa o passado comum, de luta, o qual, por isso
mesmo, não deveria ser mudado. Enfim, uma terceira leitura: os reassentados
foram alvos de desconfiança dos moradores da região e muitas vezes tiveram o
direito de receber terras questionadas. Por isso precisam demonstrar, aos que
desconhecem sua trajetória, que ser reassentado significa ser também colono. As falas de Adilson, seu Zequinha, seu Agenor, de seu Alcideo e dona Ana
são significativas, principalmente, quando nos levam a considerar os lugares
que ocupam dentro do reassentamento. Todos estão ou já estiveram próximos
à coordenação e administração local. Suas queixas sobre as confusões dos
moradores da cidade sobre suas trajetórias, ou sobre o risco de esquecimento Revista NUPEM, Campo Mourão, v. 3, n. 4, jan./jul. 2011 123 123 Colonos ou reassentados: disputas políticas e identitárias em torno da construção da prainha do reassentamento do passado de luta, são os principais argumentos favoráveis ou contrários à
denominação Colônia São Francisco de Assis. A prainha e a hípica: lugares de encontros e desencontros Estabelecida administrativamente e simbolicamente a colônia, o
convívio entre os reassentados e os moradores da região dentro do
reassentamento ou colônia evidencia a atual polêmica sobre o uso do local. A
utilização do lago existente na sede do reassentamento e a construção de uma
hípica gera discussões. Abrir ou não as portas para moradores das cidades
vizinhas é o eixo da polêmica. As opiniões divergem e, além da questão de se
sentir ou não à vontade diante de pessoas estranhas, a problemática remete às
políticas internas. Para aprofundar a discussão necessitamos de um breve olhar
à fisionomia desse lugar. Ao lado do lago encontramos uma lanchonete estruturada,
principalmente, para atender aos visitantes. Pouco acima existe um bar, este
mais frequentado pelos moradores do local. O primeiro espaço é o que se
tornou para os de fora; já o segundo, com infraestrutura mais simples, é onde
ocorrem as confraternizações entre as pessoas que preferem não frequentar o
mesmo lugar ocupado pelos visitantes. Alguns moradores questionam quem
teria o direito de administrar a lanchonete do lago. Já em relação ao bar, uma
comissão de pessoas é escolhida e/ou convidada para administrá-lo. A renda
do bar é investida na localidade e, parte dela, paga a moradores que nele
trabalham. Essa renda não se compara à da lanchonete, local muito mais
procurado devido ao movimento das visitas. Todas as verbas arrecadadas
nesses espaços devem ser investidas na manutenção e no melhoramento da
infraestrutura do local. A existência desses dois locais indica uma separação
entre reassentados e visitantes e, mais que isso, conflitos sobre a administração
local, revelando mais disputas pelo lugar. Um dos assuntos que é mais divergente nas entrevistas é exatamente o
uso desses espaços. O ressentimento em relação às disputas pelo uso e
administração do reassentamento marca as falas. Seu Agenor se posiciona a
respeito: Então, quando a gente veio pra cá, a Copel mandou
primeiro recursos pra limpar aquele lago, e foi aberto o lago,
foi, a gente abriu ele, secou, limpou, tirou o que tinha
dentro, pauleira, coisa, foi limpado, aí foi investido, ponhado
areia pra construir uma praia, foi construída aquela
lanchonete, se pode ver que ela tá dentro do lago ali, foi
feito um aterro ali. E construído aquela lanchonete, isso foi
uma conquista, eu, a gente teve, foi dinheiro público, não é
um dinheiro particular, público que a Copel mandou... A prainha e a hípica: lugares de encontros e desencontros [...] Aí
entrou um outro pessoal ali, e privatizou [critica à abertura
do espaço a visitas] (DANTAS, Agenor. Entrevista. A/A). Então, quando a gente veio pra cá, a Copel mandou
primeiro recursos pra limpar aquele lago, e foi aberto o lago,
foi, a gente abriu ele, secou, limpou, tirou o que tinha
dentro, pauleira, coisa, foi limpado, aí foi investido, ponhado
areia pra construir uma praia, foi construída aquela
lanchonete, se pode ver que ela tá dentro do lago ali, foi
feito um aterro ali. E construído aquela lanchonete, isso foi
uma conquista, eu, a gente teve, foi dinheiro público, não é
um dinheiro particular, público que a Copel mandou... [...] Aí
entrou um outro pessoal ali, e privatizou [critica à abertura
do espaço a visitas] (DANTAS, Agenor. Entrevista. A/A). Revista NUPEM, Campo Mourão, v. 3, n. 4, jan./jul. 2011 Revista NUPEM, Campo Mourão, v. 3, n. 4, jan./jul. 2011
124 Revista NUPEM, Campo Mourão, v. 3, n. 4, jan./jul. 2011 124 Colonos ou reassentados: disputas políticas e identitárias em torno da construção da prainha do reassentamento Para apresentar uma contraproposta à utilização do local por não
moradores, seu Agenor destaca a proposta da criação de um clube: Para apresentar uma contraproposta à utilização do local por não
moradores, seu Agenor destaca a proposta da criação de um clube: Exatamente, e se você fosse inscrito, tivesse uma carteira,
uma carteirinha como se fosse de identificação, que você
fosse sócio daqui e tava contribuindo pra manter aquilo ali
limpo, ter pessoas, zelador, tudo ali, né, com aquela
contribuição de cada um, entendeu, pra evitar de vir
pessoas lá de Cascavel. Inclusive até drogas traz ali, sabe, é... Ali, digamos, que não é do nosso causo ali, o cara vem ali e
leva a menina fazer sexo lá dentro... Então, a gente não queria isso, a gente queria até abrir
espaço, não só pro reassentado, mas pra pessoas de fora
também, de Cascavel, de Corbélia, de onde for, mas pessoa
de bem, pessoas que contribuísse com um valor
diferenciado, entendeu, se nós aqui, digamos, que são
realmente os proprietário, se nós paga dez real ou quinze
por semana, o outro o que vem de fora teria que pagar o
dobro, até porque evita desses mal acabado vir pra cá. Se
você põe um precinho muito baixo, não evita desse povo vir
aí. A prainha e a hípica: lugares de encontros e desencontros Sem dúvida, não me sinto à vontade, como é que você vai tá
num ambiente que você vai tá se cuidando de um indivíduo
ali, que não sabe quem é (DANTAS, Agenor. Entrevista. A/A). Cobrar um preço maior garantiria que somente fossem até lá pessoas
responsáveis pela organização do local. Um clube reúne pessoas dispostas a
pagar por um ambiente controlado, organizado, vigiado, com regras sobre
proibição do uso de drogas, embriaguez, atitudes que não condizem com certa
moral, como é o exemplo trazido das relações sexuais em lugar público. Ou
ainda, um clube permitiria que houvesse um controle sobre os participantes e
garantiria um predomínio de frequentadores locais. Até poderia ter gente de
fora, mas, nessa lógica, a maioria seria reassentados. Sua opinião sobre a
construção da hípica segue sugerindo conflitos: É que todas as hípicas que tinha lá onde a gente morava
sempre morreu gente... Sempre dava rolo. Eu cheguei ver gente, o cara derrubar,
desciam de um cavalo, entendeu, matar assim, a tiro, o outro
por causa de aposta lá e... Pergunta: E o “rolo” que dava, geralmente era com pessoas
de raias ou... Com os de fora com os dali? Agenor: Com os de fora e com os dali. Mas, envolve todo
mundo e a fama, a fama do que não presta, por aí abrange
tudo, quem não tinha nada a ver com a história, acaba
atingido também, entendeu. Aconteceu em tal lugar, o cara,
o povo dali que vai levar a fama, então, eu acho... Olha, briga
de galo, rinha de galo e hípica de cavalo, isso pra mim não Revista NUPEM, Campo Mourão, v. 3, n. 4, jan./jul. 2011 Revista NUPEM, Campo Mourão, v. 3, n. 4, jan./jul. 2011
125 125 Colonos ou reassentados: disputas políticas e identitárias em torno da construção da prainha do reassentamento serve. Tem outras maneiras de você se divertir (DANTAS,
Agenor. Entrevista. A/A). serve. Tem outras maneiras de você se divertir (DANTAS,
Agenor. Entrevista. A/A). Além das divergências quanto ao aproveitamento do lugar pela
comunidade, a reputação do local estaria em questão. De acordo com o
entrevistado, essa atividade sempre deixa a comunidade mal falada, pois
envolve apostas. Além disso, não é uma atividade que, tipicamente, os
moradores frequentam, ou seja, é uma atividade que serve, basicamente, para
corredores de fora. Próximas às opiniões de seu Agenor são as de dona Ivani e
Adilson. Primeiro, dona Ivani questiona o funcionamento: Você sabe quantas vezes eu fui lá nessa praia? Duas vezes
[...].Vem maconheiro, ficam aqui no encruzo esperando,
aqui ó, fumando no meio da rua aqui, como você vai passar,
que nem o namorado dela [de sua filha] veio e teve que
voltar, 10 horas da noite, que tavam fumando no meio da
rua e não queriam que passasse (MORAIS, Ivani de. Entrevista. A/A). Em seguida, Adilson complementa a fala de sua mãe: Em seguida, Adilson complementa a fala de sua mãe: Os cara saíam ali de noite, faziam fogo no meio do calçado
ali, e ficam... Pessoas, fica perigoso de você sair sozinha de
noite até. Não é fácil, porque você quando tem algo que tráz pessoas
de fora, desconhecidas, que não é familiar, e você vê que
tem algo de errado, com as pessoas, daí só os cegos que não
vêem [...] São mil pessoas por fim de semana, pode pegar e vim um
dia ali num sábado, pode hoje à tarde, o senhor vir ali [...] São mil pessoas por fim de semana, pode pegar e vim um
dia ali num sábado, pode hoje à tarde, o senhor vir ali [...]
Porque se você vai ali, você vê pessoas aí que você nem... Porque se você vai ali, você vê pessoas aí que você nem... As pessoas até vão participar ali, olhar, só que apostar... Que
isso aí é um jogo, que ninguém aposta quase, que benefício
trás um haras? Que beneficio traz um haras pra sociedade,
sendo que não dá condição nenhuma pra ninguém daqui
de dentro, dá condição pra quem tem lá, as estrebarias é
pros cavalos velhos de fazendeiros da região? Não, eu sei, é a cultura, se você vai em Cafelândia, tal tem, se
vai tem um haras. Mas, nós aqui não é cultura, mesmo que
tinha cultura de alguém, lá era particular, na propriedade do
cara, só que aqui não é da cultura do povo... Você não vê
nada construído pra sociedade (MORAIS, Ivani de. Entrevista. A/A). As preocupações não envolvem somente disputas em torno da
administração, mas também questões de cunho moral. Os de fora seriam
ameaça à integridade moral dos reassentados. E, nessa classificação do outro,
encontramos também a crítica ao latifundiário, afinal, os cavalos são de Revista NUPEM, Campo Mourão, v. 3, n. 4, jan./jul. 2011 126 126 Colonos ou reassentados: disputas políticas e identitárias em torno da construção da prainha do reassentamento grandes fazendeiros, não são de pequenos agricultores ou dos reassentados. Outro aspecto que envolve as críticas de Adilson e de dona Ivani diz respeito ao
próprio hábito de colocar cavalos para correr, associado a uma prática de
latifundiário. As disputas pelos lugares, denominada por Arantes de guerra dos
9
lugares , implicam a aceitação de territórios flexíveis, marcados pela ordem
simbólica e moral, encontrada nos discursos oficiais desses lugares, e pelos
usos e disputas por esses lugares (Cf. Em seguida, Adilson complementa a fala de sua mãe: [...] Por isso que fechou, daí eles
não queriam abrir, daí na época foi que dali um ano era pra
se pensar e dali um ano o homem começou de novo a... e
então, dali um ano foi feito uma assembléia pra ver se abria
ou não, como numa assembleia sempre a maioria ali
presente decide... É, eu mesmo fui até ali e falei: oh, da minha parte pode abrir,
não tem problema nenhum. Depois passaram ali com um
abaixo-assinado pra não deixar abrir e eu no dia, votei lá que
no dia, abrir, se vocês conseguir não abrir, eu não tenho
nada contra e se abrir também não (VOESE, Alcideo. Entrevista. A/A). O fato de a hípica ter sido fechada indica que as divergências tomaram
forma e mobilizaram os moradores, que exigiram um posicionamento da
administração. Adilson nos falou sobre essas confusões que levaram ao
fechamento da hípica e também classificou os “cuidadores de cavalo” como
“malandros”: Não, fechou tava dando incômodo com os próprios
cuidadores dos cavalos... Não, fechou tava dando incômodo com os próprios
cuidadores dos cavalos... Tinha casos aqui de colegas meus de sair corrido, colega
meu sair corrido, deixar a moto, abandonar a moto e sair
corrido e os caras correndo de atrás com faca e tudo,
escapou não sei como. E além de tudo o cara nem pagou lá
que precisava, o cara gastou mais de 1.000 reais com a moto,
ali na frente do colégio os cara vinha ali incomodar as
meninas, os pais não podem acompanhar as meninas 24
horas por dia. O cuidador, malandro, pra mim é malandro, vem ali daí as
meninas não tem cabeça, né, vai na conversa de qualquer
um e sai com muita meninada aí, isso gerava muita
polêmica (MORAIS, Adilson de. Entrevista. A/A). Tivemos o cuidado de perguntar para seu Agenor, seu Pedro, Alcideo e
dona Ana sobre como seus filhos usam os espaços apresentados e eles nos
disseram que estes também pouco frequentam o lago e a Prainha. Adilson e
Vilmar, mais jovens, pouco vão à Prainha. Adilson diz o mesmo sobre seus
irmãos. Logo, não se trata de uma rejeição que se remeta a uma questão
geracional. Em seguida, Adilson complementa a fala de sua mãe: ARANTES NETO, 2000). Implícito nessa discussão temos o exemplo de como os reassentados
procuram recriar territórios pautados, de alguma maneira, na vida de antes. E
jogam com essas possibilidades, aceitando ou não modificações de acordo
com as diferentes situações. Na fala de Adilson, o haras (hípica) não se enquadra como prática comum
dos moradores das cidades do Sudoeste atingidas pelas águas do Rio Iguaçu:
mesmo que tinha cultura de alguém, lá era particular, na propriedade do cara
(MORAIS, Adilson de. Entrevista. A/A). O fato de alegar que essa prática não era
típica da cultura do povo justificaria a crítica ao funcionamento da hípica. O lá e
o aqui são trazidos na narrativa e diferentes lugares e temporalidades servem
para que, através de uma explicação cultural, Adilson se posicione. De fato, a Prainha é mais procurada pelos visitantes do que pelos
moradores reassentados. Mesmo assim, com exceção da sugestão de seu
Agenor, os entrevistados não propuseram o fechamento do local para visitas. Já
a hípica, essa já foi fechada no passado, isso devido a certos desentendimentos
gerados pelo seu uso. Após certo período, uma assembleia optou por reabri-la. Foi o que nos contou seu Alcideo: Aquilo ali, a raia mesmo, virou uma polêmica muito grande,
porque aquilo ali tava meio abandonada, porque sempre
alguém tem que tomar a frente, daí conseguiu tomar a
frente, pela associação, daí da diretoria foi tirado uma
diretoria que chama Conselho Unificado, só que as 8
diretorias faz parte, daí foi decidido então de alugar essa
lanchonete. Tinha outras pessoas, daí foi aberto pras
pessoas trazer as propostas, daí ia ser analisado e quem
trouxesse a proposta melhor conseguia, então, daí foi
alugado pro rapaz lá, ele já queria logo, que fosse fazer a raia. Daí como nós não queria, eu aqui e o seu Pedro, nos já sabia
que o nosso pessoal aqui não ia aceitar a raia, né? (VOESE,
Alcideo. Entrevista. A/A.) Ao ser perguntado se não haveria controle, respondeu comentando
sobre uma interdição que ocorrera: É, não tem controle, tem meio malandrão, que já aconteceu
isso no passado [...] Aconteceu ali briga com o pessoal daqui Revista NUPEM, Campo Mourão, v. 3, n. 4, jan./jul. 2011 Revista NUPEM, Campo Mourão, v. 3, n. 4, jan./jul. 2011
127 Colonos ou reassentados: disputas políticas e identitárias em torno da construção da prainha do reassentamento com gente que cuidava... Em seguida, Adilson complementa a fala de sua mãe: Em relação à hípica, a rejeição está mais explicita, afinal, as corridas de
cavalo envolvem apostas inacessíveis à situação econômica dos reassentados,
além de alguns, como vimos, apontarem problemas ligados à utilização da
hípica como, por exemplo, o movimento de pessoas estranhas na localidade e
possíveis brigas. Nesses casos de contradições internas, os “transtornos” são
mais lembrados do que o lazer. Podemos, então, trazer um resumo sobre como a sede do Revista NUPEM, Campo Mourão, v. 3, n. 4, jan./jul. 2011 128 Colonos ou reassentados: disputas políticas e identitárias em torno da construção da prainha do reassentamento reassentamento passa a ser entendida nesses conflitos. Os reassentados com
quem conversamos pouco usam o lago e a hípica. A polêmica gira em torno do
contato que teriam nesses locais com pessoas desconhecidas e,
principalmente, com transtornos causados por parte dessas pessoas, que
procuram esses espaços para transgredir as condutas legais e morais. Essa
aceitação ou não do visitante envolve disputas políticas e administrativas. O coletivo hoje: só no futebol e na igreja? 2011 129 129 Colonos ou reassentados: disputas políticas e identitárias em torno da construção da prainha do reassentamento É que nós aqui quase não tamo indo [à igreja], até porque a
gente tem as vacas pra tratar, pra cuidar, não dá tempo, às
vezes. Tem ali a igrejinha, mais pra frente, daqui lá dá 5
quilômetros. Não, não é mesma coisa.( DANTAS, Agenor. Entrevista. A/A). Talvez essa afirmativa de seu Agenor esteja relacionada aos amigos que
ficaram e, ainda, a toda a mudança vivida: Isso foi implantado aqui [sobre a política de se manter as
comunidades e atividades coletivas de antes], mas, na
verdade, bastante do companheirismo a gente deixou pra
lá, a gente tem aqui, mas a gente não tem o suficiente,
pessoas de confiança, como se fosse um irmão, uma pessoa
da família, a gente deixou pra lá, porque eles não foram é... Atingidos. O terreno deles não foi diretamente atingido
pelas obras, mas na verdade foi, porque eles perderam a
gente lá, a vizinhança, quem foi atingido veio pra cá, e eles
ficaram, né, então no começo a gente teve uma briga com a
empresa, Nós queria que todos que estivessem ali fossem
indenizados (DANTAS, Agenor. Entrevista. A/A). Isso foi implantado aqui [sobre a política de se manter as
comunidades e atividades coletivas de antes], mas, na
verdade, bastante do companheirismo a gente deixou pra
lá, a gente tem aqui, mas a gente não tem o suficiente,
pessoas de confiança, como se fosse um irmão, uma pessoa
da família, a gente deixou pra lá, porque eles não foram é... Atingidos. O terreno deles não foi diretamente atingido
pelas obras, mas na verdade foi, porque eles perderam a
gente lá, a vizinhança, quem foi atingido veio pra cá, e eles
ficaram, né, então no começo a gente teve uma briga com a
empresa, Nós queria que todos que estivessem ali fossem
indenizados (DANTAS, Agenor. Entrevista. A/A). Essas atividades são significativas para os entrevistados na reconstrução
e afirmação de vida, no entanto muito se modificou com a mudança espacial. Podem até jogar com moradores de Aliança do Oeste (cidade atingida pela
barragem e de onde vieram muitos dos moradores do São Francisco de Assis) e
esse jogo ocorre anualmente, como nos informou outro morador, mas não é
mais a mesma coisa. Assim, também ocorre com a igreja católica. O coletivo hoje: só no futebol e na igreja? O que se faz nos fins de semana aqui no reassentamento? Ao se esforçar
para responder a essa pergunta, Zequinha falou da organização interna do São
Francisco de Assis. Para tanto, trouxe exemplos de como se dá a união dos
moradores em torno de comunidade: Mas era o objetivo nosso. Em vez de deixar um filho nosso ir
lá pra cidade, que fique aqui, oh. Oh, nós temos, agora no
outro domingo nós começamos outro campeonato de
futebol, domingo vai ter jogo. Eu sou treinador até. Mas, daqui a pouco nós vamos ter um
treininho ali. E no caso, domingo vamos jogar contra o
Juvinópolis e o Alto Alegre também tá no campeonato, mas
tão só com aspirante, nós temos titular e aspirante
(MEURER, José Rossi. Entrevista. A/A). Apesar da rivalidade esportiva com a comunidade do Alto Alegre, o
interesse do entrevistado era demonstrar a união que o futebol propicia no
reassentamento. Essas atividades são tão significativas que foram eleitas pelo
entrevistado como introdução de sua fala sobre a importância da construção
comunitária a partir das iniciativas de produção interna. O futebol e a religiosidade não se separam quando tratamos com os
entrevistados da organização social das comunidades de onde saíram e da
organização nas comunidades onde vivem agora. Estas práticas foram trazidas
e isso foi facilitado pela decisão de, dentro do possível, se manter as
vizinhanças. Todavia, para seu Agenor, o envolvimento de agora não é o mesmo
de antes: A única coisa que a gente tá faltando mais, eu acho até que é
porque a gente é mais velho, porque lá eu me envolvia
muito com diretoria de igreja e esporte. Você sai jogar e
coisa, então, aqui a gente já não tem muito, mas um pouco
eu acho que é porque que a gente não quer (DANTAS,
Agenor. Entrevista. A/A). A única coisa que a gente tá faltando mais, eu acho até que é
porque a gente é mais velho, porque lá eu me envolvia
muito com diretoria de igreja e esporte. Você sai jogar e
coisa, então, aqui a gente já não tem muito, mas um pouco
eu acho que é porque que a gente não quer (DANTAS,
Agenor. Entrevista. A/A). A comunidade religiosa e as relações de vizinhança, em parte, ainda
existem, mas não é a mesma coisa: Revista NUPEM, Campo Mourão, v. 3, n. 4, jan./jul. O coletivo hoje: só no futebol e na igreja? Ela foi
reconstruída, continua a paróquia com o mesmo nome, São Francisco de Assis,
os moradores entrevistados na sua maioria são católicos, mas o morador já não
vai à missa com a mesma frequência. As diferenças entre as celebrações religiosas de antes e de hoje, assim
como o jogo que disputavam lá e as partidas de agora, provavelmente estejam
no fato, entre outros, de que nem todos os antigos participantes dos locais de
origem estejam agora presentes nessas atividades no reassentamento. Além
da falta desses vizinhos, sua fala ainda reflete uma crítica à organização atual da
“comunidade”. Quando seu Agenor diz todos dali, na verdade, refere-se a todos
os pequenos agricultores e apresenta a “comunidade” enquanto um lugar,
espaço físico e reunião de trajetórias de iguais. Com esse raciocínio é necessário voltarmos às críticas já citadas de seu
Agenor sobre a venda de terras e transformação do local numa colônia, fato
que favoreceria, em sua opinião, a entrada de agricultores que não
participaram do processo de luta. Foi diante dessa postura que lemos sua fala
sobre o fato de as coisas já não serem as mesmas. De certa forma, ele segue com
a crítica à administração atual, que favorece a entrada de mais pessoas Revista NUPEM, Campo Mourão, v. 3, n. 4, jan./jul. 2011 130 130 Colonos ou reassentados: disputas políticas e identitárias em torno da construção da prainha do reassentamento estranhas na comunidade. Assim, portanto, nesse raciocínio, cada vez mais as
celebrações religiosas e os jogos seriam compostos por outros. ç
g
j g
p
p
E mais coisas mudaram, de acordo com o entrevistado, além do futebol e
da religião, na comparação entre ontem e hoje, ou entre lá e cá: E mais coisas mudaram, de acordo com o entrevistado, além do futebol e
da religião, na comparação entre ontem e hoje, ou entre lá e cá: Pergunto: Inclusive, aqui tem uma questão interna, é... Nem
sempre os nomes que estão no papel, é como as pessoas se
chamam. Por exemplo, acho que a Vargem Bonita, era
Aliança do Oeste, não? Agenor: Como é que é... A Aliança do Oeste era Barra Bonita
[...]. É aqui onde que o Genivaldo mora, na época era Maroto
e agora é Vargem Bonita. [...] Lá era Maroto. O São Lucas, não
era São Lucas, era Rato. [...] Pergunto: Quem que propôs esses nomes todos aí, Aliança,
Vargem Bonita... O coletivo hoje: só no futebol e na igreja? Agenor: Aqui foi o seguinte, quando a gente veio pra cá,
porque cada um era de uma localidade e cada localidade
tinha aquele nome, daí quando vem pra cá, aí mudaram, né,
o São Lucas que era Rato, mudou pra São Lucas... [...] O padroeiro era São Lucas, eles ponharam o nome da
comunidade deles de São Lucas. Aqui na Santa Luzia não foi
diferente: Santa Luzia era o Laranjal no começo, lá era o
Laranjal, daí a padroeira da igrejinha deles lá era Santa Luzia,
daí eles vieram pra cá e mudaram pra Santa Luzia. Pergunto: O senhor ainda fala: vamos ali no pessoal do
Laranjal, ou hoje em dia já...? Agenor: Hoje não falo porque fica eu falando e às vezes tem
gente que nem tem noção (DANTAS, Agenor. Entrevista. A/A). Agenor: Hoje não falo porque fica eu falando e às vezes tem
gente que nem tem noção (DANTAS, Agenor. Entrevista. A/A). Novamente as mudanças vividas são trazidas criticamente. Reparamos
que, ao citar o jogo que faziam contra a comunidade do São Lucas, o
entrevistado referia-se a ela com o nome como ela era conhecida em Boa Vista
da Aparecida: o Rato (esta foi nomeada por São Lucas no reassentamento, em
homenagem ao padroeiro da comunidade). Somente os que já moravam lá
conheceriam os nomes das comunidades que passaram pelo processo de
alagamento. Dessa maneira, podemos seguir a ideia de que nem o futebol, nem
as reuniões na igreja e nem mesmo o tratamento entre os membros de
comunidades vizinhas tem agora o mesmo significado. Provavelmente, ao
destacar essa outra diferença entre a vida em comunidade, e que poucos
conhecem o nome original dessas comunidades submersas, seu Agenor
novamente tece uma crítica à venda de terras para estranhos à luta dos
reassentados, pois, de acordo com ele: “hoje não fala porque fica eu falando e às
vezes tem gente que nem tem noção”. As entrevistas nos falam de ausências e de mudanças. Mesmo a igreja,
estando próxima de sua casa, mesmo os jogos ocorrendo e mesmo que as
vizinhanças tenham sido, dentro do possível, mantidas, já não é a mesma coisa. Revista NUPEM, Campo Mourão, v. 3, n. 4, jan./jul. 2011 131 131 Colonos ou reassentados: disputas políticas e identitárias em torno da construção da prainha do reassentamento A mudança espacial significou mudanças nas sociabilidades. O coletivo hoje: só no futebol e na igreja? Nem por isso,
porém, as divergências são novidades para os moradores que vieram para o
reassentamento, pois antes elas existiam também. Percebemos que a vida nas barrancas do Rio Iguaçu e na região próxima
não era desprovida de conflitos, por isso as discordâncias de agora não são algo
novo para seu Pedro. Elas foram apenas intensificadas diante das novas
situações que se apresentaram: É que maioria é com o grupinho, confusão, a outra maioria é
dívida... É que maioria é com o grupinho, confusão, a outra maioria é
dívida... Pergunto: Mas, por que que lá não havia brigas se eram os
mesmo vizinhos? Ressentimentos e conflitos existiam, mas a “necessidade” de agora, de se
demonstrar uma origem na terra e de ser um “empreendedor” gera novas
relações e outras possibilidades. Não teria sentido afirmar que as coisas não mudaram, até porque ficou
explícito nas reivindicações dos moradores o fato de que algo foi “deixado para
trás” e, a partir dessa perda, o processo de reassentamento iniciou-se. Mesmos
nos momentos em que, ao invés de saudosismo, aparecia presente nas
memórias a descrição de um processo de mudança marcado pelas dificuldades
e conquistas, ou mesmo de luta, o hoje não poderia ser mais o mesmo que o
vivido nas terras submersas. Nos diálogos anteriores podemos ter uma noção geral da sociabilidade
de antes e da de agora. Nessa apresentação de atividades coletivas, chegamos
a algumas considerações que “amarram” a problemática deste tópico. Primeiramente, o futebol e religiosidade geravam as principais formas de
sociabilidade das famílias antes de migrarem. Também percebemos que foi
com essas atividades que se buscou formar uma comunidade também no
reassentamento, dificilmente alcançada a partir de reuniões sobre a
administração local. Outra questão importante: tanto o futebol quanto a igreja
já não são a mesma coisa e essa observação está permeada pelas disputas pelo
território. Seu Agenor reforçou essa crítica ao narrar como as disputas
esportivas entre as comunidades não têm mais a mesma rivalidade de antes. Revista NUPEM, Campo Mourão, v. 3, n. 4, jan./jul. 2011 Revista NUPEM, Campo Mourão, v. 3, n. 4, jan./jul. 2011 Revista NUPEM, Campo Mourão, v. 3, n. 4, jan./jul. 2011
132 132 Colonos ou reassentados: disputas políticas e identitárias em torno da construção da prainha do reassentamento Provavelmente, a ausência de algum companheiro e a compra de terras por
pessoas da região influencia nessa diferença. O coletivo hoje: só no futebol e na igreja? Por fim, as divergências de hoje
não “controladas” pelo futebol e pela igreja não significam algo novo
socialmente falando. As confusões, como nos lembra seu Pedro, sempre
existiram e, agora, no reassentamento, ganham novas formas e novos arranjos,
seja na hora do futebol ou em qualquer atividade que vise o bem da
coletividade. Algumas considerações Nossa proposta foi dialogar com memórias para então apresentarmos
possibilidades de uma escrita sobre reconstrução de territórios. Tal proposta foi
permeada pelo estudo de identidades. Essas disputas podem ser entendidas a
partir do estudo da própria materialidade do reassentamento, nesse caso em
especifico com os projetos ligados ao ecoturismo, representado pela
construção e utilização da Prainha e da hípica, no reassentamento. As narrativas dos moradores demonstraram como as apropriações
simbólicas estão relacionadas com as disputas pelos lugares. Por sua vez, essas
disputas recorrem às disputas pelo passado. Afinal, dizer-se próximo ao
passado de luta, procedimento comum do grupo reassentado, ou ainda a toda
uma tradição que os liga ao homem do campo, autoriza os entrevistados a se
posicionarem e a reafirmarem lugares sociais no reassentamento e nas cidades
vizinhas. Dessa forma, as memórias nos ajudaram a compreender parte desse
processo de construção de territorialidades muitas vezes organizadas em
torno do dilema da construção comunitária, no qual a segurança presente na
idealização comunitária pouco pode ser alcançada diante das políticas atuais
de trabalho e talvez se aproxime de sua efetivação em práticas como o futebol. Construir um território remete ao entendimento de viveres marcados por
disputas, resistências e cooperação, e remete, também, ao entendimento dos
lugares sociais ocupados e disputados, das identidades disputadas e
negociadas, das políticas agrárias, energéticas e econômicas e da própria
estrutura regional. Notas * Mestre em História pela Universidade Estadual do Oeste do Paraná (UNIOESTE). E-mail: palhajr@yahoo.com.br. * Mestre em História pela Universidade Estadual do Oeste do Paraná (UNIOESTE). E-mail: palhajr@yahoo.com.br. 1 Este artigo apresenta parte das problematizações da pesquisa de mestrado
defendida em 2009 e intitulada: Memórias de Luta, Lutas pela Memória: O
Reassentamento São Francisco de Assis (1995- 2008). 2 Adotaremos, no decorrer de toda a escrita, a denominação “reassentado” e/ou
“atingido”. O uso destes termos metodologicamente ajuda-nos a apresentar os
sujeitos ou “agentes” sociais que interagem no decorrer de nossa pesquisa. Assim,
reassentado e atingido referem-se a pessoas que têm uma trajetória comum: o Revista NUPEM, Campo Mourão, v. 3, n. 4, jan./jul. 2011 Revista NUPEM, Campo Mourão, v. 3, n. 4, jan./jul. 2011
133 133 Colonos ou reassentados: disputas políticas e identitárias em torno da construção da prainha do reassentamento deslocamento forçado de suas terras devido ao processo de submersão desses locais
quando da construção da hidrelétrica de Salto Caxias; participam de um movimento
social em torno de luta na e pela terra e têm seus relacionamentos tanto com
moradores das cidades de Cascavel e Corbélia, por exemplo, como recebem outros
agricultores que compraram terras no reassentamento e não participam desse
processo vivido pelos reassentados. Um dos fatores que nos levaram a adotar esse
termo, reassentado, foi o fato de os próprios moradores se designarem dessa forma. Essas outras denominações e a razão do uso de outros termos serão citadas e
analisadas no decorrer do trabalho, visto que considerados o realce nos processos de
autoidentificação importantes para analisar as narrativas. 3 Os embates diante do Estado, representado pela Copel, significaram reunir em torno
de uma causa e com diferentes graus de adesão, políticos, grandes proprietários,
médios e pequenos agricultores, meeiros, posseiros e agregados. Os resultados desses
embates, apresentados no decorrer deste trabalho, significaram, a partir de 1995,
mudanças nas vidas de 1.322 famílias destituídas em nove municípios da região
Sudoeste do Paraná, sendo eles: Capitão Leônidas Marques, Boa Vista da Aparecida,
Três Barras do Paraná, Quedas do Iguaçu, Nova Prata do Iguaçu, Salto do Lontra, Boa
Esperança do Iguaçu, Cruzeiro do Iguaçu e São Jorge do Oeste. Dessas famílias, 612
foram reassentadas em 10 reassentamentos. As famílias atingidas pela água da
barragem, possuidoras de propriedades compostas por mais de 5 alqueires, foram
indenizadas com dinheiro. Notas Foram incluídas no projeto de reassentamento as famílias
que possuíam área de até 5 alqueires, juntamente com meeiros, posseiros e agregados. Assim, das 612 famílias reassentadas, 483 optaram pela indenização de terra por terra e
159 optaram pela indenização em dinheiro para comprarem terras em outras
localidades. Das famílias indenizadas por terra, 76% eram de pequenos proprietários. De acordo com: Relatório da Companhia Paranaense de Energia (COPEL). Departamento do Reservatório Usina Hidrelétrica de Salto Caxias. Programa de
reassentamento: determinação do público. Curitiba, maio de 1995. 4 Não pretendemos responder se a criação da colônia representa ou não um estágio
final do reassentamento, resultado dos movimentos de lutas por terra, inserção
regional, reivindicações de um movimento social e da construção de identidades. Da
mesma maneira, não podemos concluir se o nome colônia representa, de fato, as
expectativas da maioria dos sujeitos envolvidos no processo, nem, por outro lado,
defender que esta designação seja uma estratégia momentânea da administração
local para a inserção nos discursos e políticas do agronegócio ou das iniciativas de
agregar valor à mão de obra familiar. O que pretendemos apresentar são as disputas
sociais, administrativas e identitárias envolvidas nesse processo. 5 Aqui, ao utilizarmos o termo comunidade, referimo-nos à divisão espacial e
administrativa do reassentamento, sendo oito as comunidades do São Francisco de
Assis. 6 Pertencer a uma comunidade, no mundo atual, de acordo com Bauman, significa estar
diante de um dilema: a busca por segurança em meio à individualização típica das
sociedades capitalistas. O dilema de se viver em sociedade está marcado, nesse estudo
genealógico do autor, pelo poder de secessão dos “bem sucedidos”, elite que
sobreviveu à recessão do capital e que hoje vive fechada em redomas capitalistas. Protegidos do convívio em comunidades por seus condomínios e pelas possibilidades
cotidianas do mundo capitalista, construído em viagens a negócios e atividades
cotidianas realizadas virtualmente, pela internet, de maneira que não necessite de Revista NUPEM, Campo Mourão, v. 3, n. 4, jan./jul. 2011 Revista NUPEM, Campo Mourão, v. 3, n. 4, jan./jul. 2011
134 134 Colonos ou reassentados: disputas políticas e identitárias em torno da construção da prainha do reassentamento maior envolvimento comunitário. As contradições do convívio em comunidade ainda
foram bem destacadas pelo autor nas discussões sobre diferenças entre comunidades
fortes e fracas, com a observação das relações de força e desigualdades entre os
sujeitos, discursos e políticas que envolvem essas relações sociais. Notas Nesse sentido, um
exemplo apresentado por Bauman destaca maneiras como, em muitas relações
desiguais do capitalismo, os discursos sobre o multiculturalismo acabam sendo
apontados enquanto uma espécie de justificativa das elites para as desigualdades
sociais e históricas. Nessa perspectiva, a diferença cultural deixa de ser o ponto de
partida para o estudo do social e acaba corroborando a naturalização das diferenças. 7 Valor cobrado na temporada de 2007/2008. 8 Seu Zequinha, morador advindo do município de Três Barras, acompanhou o
processo de mobilização dos moradores e adquiriu terras no reassentamento em
meados de 2001. 9 Na obra em destaque, o passeio que Arantes nos convida a fazer pela Praça da Sé, em
São Paulo, ajuda-nos a compreender como os sujeitos se utilizam e atravessam lugares
e marcos de cenários híbridos. Fontes DANTAS, Agenor. Entrevista. Reassentamento São Francisco de Assis, em 5 de
março de 2008. (A/A). MEURER, José Rossi. Entrevista. Reassentamento São Francisco de Assis, em 25
de julho de 2007. (A/A). MORAIS, Adilson de. Entrevista. Reassentamento São Francisco de Assis, em 8
de março de 2008. (A/A). OLIVEIRA, Djair Márcio. Entrevista. Reassentamento São Francisco de Assis, 27
de julho de 2007. (A/A). ROCHA, Pedro. Entrevista. Reassentamento São Francisco de Assis, em 1º de
março de 2008. (A/A). VOESE, Ana e VOESE, Alcideo. Entrevista. Reassentamento São Francisco de
Assis, em 3 de março de 2008. (A/A). Referências ARANTES NETO, Antonio Augusto. Paisagens paulistanas: transformações
do espaço público. São Paulo: Ed. Unicamp, 2000. BAUMAN, Zygmunt. Comunidades: a busca por segurança no mundo atual. Rio de Janeiro: Jorge Zahar, 2003. HALL, Stuart. Identidade cultural na pós-modernidade. 7. ed. Rio de Janeiro:
DP & A, 2002. HALL, Stuart. Identidade cultural na pós-modernidade. 7. ed. Rio de Janeiro:
DP & A, 2002. HOBSBAWM, Eric. O sentido do passado: o que os historiadores devem a Karl
Marx?; A história vista de baixo para cima. In: _____. Sobre história. São Paulo:
Companhia das Letras, 1998. Revista NUPEM, Campo Mourão, v. 3, n. 4, jan./jul. 2011 Revista NUPEM, Campo Mourão, v. 3, n. 4, jan./jul. 2011 Revista NUPEM, Campo Mourão, v. 3, n. 4, jan./jul. 2011 Revista NUPEM, Campo Mourão, v. 3, n. 4, jan./jul. 2011
135 135 Colonos ou reassentados: disputas políticas e identitárias em torno da construção da prainha do reassentamento POLLAK, Michael. “Memória, esquecimento, silêncio”. Revista Estudos
históricos, 2 (3). Rio de Janeiro, 1989. PORTELLI, Alessandro. Tentando aprender um pouquinho. Algumas reflexões
sobre a ética na história oral. Revista Projeto História, São Paulo, PUC/SP, n. 15, 1997. WILLIAMS, Raymond. O campo e a cidade na história e na literatura. São
Paulo: Companhia das Letras, 1988. WILLIAMS, Raymond. O campo e a cidade na história e na literatura. São
Paulo: Companhia das Letras, 1988. Recebido em: fevereiro de 2010. Aprovado em: agosto de 2010. Revista NUPEM, Campo Mourão, v. 3, n. 4, jan./jul. 2011 136
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Spatial proximity matters: A study on collaboration
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PLOS ONE a1111111111
a1111111111
a1111111111
a1111111111
a1111111111 RESEARCH ARTICLE Editor: Jim Uttley, University of Sheffield, UNITED
KINGDOM Editor: Jim Uttley, University of Sheffield, UNITED
KINGDOM Received: February 25, 2021
Accepted: November 1, 2021
Published: December 1, 2021 Arianna Salazar MirandaID1*, Matthew Claudel2 1 Department of Urban Studies and Planning, Massachusetts Institute of Technology, Cambridge, MA, United
States of America, 2 Department of Geography, Portland State University, Portland, Oregon, United States of
America a1111111111
a1111111111
a1111111111
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a1111111111 * ariana@mit.edu * ariana@mit.edu a1111111111
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a1111111111 OPEN ACCESS OPEN ACCESS
Citation: Salazar Miranda A, Claudel M (2021)
Spatial proximity matters: A study on collaboration. PLoS ONE 16(12): e0259965. https://doi.org/
10.1371/journal.pone.0259965 Abstract As scientific research becomes increasingly cross-disciplinary, many universities seek to
support collaborative activity through new buildings and institutions. This study examines
the impacts of spatial proximity on collaboration at MIT from 2005 to 2015. By exploiting a
shift in the location of researchers due to building renovations, we evaluate how discrete
changes in physical proximity affect the likelihood that researchers co-author. The findings
suggest that moving researchers into the same building increases their propensity to collab-
orate, with the effect plateauing five years after the move. The effects are large when com-
pared to the average rate of collaboration among pairs of researchers, which suggests that
spatial proximity is an important tool to support cross-disciplinary collaborative science. Fur-
thermore, buildings that host researchers working in the same or related fields and from mul-
tiple departments have a larger effect on their propensity to collaborate. Spatial proximity matters: A study on
collaboration Arianna Salazar MirandaID1*, Matthew Claudel2 PLOS ONE PLOS ONE PLOS ONE PLOS ONE Spatial proximity matters: A study on collaboration Funding: The author(s) received no specific
funding for this work. Scholarship in several fields offers theoretical perspectives that explain the effect of physical
proximity on collaboration, even in an era of fast and cheap long-distance communication. One prevailing view emphasizes that geographical proximity leads to collaboration because the
so-called ‘tacit’ character of knowledge requires face-to-face interaction [9]. This is most often
explained by the fact that tacit knowledge is hard to transmit through writing and is best-
exchanged face-to-face through a range of interactions between individuals [10]. Other com-
plementary research argues that physical proximity influences social ties because of exposure:
the more proximate people are, the more likely they are to be exposed to one another, and the
higher the likelihood of a new social tie between them [5, 11]. Physical proximity is particularly
important for solving complex problems and promoting innovation, between new colleagues
as well as prior collaborators [12–14]. Competing interests: The authors have declared
that no competing interests exist. Competing interests: The authors have declared
that no competing interests exist. This paper explores whether physical proximity is an important determinant of collaboration
within organizations. We do so by studying over 10 years of collaborative activity among
researchers in the Massachusetts Institute of Technology (MIT). The MIT campus is particu-
larly well-fit to evaluating collaboration because the body of faculty and researchers are orga-
nized into discrete departments, labs, and research groups, which may be co-located or
physically separate. Furthermore, as a technical institute, MIT prioritizes technology transfer
from basic science, which requires increasingly diverse teams with varied skill sets [6, 15–20]. Researchers must confront a fundamental challenge when studying the effect of physical
distance on collaboration: location is not random. In particular, researchers collaborating or
seeking to collaborate might decide to locate close to one another. We address this identifica-
tion challenge by exploiting a natural experiment. As a result of centralized administrative
decisions about office renovations and new building openings, faculty were relocated across
MIT buildings. This discrete shift allows us to estimate the causal effect of physical proximity
on collaboration by looking at pairs of researchers who were moved to the same building. We
exploit this variation using fixed effects models and a treatment effects framework. 1 Introduction A longstanding view in urban planning and management science maintains that geography
and distance play a role in shaping the exchange of ideas and collaboration [1–5]. Motivated
by this conviction, building and campus managers have invested vast amounts of resources in
their physical spaces, transforming traditional office spaces into open floor plans with fewer
walls and doors and inviting common areas with the objective to create dense and attractive
environments that support interactions and chance encounters. Meanwhile, the ubiquity of
communication technologies has led many to question whether proximity will continue to
play a critical role. Scholarship is increasingly collaborative, but also increasingly spread across
universities [6]. Some hypothesize that telecommunication technologies negate the value of
physical proximity [7]. Copyright: © 2021 Salazar Miranda, Claudel. 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 underlying
the results presented in the study is not
immediately public. However, the Office of
Institutional Research (OIR) is willing to provide
the data set to a third party if requested individually
by that party. Data are available from the OIR in the
Office of the Provost for researchers who meet the
criteria for access to confidential data. Please
contact the OIR at the following address:
Massachusetts Institute of Technology; 77
Massachusetts Avenue, Room 11-268; Cambridge,
MA 02139-4307 or email them at: ir@mit.edu. The recent COVID-19 pandemic caused a dramatic shift in workplace practices and modes
of collaboration. Millions worldwide have been forced to abandon the physical office and
work, study, and collaborate remotely. Some companies like Twitter have gone as far as to
allow their employees to work remotely permanently [8]. The value of in-person collaboration,
serendipity, and workplace environment has become a crucial question, both for knowledge
organizations and cities. 1 / 18 PLOS ONE | https://doi.org/10.1371/journal.pone.0259965
December 1, 2021 December 1, 2021 PLOS ONE Complementary evidence has shown that being proxi-
mate enhances communication [27, 28], and amplifies the quality of collaborative outcomes
[29]. This finding has been replicated in various settings, including engineering offices [30,
31], and scientific offices [4]. Not only does geographical proximity play an important role in
facilitating collaboration [32–34], but the inverse has also been studied: collaborators tend to
be located more geographically proximate [35]. Within this literature, we most closely follow [5, 36–38]. [36] analyzes data from teachers
working at five public schools and documents greater collaboration and ties among school
teachers who are assigned to classrooms on the same floor. Our findings complement the
work of [5], who uses similar exogenous variation to demonstrate the effect of sudden co-loca-
tion on likelihood of collaboration between knowledge workers. [37] run a field experiment at
the Harvard Medical School and show that researchers randomly assigned to share an infor-
mation session are more likely to co-apply to grants. In line with these papers, our findings
suggest a key role for proximity in facilitating collaboration within organizations. We comple-
ment these studies by emphasizing the role of physical co-location (a point we share with [36])
in fostering collaboration even among researchers in different fields. In addition, we trace col-
laboration patterns over time and document a persistent positive effect on the propensity to
co-author (a point we share with [5]). In addition, [38] studies collaboration patterns in MIT,
describing how these depend on networks, departments, and the location of researchers. Because we exploit a discrete shift in the location of researchers, our analysis brings greater
explanatory power as to the causal effect of proximity on collaboration patterns. This paper is also related to scholarship that analyzes the relationship between the physical
layout and characteristics of spaces, on one hand, and interaction between individuals, on the
other [39, 40]. Linear measures of distance alone miss important aspects of spatial layouts. [41]
developed space syntax techniques to explicitly quantify built spaces by measuring the distance
between and the experiential qualities of rooms, passageways, and public spaces. This has led
to ongoing research explaining how the nuances of spatial design affect collaboration [42]. Related studies use location-tracking devices to follow individuals’ specific location within a
room [40]. Architecturally sophisticated characterizations of the physical environment and
detailed tracking provide rich insights, and can inform architectural design [43]. PLOS ONE The treat-
ment group includes the pairs of researchers who moved to the same building for the first time
as MIT affiliates, and the control group includes pairs of MIT affiliates who have never shared
the same building. To measure collaboration, we combine research publications spanning the 2005–2015
period with MIT’s directory data. Using these data, we construct two measures of collabora-
tion: the number of papers co-authored by a pair of researchers in each given year and a
dummy for whether the pair collaborated at all in each year. We find that moving two researchers to the same building increases their collaboration rate
up to 2.7 on the third year after moving. We explain the time delay as a standard cycle of aca-
demic publication [21, 22]. The effect plateaus at 1.85 more papers per hundred pairs five
years after the move. To put this in context, this means that moving researchers to a new build-
ing where they share space with 100 new colleagues increases their collaboration with these
new colleagues by 0.8 papers per year. This is a large number relative to the average rate at
which MIT researchers co-author papers, which is around 1 paper per year. Reassuringly, we
find no evidence of pretrends in collaboration among researchers that were moved to the same
building, which suggests that people who got moved to the same building were not already col-
laborating before the move. One advantage of our data is that we can also explore the organizational characteristics that
mediate the increase in collaboration documented above. In the second part of the paper, we
explore the role of the density of researchers, the number of departments and their distribution
across buildings, and the discipline affinity of researchers in a given building. We find that
moving researchers into buildings that host researchers working in the same or related fields
and from multiple departments can foster more collaboration. 2 / 18 PLOS ONE | https://doi.org/10.1371/journal.pone.0259965
December 1, 2021 PLOS ONE Spatial proximity matters: A study on collaboration Literature has examined the importance of (and difference between) physical proximity
and organizational proximity as an influence on the likelihood of collaboration between indi-
viduals in organizations [23–25]. One explanation holds that physical proximity influences
social ties because less effort is required to connect with physically closer individuals relative to
individuals who are more distant [26]. PLOS ONE | https://doi.org/10.1371/journal.pone.0259965
December 1, 2021 PLOS ONE However, in
this paper, we measure spatial proximity using researchers’ co-location in the same building
rather than physical distance, and without great detail about the designed qualities of the
space. Although it offers less nuance, our approach can be more easily replicated with large
datasets from businesses or campuses. In addition, the ‘functional’ approach to proximity proposed by [44] is particularly well
suited to identify the conditions under which unexpected collaboration might happen. A study
that examined an academic setting found that faculty whose offices were located along central
corridors had greater co-authorship rates than did colleagues whose offices were more periph-
eral [39]. Other studies have explored the spatial layout designs that support collaboration in
the context of the workspace. For example, recent work suggests that layout characteristics
such as the percentage of floor space dedicated to shared services and amenities [45] and the
visibility across different spaces [46] are associated with knowledge sharing. Although we
acknowledge the value of implementing a more sophisticated characterization of the physical
environment, in this paper, we measure spatial proximity using researchers’ co-location in the
same building rather than physical distance. We follow this approach because offices and PLOS ONE | https://doi.org/10.1371/journal.pone.0259965
December 1, 2021 3 / 18 December 1, 2021 PLOS ONE Spatial proximity matters: A study on collaboration researchers’ allocation within buildings could be subject to particular research agendas or
space allocation constraints. This is especially relevant at MIT, where office space can be desig-
nated to entire labs instead of individual researchers. For this reason, we focus on the move-
ment of researchers to the same building rather than the precise position that researchers
occupy within a given building. Finally, this paper is related to a large body of literature documenting collaboration patterns
using citations received by scientific articles and co-authorships [47, 48]. Among the strongest
conclusions drawn by this line of research is the trend towards collaboration [17, 18] and an
increase in scientific publication co-authorships across nearly all disciplines [19]. Science
across many fields is becoming more interdisciplinary, drawing on a greater variety of skills
and expertise [15, 47], and producing work with a higher impact that spans many different
institutions and crosses national boundaries [49]. This paper provides evidence using scholarly
output as a proxy for collaboration, to shed light on the spatial dimensions of the knowledge
creation process. PLOS ONE The remainder of the paper is organized as follows. Section II describes the data sources
and outlines how we construct the building measures for our empirical analysis. Section III
introduces our empirical strategy and presents our results. Section IV is a brief discussion, and
section V concludes. 2 Data and measurement This section describes the data sources and main variables used in the empirical analysis. We
use directory information that describes MIT affiliated faculty and combine it with data on
publications from MIT affiliated faculty for the 2005-2015 period. The two databases are linked
using the MIT Identification Number: a unique 9-digit numerical value assigned to each MIT
affiliate, which persists through changes in affiliation over time. The MIT Directory database includes organizational affiliation, such as school, department,
or lab, as well as the location-attributes of the offices, such as building, floor, and room. To cal-
culate the geographical proximity between MIT affiliates, we extract the office number from
the MIT Directory and use it as an indicator that distinguishes if researchers share the same
building or not for every year in the sample. Because publications in peer-reviewed scientific journals are the most common form of
scholarly output in a research institution, and because co-authorship is a common mode of
scholarly collaboration, we use papers as a proxy for collaboration. Here, collaboration is
defined as the co-authorship between any two or more affiliates during a given year. We use
co-authorships as a proxy for intellectual engagement [47, 50]. The dataset includes papers
published by MIT-affiliated individuals in peer-reviewed journals with DOI number identifi-
ers, as well as the date, and authorship. This publication information is available from a com-
prehensive list aggregated by Academic Analytics—a non institutional affiliated data analytics
company. Academic Analytics aggregates publication data from scholarly journals, for the pur-
poses of evaluation, strategic decision-making, and benchmarking in universities. A publica-
tion may contain multiple inter-department or intra-department pairs. All department pairs
are counted according to their rate of occurrence and every co-publication of two or more
individuals is counted as a co-authorship. There are 878,337 MIT co-authorship instances and
38,211 papers (with unique DOIs) spanning the years 2005 to 2015. In this dataset, there are
1,417 total MIT authors; including faculty and non-faculty. Fig 1 plots the average number of publications among MIT affiliates over time, as well as
the average number of papers that are coauthored, both with researchers within and in a PLOS ONE | https://doi.org/10.1371/journal.pone.0259965
December 1, 2021 4 / 18 PLOS ONE Spatial proximity matters: A study on collaboration Fig 1. The average number of publications, co-authorships, intra-department co-authorships, and inter-
department co-authorships by year. Fig 1. 2 Data and measurement The average number of publications, co-authorships, intra-department co-authorships, and inter-
department co-authorships by year. https://doi.org/10.1371/journal.pone.0259965.g001 different department. These figures reveal a rise in collaboration from 2005 to 2012, followed
by a decline since then. PLOS ONE | https://doi.org/10.1371/journal.pone.0259965
December 1, 2021 2.1 Measuring organizational attributes in buildings The composition of researchers in buildings can contribute to the frequency and volume of
communication and subsequent collaboration [4, 5, 39, 40]. Following this literature, we focus
on measuring four organizational attributes that are suggested as important mediators of col-
laboration among researchers: the density of researchers per building, the number of depart-
ments hosted in each building and the distribution across buildings, and the discipline affinity
of researchers in each building. We measure the density of researchers in each building by the
number of different researchers per 100 square meters. To measure the distribution of depart-
ments across buildings we first compute for each department d the shares of researchers in
each building, sb
d, so that P
bsb
d ¼ 1. We then compute the sum of these departmental shares
for each building, given by P
dsb
d. By construction, this measure is low when a building hosts
departments that are spread across multiple buildings. Conversely, this measure is high when a
building hosts departments that are concentrated in that building. To measure the discipline
affinity among researchers, we match the department represented by each faculty to a set of 11
high-level disciplines. In particular, we use the network in Fig 2b proposed by [51] to define
the related and unrelated disciplines. For instance, humanities are directly linked to social sci-
ences, and social sciences are linked to mathematics and engineering. Similarly, computer sci-
ence is more closely related to physics, and physics is also closely related to engineering. The
disciplines included in our analysis are mathematics, computer science, physics, chemistry,
engineering, earth sciences, biology, brain research, health, social science, and humanities. 5 / 18 PLOS ONE | https://doi.org/10.1371/journal.pone.0259965
December 1, 2021 December 1, 2021 PLOS ONE Spatial proximity matters: A study on collaboration Fig 2. The maps show the density of researchers in each building (top left), the number of departments (top right), the distribution of
departments across buildings (bottom left), and the research affinity (bottom right). Buildings shown in grey do not host researchers and are
therefore not included in our sample. Fig 2. The maps show the density of researchers in each building (top left), the number of departments (top right), the distribution of
departments across buildings (bottom left), and the research affinity (bottom right). Buildings shown in grey do not host researchers and are
therefore not included in our sample. 2.1 Measuring organizational attributes in buildings https://doi.org/10.1371/journal.pone.0259965.g002 https://doi.org/10.1371/journal.pone.0259965.g002 Since categories such as Psychology/psychiatry and Medical specialties don’t exist at MIT, we
exclude them from our categorization. Fig 2 shows the resulting four measures for each building across campus. The top panel
shows the density of researchers per 100 square meters and the number of departments per
building. The bottom panel shows the distribution of departments across buildings and the
researchers’ discipline affinity in each building. For each measure, buildings shown in red are
the highest values, and values represented in blue are the lowest. PLOS ONE | https://doi.org/10.1371/journal.pone.0259965
December 1, 2021 3.1 Fixed effects estimator To study the effect of proximity on collaboration, we first present fixed-effect estimates that
exploit variation in the location of researchers across buildings over time. In particular, we
estimate the regression model of collaboration among researcher pairs: Collaborationt;p ¼ b Same Buildingt;p þ dp þ lt þ εt;p
ð1Þ ð1Þ Here, each observation corresponds to a pair of affiliated MIT researchers, p, in year t = 2005,
2006, . . ., 2015, independently of whether they collaborated on that year or not. Our estima-
tion sample includes 961 researchers and 449,055 pairs of researchers. To ensure that we are
only identifying the effects of proximity by comparing existing researchers who moved to the
same building, we exclude new MIT affiliates that enter the sample after 2005. The dependent
variable, Collaborationt,p denotes the number of papers co-authored by pair p during year t. This variable is set to zero for pairs that did not collaborate in year t. To facilitate the interpre-
tation of the point estimates, we multiply the variable by 100, such that we observe papers per
hundred pairs of researchers produced each year. The key explanatory variable is Same Buil-
dingt,p, a dummy variable that indicates whether the pair of researchers are located in the same
building in year t. β is the coefficient of interest, which captures the relationship between prox-
imity and collaboration. Eq (1) assumes that collaboration by pair p in year t also depends on
pair fixed effects, δp, year fixed effects, λt, and an error term, εt,p. 6 / 18 PLOS ONE | https://doi.org/10.1371/journal.pone.0259965
December 1, 2021 December 1, 2021 PLOS ONE Spatial proximity matters: A study on collaboration The inclusion of pair fixed effects ensures that β is identified from the change in collabora-
tion following the movement of a pair of researchers to the same building, after accounting for
their baseline rate of collaboration. Treating δp as a fixed effect that must be controlled for (as
opposed to a random effect) is important because one could imagine pairs with a greater pro-
pensity to collaborate sorting into the same building, which would bias our estimates of β. Pair fixed effects allow for a different intercept for each pair of researchers in our sample
and also control for permanent differences in collaboration across pairs. 3.1 Fixed effects estimator For example, pair
fixed effects account for the possibility that a given pair of researchers who share a common
past, have similar interests, and have compatible personalities will tend to build long-lasting
collaboration relationships independently of whether they are in the same building. Pair fixed
effects are also more general than a specification that explains collaboration as a function of
individual researcher fixed effects. A specification with individual researcher fixed effects
assumes that collaboration is given by Collaborationt;p ¼ b Same Buildingt;p þ giðpÞ þ gjðpÞ þ lt þ εt;p; where i(p) and j(p) denote the identity of the two researchers in pair p and γi(p) and γj(p) denote
their respective fixed effects. Note that one can always define δp = γi(p) + γj(p), which implies
that pair fixed effects provide a more general functional form for collaboration patterns. In
particular, specifications with researcher fixed effects require their effects to be additive, ruling
out complementarities or pair-specific differences in collaboration. As such, pair fixed effects
account for the fact that some researchers will collaborate more with others independently of
their proximity (i.e., δp is high for all pairs that include these highly collaborative individuals). Regarding inference, we report standard errors that are two-way clustered by each
researcher in a pair, p. This procedure recognizes that the error term εt,p might be correlated
across pairs of researchers that have at least one researcher in common. For example, one par-
ticular researcher might have a very productive year, increasing the number of papers coau-
thored with some of her colleagues, and generating correlation across some of the pairs that
include her. Intuitively, despite having a large number of pairs in our data, these are formed by
the same 961 researchers who appear repeatedly in multiple pairs. For this reason, we cluster
at the researcher level. Note that the inclusion of pair fixed effects does not ensure that the
error term εt,p is independent across pairs nor overtime. Pair fixed effects only remove the per-
manent collaboration component of a pair but cannot account for other forms of correlation
between pairs that have a researcher in common. For example, a positive collaboration shock
might improve collaboration between A and B and also cause A to collaborate more with C in
a given year, inducing correlation in the collaboration patterns of the pairs (A, B) and (A, C). PLOS ONE | https://doi.org/10.1371/journal.pone.0259965
December 1, 2021 PLOS ONE Spatial proximity matters: A study on collaboration Table 1. Estimates of the effect of proximity on collaboration. DEPENDENT VARIABLE: COLLABORATION
(1)
(2)
(3)
(4)
(5)
(6)
PANEL I. COLLABORATION RATE (PAPERS PER HUNDRED PAIRS EACH YEAR)
Same Building
1.765
0.786
0.382
0.381
0.381
0.374
(0.560)
(0.263)
(0.119)
(0.119)
(0.119)
(0.120)
Lagged Collaboration
0.348
0.348
0.348
0.348
(0.067)
(0.067)
(0.067)
(0.067)
Implied Long-Run Effect
0.586
0.585
0.584
0.574
Observations
3357979
3325534
2874249
2874249
2874249
2874249
Number of Researchers
961
925
887
887
887
887
Number of Pairs
449055
416610
383361
383361
383361
383361
R-squared
0.00
0.49
0.61
0.61
0.61
0.61
PANEL II. DUMMY FOR COLLABORATION (MULTIPLIED BY 100)
Same Building
0.707
0.208
0.197
0.197
0.196
0.186
(0.114)
(0.061)
(0.064)
(0.064)
(0.064)
(0.064)
Lagged Collaboration
0.011
0.011
0.011
0.011
(0.014)
(0.014)
(0.014)
(0.014)
Implied Long-Run Effect
0.199
0.199
0.199
0.188
Observations
3357979
3325534
2874249
2874249
2874249
2874249
Number of Researchers
961
925
887
887
887
887
Number of Pairs
449055
416610
383361
383361
383361
383361
R-squared
0.00
0.35
0.36
0.36
0.36
0.36
Covariates:
Pair Fixed Effects
✓
✓
✓
✓
✓
Lagged Collaboration
✓
✓
✓
✓
Year Fixed Effects
✓
✓
✓
Department Fixed Effects
✓
✓
Same Department
✓ Table 1. Estimates of the effect of proximity on collaboration. Notes: The table presents OLS estimates of the relationship between being in the same building and the collaboration rate between pairs of MIT researchers. Panel I
shows results defining collaboration rates in terms of papers per hundred pairs per year. Panel II uses a dummy variable for collaboration. Column 1 presents the
estimates for the baseline specification with no controls. Column 2 controls for researcher-pair fixed effects. Column 3 controls for lagged collaboration among pairs. Column 4 controls for year fixed effects. Column 5 controls for a full set of department fixed effects for both researchers in a pair. Column 6 controls for a dummy of
whether the two researchers in a pair are affiliated with the same department. In parentheses, we report standard errors that are robust against heteroskedasticity and
correlation within researchers across pairs. Notes: The table presents OLS estimates of the relationship between being in the same building and the collaboration rate between pairs of MIT researchers. Panel I
shows results defining collaboration rates in terms of papers per hundred pairs per year. Panel II uses a dummy variable for collaboration. 3.1 Fixed effects estimator This example also shows why two-way clustering at the individual researcher level is more
appropriate in our context than clustering at the pair level. Table 1 presents the estimates of Eq (1). The first panel reports the coefficients for our con-
tinuous collaboration measure (defined as papers per hundreds of pairs per year) as the depen-
dent variable. The second panel repeats the same specifications but using a dummy variable of
collaboration as the dependent variable. Here we also multiply the dependent variable by 100
to facilitate its interpretation. Column 1 presents the estimates for the baseline specification with no controls. The esti-
mates in column 1 show that researchers located in the same building produce 1.765 more
papers per hundred of pairs each year than researchers in different buildings. Panel B shows
that this is to a large extent driven by a 0.707 percentage point increase in the likelihood of col-
laboration among researchers in the same building relative to others. Column 2 goes one step
further and controls for pair fixed effects, which ensures that our estimates are identified from 7 / 18 PLOS ONE | https://doi.org/10.1371/journal.pone.0259965
December 1, 2021 https://doi.org/10.1371/journal.pone.0259965.t001 PLOS ONE | https://doi.org/10.1371/journal.pone.0259965
December 1, 2021 PLOS ONE Column 1 presents the
estimates for the baseline specification with no controls. Column 2 controls for researcher-pair fixed effects. Column 3 controls for lagged collaboration among pairs. Column 4 controls for year fixed effects. Column 5 controls for a full set of department fixed effects for both researchers in a pair. Column 6 controls for a dummy of
whether the two researchers in a pair are affiliated with the same department. In parentheses, we report standard errors that are robust against heteroskedasticity and
correlation within researchers across pairs. denote a coefficient significant at the 1% level, denote a coefficient significant at the 1% level,
at the 5% level, and
at the 10% level. https://doi.org/10.1371/journal.pone.0259965.t001 at the 5% level, and at the 10% level. the variation of researcher pairs being moved to the same building. The estimated increase in
collaboration rate is now of 0.786 papers per hundred pairs. Column 3 controls for the lag
value of the collaboration rate among pairs of researchers the year before they moved to the
same building. This accounts for the possibility that researchers were collaborating before
moving to the same building, which would bias our estimates. We find evidence of persistence
in collaboration, with a coefficient of ρ = 0.384. The immediate effect of moving researchers to
the same building is an increase in their collaboration rate of 0.382 papers per hundred pairs. Because this increase in collaboration persists, the estimates in Column 3 imply larger long-
run effects of collaboration given by β/(1 −ρ) = 0.586 papers per hundred pairs, and reported PLOS ONE | https://doi.org/10.1371/journal.pone.0259965
December 1, 2021 8 / 18 PLOS ONE Spatial proximity matters: A study on collaboration at the bottom rows of the table. Finally, Columns 4, 5, and 6 control for year and building fixed
effects, a full set of department fixed effects for both researchers in a pair p, and a dummy of
whether the researchers are affiliated with the same MIT department, respectively. The inclu-
sion of these controls does not affect our findings from Column 3. An alternative framework for estimating Eq (1) is a random effects model. Different from a
fixed effects model, in random effects, the key assumption is that the unobserved pair compo-
nent δp is orthogonal to whether researchers share the same building. PLOS ONE Random effects models
are more efficient and precise but rely on this stronger assumption. Estimates of the model in
column 2 via random effects deliver a point estimate of 1.133 (s.e = 0.043). This is larger than
the fixed effects estimate reported in Table 1, column 2, and their difference is statistically sig-
nificant at all traditional levels. This suggests that the assumptions for random effects might be
violated in our context. In particular, the difference between these models suggests that pairs
with a higher permanent collaboration component δp tend to sort into the same buildings
underscoring the importance of controlling for pair fixed effects. 3.2 Treatment effects framework To be more explicit about the control and treatment groups, we present an in-depth analysis
of pairs of researchers who moved to the same building in a given year. This analysis shows
how collaboration changes over time. For each year between 2006 and 2014, we define treat-
ment and control groups as follows: Tt;p ¼
1
if the pair moved to the same building for the first time in year t
and both were affiliated with MIT in previous years:
0
if the pair has never shared the same building but are both
affiliated with MIT:
8
>
>
>
>
<
>
>
>
>
:
ð2Þ ð2Þ The treatment group comprises all pairs of researchers who were not located in the same build-
ing initially but moved to the same building a given year t. The control group comprises all pairs
of researchers who never shared the same building during the 10-year period between 2005 and
2015. Our sample excludes pairs observed in 2005 (since we do not know if this is their first year
at MIT), and pairs in 2015 because we cannot trace their subsequent collaboration patterns. Table 2 shows the number of researcher pairs by year in the treatment and control groups. The treatment group (pairs who moved to the same building in the year t) represents approxi-
mately 3% of the total pairs. Using this treatment and control assignment, we estimate the following regression model: Collaborationtþh;p ¼ bh Tt;p þ lh;t þ εt;h;p:
ð3Þ ð3Þ Here, Collaborationt+h,p denotes the number of papers co-authored by pair p during year
t + h. We allow h to vary from -4 to 4 to understand how co-location relates not only to the
current collaboration but also to past and future collaboration patterns. Scholarly publications
are characterized by long delays to publish. The choice to focus on a 4 year time span is moti-
vated by the fact that total average time delay from submission to publication in any field jour-
nal is 12.2 months [22]. βh is the main coefficient of interest, which captures the relationship
between co-location and collaboration. λh,t is a full set of year fixed effects capturing trends in
collaboration over time, t. Finally, εt,h,p is the error term, which we again allow to be correlated
within researchers across pairs. PLOS ONE | https://doi.org/10.1371/journal.pone.0259965
December 1, 2021 PLOS ONE Spatial proximity matters: A study on collaboration Table 2. Number of researcher pairs assigned to treatment and control groups by year. NUMBER OF TREATED AND CONTROL PAIRS BY YEAR
2006
2007
2008
2009
2010
2011
2012
2013
2014
Number of Researcher
453,827
460,818
468,738
470,375
484,885
474,807
474,676
478,970
485,652
Pairs in Control Group (0)
Number of Researcher
760
287
287
524
285
2,518
810
221
1,321
Pairs in Treated Group (1) Table 2. Number of researcher pairs assigned to treatment and control groups by year. Notes: The table presents the number of MIT researcher pairs assigned to the treatment and control groups by year. The first row summarizes the control group, which
corresponds to the number of MIT researcher pairs that have never shared the same building in the 2005-2015 period. The second row summarizes the treatment group,
which corresponds to the number of MIT affiliated pairs that moved to the same building for the first time in each year and were already affiliated with MIT before the
move. Notes: The table presents the number of MIT researcher pairs assigned to the treatment and control groups by year. The first row summarizes the control group, which
corresponds to the number of MIT researcher pairs that have never shared the same building in the 2005-2015 period. The second row summarizes the treatment group,
which corresponds to the number of MIT affiliated pairs that moved to the same building for the first time in each year and were already affiliated with MIT before the
move Notes: The table presents the number of MIT researcher pairs assigned to the treatment and control groups by year. The first row summarizes the control group, which
corresponds to the number of MIT researcher pairs that have never shared the same building in the 2005-2015 period. The second row summarizes the treatment group,
which corresponds to the number of MIT affiliated pairs that moved to the same building for the first time in each year and were already affiliated with MIT before the
move. https://doi.org/10.1371/journal.pone.0259965.t002 https://doi.org/10.1371/journal.pone.0259965.t002 Fig 3. Figure showing the estimated treatment effect of moving to the same building for each year before and
after the move with 90% confidence intervals. Left panel shows the estimates for βh of Eq (3) and the right panel the
estimates controlling for the lag of collaboration the year before treatment. https://doi.org/10.1371/journal.pone.0259965.g003 Fig 3. 3.2 Treatment effects framework The left panel in Fig 3 plots the estimates for βh for h = −5 to h = 5. Moving to the same
building increases the collaboration rate between researchers by 0.8 papers per hundred pairs PLOS ONE | https://doi.org/10.1371/journal.pone.0259965
December 1, 2021 9 / 18 PLOS ONE PLOS ONE Figure showing the estimated treatment effect of moving to the same building for each year before and
after the move with 90% confidence intervals. Left panel shows the estimates for βh of Eq (3) and the right panel the
estimates controlling for the lag of collaboration the year before treatment. https://doi.org/10.1371/journal.pone.0259965.g003 PLOS ONE | https://doi.org/10.1371/journal.pone.0259965
December 1, 2021 10 / 18 PLOS ONE Spatial proximity matters: A study on collaboration on the year of the move (t = 0). Three years after moving, the effect increases to 2.7 and pla-
teaus at 1.85 more papers per hundred pairs five years after the move. The right panel in Fig 3 plots the estimates for βh, but now controlling for lagged collabora-
tion rates the year before the move. Moving to the same building increases the collaboration
rate between researchers by 0.5 papers per hundred pairs on the first year. Three years after
moving, the effect increases to 2.5 and plateaus at 1.77 more papers per hundred pairs five
years after the move. The difference-in-difference results are a weighted average of all of the
treatment effects estimated in Fig 3. However, the weights vary with the sample being treated
each year, and hence, the magnitude of the difference-in-difference estimate is not necessarily
comparable to Fig 3. This explains why the long run effect of 0.6 in the difference-in-difference
exercise is lower than the 1.77 long run effect in Fig 3. Importantly, we find no evidence of pretrends in any of these figures, suggesting that the
increase in collaboration starts after the move and did not precede it. To further bolster our identification, we now use an inverse probability score weighting to
account for observed differences between pairs in the control and treatment groups. In partic-
ular, we use a logistic regression model to estimate the probability that a pair is moved to the
same building as a function of the departmental affiliation of both researchers, year dummies,
and their past collaboration in t −1. Following [52], we then estimate the average treatment
effect on the treated—ATT—by estimating Eq (3) after reweighting the data by the inverse of
the propensity score. This ensures that the control group has a similar predicted probability of
treatment to treated units in the reweighted sample. Fig 4 plots the estimated coefficients for the ATT five years before (to check for pretrends)
and five years after the move. 3.3 Building heterogeneity and collaboration Some buildings are occupied entirely by a single department or lab, while others host a diverse
group of faculty from various disciplines. In this section we explore the role of organizational
arrangements in promoting collaboration. As a first step, we estimate Eq (1) for each building: the increase in collaboration as a result
of moving two researchers to the same building. We make this estimate separately for each
building on the MIT campus. Formally, this entails including a full set of interactions between
the Same buildingt,p dummy and indicators for the building hosting that pair. The point esti-
mate on each interaction gives the gains in collaboration from moving researchers to each
building. Although the building-specific estimates must be interpreted with caution due to the
small sample of researchers in each building used to estimate their effect on collaboration, the
estimates nevertheless suggest some heterogeneity across buildings. Fig 5 summarizes our
results by plotting the building-specific estimates on collaboration using different colors. Buildings shown in dark purple have an estimated impact on collaboration above 1. Buildings
in orange have an estimated impact on collaboration between 0 and 0.5. In addition, there are
21 buildings with negative but generally imprecise estimates (shown in yellow). We now explore the differences across buildings more systematically, in order to evaluate
the role of their distinct institutional arrangements. We estimate a variant of Eq (1) that allows
the effect of being in the same building to vary with organizational attributes of buildings. In
particular, we consider the role of four variables: the density of researchers, the number of
departments and their distribution across buildings, and the affinity of fields hosted in a build-
ing. In addition, to ensure that these interactions are not confounding other differences across
buildings, we control for the interaction between being in the same building and specific
physical attributes of buildings, using three variables: total building area, the share of area Fig 5. Figure showing the estimated collaboration effect for each building (Eq (1)). Buildings shown in dark purple have the highest estimated values
of collaboration, and buildings shown in yellow correspond to the lowest. Buildings shown in grey do not host researchers and are therefore not Fig 5. Figure showing the estimated collaboration effect for each building (Eq (1)). Buildings shown in dark purple have the highest estimated values
of collaboration, and buildings shown in yellow correspond to the lowest. PLOS ONE Reassuringly, the coefficients before treatment are precisely
estimated zeros, which suggests that people who got moved to the same building where not
already collaborating more and thus the control group is a suitable one. Fig 4. Figure showing the results from inverse propensity score reweighting. It shows the estimated treatment effect of moving to the same building
for 5 years before and five years after the move (ATT) with 90% confidence intervals. results from inverse propensity score reweighting. It shows the estimated treatment effect of moving to the same building
years after the move (ATT) with 90% confidence intervals. Fig 4. Figure showing the results from inverse propensity score reweighting. It shows the estimated treatment effect of moving to the same building
for 5 years before and five years after the move (ATT) with 90% confidence intervals. https://doi.org/10.1371/journal.pone.0259965.g004 PLOS ONE | https://doi.org/10.1371/journal.pone.0259965
December 1, 2021 11 / 18 PLOS ONE Spatial proximity matters: A study on collaboration Moving to the same building increases the collaboration rate between researchers by 0.37
papers per hundred pairs on the year they are moved to the same building. Three years after
moving, the effect increases to 2.29 and plateaus at 1.67 more papers per hundred pairs five
years after the move. Notes: The table presents OLS estimates of the relationship between collaboration and the organizational attributes of buildings. In all models, we measure collaboration
rates in terms of papers per hundred pairs per year. All models control for pair fixed effects, the lag of collaboration, and include year and building fixed effects. The text
provides details on the construction of the organization attributes and building controls used as interactions. In parentheses, we report standard errors that are robust
against heteroskedasticity and correlation within researchers across pairs.
denote a coefficient significant at the 1% level,
at the 5% level, and
at the 10% level. 3.3 Building heterogeneity and collaboration Buildings shown in grey do not host researchers and are therefore not
included in our sample. Fig 5. Figure showing the estimated collaboration effect for each building (Eq (1)). Buildings shown in dark purple have the highest estimated values
of collaboration, and buildings shown in yellow correspond to the lowest. Buildings shown in grey do not host researchers and are therefore not
included in our sample. https://doi.org/10.1371/journal.pone.0259965.g005 https://doi.org/10.1371/journal.pone.0259965.g005 PLOS ONE | https://doi.org/10.1371/journal.pone.0259965
December 1, 2021 12 / 18 Spatial proximity matters: A study on collaboration https://doi.org/10.1371/journal.pone.0259965.t003 PLOS ONE Table 3. Interactions of the effect of proximity on organizational attributes. DEPENDENT VARIABLE: COLLABORATION RATE
(1)
(2)
(3)
(4)
Same Building
0.387
0.380
0.382
0.386
(0.123)
(0.121)
(0.119)
(0.123)
Organizational Attributes:
Same Building x Same Field
0.538
0.790
0.860
0.880
(0.248)
(0.339)
(0.347)
(0.350)
Same Building x Related Field
0.298
0.275
0.467
0.454
(0.255)
(0.253)
(0.256)
(0.262)
Same Building x 4-7 Departments
0.589
0.792
0.730
(0.322)
(0.336)
(0.376)
Same Building x 7+ Departments
0.828
1.042
0.982
(0.589)
(0.594)
(0.588)
Same Building x Distribution of Departments
-0.503
-0.423
(0.255)
(0.261)
Same Building x Researcher Density
-0.292
(0.587)
Building Controls:
Same Building x Log Total Building Area
0.216
0.080
0.122
0.091
(0.287)
(0.242)
(0.246)
(0.267)
Same Building x Circulation Space
2.839
1.518
1.830
1.843
(2.109)
(1.956)
(1.897)
(1.895)
Same Building x Number of Floors
-0.042
-0.029
-0.026
-0.026
(0.064)
(0.061)
(0.063)
(0.063)
Observations
2874147
2874147
2874147
2874147
Number of Researchers
887
887
887
887
Number of Pairs
383361
383361
383361
383361
R-squared
0.61
0.61
0.61
0.61
Covariates:
Pair Fixed Effects
✓
✓
✓
✓
Lagged Collaboration
✓
✓
✓
✓
Year and Building Fixed Effects
✓
✓
✓
✓ Table 3. Interactions of the effect of proximity on organizational attributes. at the 5% level, and at the 10% level. designated for circulation, and the number of floors. These building-level attributes refer to
the concept of functional zones proposed by [4]. Table 3 presents the estimates of these interactions. Column 1 explores whether the effect of
being in the same building varies for pairs of researchers in the same field, related fields, or
unrelated fields. Our results suggest that moving researchers to the same building produces
more collaboration when they are in the same or in related fields. In particular, moving
researchers to a building with others who share their same academic field increases their PLOS ONE | https://doi.org/10.1371/journal.pone.0259965
December 1, 2021 13 / 18 PLOS ONE Spatial proximity matters: A study on collaboration collaboration rate by 0.558 papers per hundred pairs relative to moving to a building with
scholars working on unrelated fields (the excluded category). We also find a similar effect for
researchers in related fields, although the magnitude is smaller and less precise. Column 2 explores the role of having multiple departments within the same building. PLOS ONE We
separately estimate the effect of moving to the same building for buildings with 1–3 depart-
ments, 4–7 departments and more than 7 departments. The effects increase monotonically
with the number of departments. Moving to a buildings hosting between 4 and 7 departments
increases collaboration rates by 0.572 papers per hundred pairs each year relative to a building
with with 1–3 departments (excluded category). Moving to a buildings hosting more than 7
departments increases collaboration rates by 0.813 papers per hundred pairs each year relative
to a building with with 1–3 departments, although this effect is not precisely estimated. Column 3 estimates the role of the distribution of departments across buildings. The esti-
mate for this variable is negative and significant at the 10% level. The results in this column
suggest that a building with multiple departments that are spread across various other build-
ings is more likely to foster collaboration. One potential interpretation is that researchers from
departments that are spread across multiple buildings are already more open to collaborate
with others. Finally, column 4 tests for the role of the density of researchers. We estimate a negative
coefficient for researcher density, but it is not statistically significant. In sum, our results suggest that buildings that host researchers who are working in the
same or related fields and from multiple departments tend to foster more collaboration. This is
particularly the case for departments that are spread across multiple buildings. Other factors
such as the density of researchers do not seem to play a significant role. These results should
be interpreted with caution since buildings with specific organizational attributes might differ
from others in terms of unobserved characteristics. 4 Discussion Our findings contribute to a growing body of evidence highlighting the importance of proxim-
ity for collaboration. As a whole, there is wide agreement that proximity fosters collaboration
and communication, but individual papers differ in their notion of proximity and the out-
comes studied, as well as the question of whether barriers to social interaction operate across
or within buildings. A first set of studies suggests that even within a building, there might be
significant barriers to collaboration and social interactions. For example, [39] identifies aspects
of office layouts that matter for social network formation within buildings. [36] shows that
school teachers interact more when they share offices in the same floor, which points to the
local nature of social interactions. Finally, [37] show that collaboration among Harvard faculty
belonging to the Medicine department increases following the assignment to a shared infor-
mation session, even though these faculty shared offices in the same department building. A
second set of studies including [5] and our work, shows that there are gains in collaboration as
a result of placing researchers in the same building. We interpret our estimates as the average
effect of reducing collaboration barriers by placing researchers in the same building. It could
well be the case that this average effect masks significant heterogeneity driven by differences in
the layout of offices, whether researchers have offices in the same floor, the availability of com-
mon spaces (such as break rooms and cafeterias), and whether researchers use these common
spaces as intended. Nonetheless, the fact that we find significant effects on collaboration just
from researchers sharing the same building suggests that there are significant search costs both
between and within buildings that could impede communication, collaboration, and the for-
mation of social networks. 14 / 18 PLOS ONE | https://doi.org/10.1371/journal.pone.0259965
December 1, 2021 December 1, 2021 PLOS ONE Spatial proximity matters: A study on collaboration There are several limitations to our approach. First, as explained above, we view our esti-
mates as an average effect, which risks missing important social interactions happening within
buildings and how these are mediated by their physical design. For example, details on the
presence of collaboration rooms or kitchens, and the size of staircases, could help provide a
better characterization of how particular spaces within buildings mediate collaboration. A
related aspect is that we measure co-location using the assigned offices in buildings. 4 Discussion However,
this definition cannot untangle between the very local effects of proximity that have been
explored using concepts like functional distance and other more nuanced ways of characteriz-
ing co-location [11, 41, 43]. A second limitation is that we study the effects of collaboration for
each building in isolation. This means that our findings and research design do not account
for spillovers across buildings and how the reorganization of research activities across campus
can affect the overall rate of collaboration at MIT. For example, some highly collaborative
researchers sharing the same building might increase collaboration in that particular building
but can reduce it elsewhere on campus. A better understanding of these global aspects and
trade-offs is important when considering how to allocate space across departments. Finally,
our research was limited to a single campus and organization, MIT. Based on our results and identified limitations, we suggest a number of promising avenues
for future research. The first is to blend our approach—using a large, long-term dataset and
observing variation across relocation events—with fine-grained approaches—considering the
architectural design of spaces, or conducting surveys with researchers to subjectively under-
stand their motivations for collaborating. Subsequent work could explore a more nuanced
characterization of proximity and how its effects are mediated by the physical design of build-
ings. Another is to do a comparative analysis of several different research institutes, or to com-
pare different campuses of the same organization. Finally, we suggest studying the effect of
full institutional closure during the COVID-19 pandemic. The effect of physical co-location
on patterns of collaboration are sure to shift dramatically when all researchers are working
remotely. 5 Conclusion In this paper, we consider whether or not physical co-location affects the likelihood that
researchers engage in scholarly collaboration. To achieve this, we exploit changes in physical
proximity caused by office renovations and new building construction at the MIT campus,
and take two different analytical approaches to our central question. First, we use a treatment effects framework to explicitly define the control (pairs of
researchers that never shared the same building between 2006 and 2014) and treatment groups
(all pairs of researchers moved to the same building in a given year). We then estimate the
treatment effect of moving to the same building on collaboration using regression methods. Second, we use inverse probability score weighting, which relies on a logistic regression model,
to estimate the probability of being moved to the same building given a researchers’ depart-
ment affiliation and past collaboration patterns. In particular, we find that moving researchers
to the same building increase collaboration between researchers. This finding suggests that
geographical co-location can help overcome barriers between departments. In the second part
of the paper, we explore the role of organizational attributes of specific buildings, such as the
density of researchers, the number of departments and their distribution across buildings, and
the discipline affinity of researchers in a given building. This paper provides strong empirical evidence to explain the relationship between physical
co-location and the likelihood of scholarly collaboration—which is fundamental to successful
scientific collaboration today. Our results suggest that buildings that host researchers who are PLOS ONE | https://doi.org/10.1371/journal.pone.0259965
December 1, 2021 December 1, 2021 15 / 18 PLOS ONE Spatial proximity matters: A study on collaboration working in the same or related fields and from multiple departments tend to foster more col-
laboration. We find that moving two researchers to the same building increases their collabo-
ration rate up to 2.7 on the third year after moving. The effect plateaus at 1.85 more papers per
hundred pairs five years after the move. Our results provide insights into how organizational
logics for allocating space might be an important tool for building and campus planners to use
as they work to design a collaborative environment—particularly relevant in the design of
post-pandemic hybrid remote/on-site space use policies. Author Contributions Conceptualization: Arianna Salazar Miranda, Matthew Claudel. Data curation: Arianna Salazar Miranda, Matthew Claudel. Formal analysis: Arianna Salazar Miranda. Methodology: Arianna Salazar Miranda. Validation: Matthew Claudel. Visualization: Arianna Salazar Miranda. Writing – original draft: Arianna Salazar Miranda. Writing – review & editing: Arianna Salazar Miranda, Matthew Claudel. Conceptualization: Arianna Salazar Miranda, Matthew Claudel. Data curation: Arianna Salazar Miranda, Matthew Claudel. Formal analysis: Arianna Salazar Miranda. Methodology: Arianna Salazar Miranda. Validation: Matthew Claudel. Visualization: Arianna Salazar Miranda. Writing – original draft: Arianna Salazar Miranda. Writing – review & editing: Arianna Salazar Miranda, Matthew Claudel. Conceptualization: Arianna Salazar Miranda, Matthew Claudel. Writing – review & editing: Arianna Salazar Miranda, Matthew Claudel. References Journal of Informetrics. 2011; 5(4):574–582. https://doi.org/10.1016/j.joi.2011.05.003 19. Wuchty S, Jones BF, Uzzi B. The Increasing Dominance of Teams in Production of Knowledge. Sci-
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Post-Drilling Changes in Seabed Landscape and Megabenthos in a Deep-Sea Hydrothermal System, the Iheya North Field, Okinawa Trough
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PloS one
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RESEARCH ARTICLE Abstract Data Availability Statement: All relevant data are
within the paper. OPEN ACCESS Citation: Nakajima R, Yamamoto H, Kawagucci S,
Takaya Y, Nozaki T, Chen C, et al. (2015) Post-
Drilling Changes in Seabed Landscape and
Megabenthos in a Deep-Sea Hydrothermal System,
the Iheya North Field, Okinawa Trough. PLoS ONE
10(4): e0123095. doi:10.1371/journal.pone.0123095 * nakajimar@jamstec.go.jp Post-Drilling Changes in Seabed Landscape
and Megabenthos in a Deep-Sea
Hydrothermal System, the Iheya North Field,
Okinawa Trough Ryota Nakajima1*, Hiroyuki Yamamoto2, Shinsuke Kawagucci2,3,4, Yutaro Takaya2,
Tatsuo Nozaki2,5, Chong Chen6, Katsunori Fujikura1, Tetsuya Miwa2,7, Ken Takai2,3,4 Ryota Nakajima1*, Hiroyuki Yamamoto2, Shinsuke Kawagucci2,3,4, Yutaro Takaya2,
Tatsuo Nozaki2,5, Chong Chen6, Katsunori Fujikura1, Tetsuya Miwa2,7, Ken Takai2,3,4 1 Department of Marine Biodiversity Research, Japan Agency for Marine-Earth Science and Technology
(JAMSTEC), 2–15 Natsushima, Yokosuka, Kanagawa, 237–0061, Japan, 2 Research and Development
Center for Submarine Resources, Japan Agency for Marine-Earth Science and Technology (JAMSTEC),
2–15 Natsushima, Yokosuka, Kanagawa, 237–0061, Japan, 3 Laboratory of Ocean-Earth Life Evolution
Research (OELE), Japan Agency for Marine-Earth Science and Technology (JAMSTEC), 2–15 Natsushima,
Yokosuka, Kanagawa, 237–0061, Japan, 4 Department of Subsurface Geobiological Analysis and Research
(D-SUGAR), Japan Agency for Marine-Earth Science and Technology (JAMSTEC), 2–15 Natsushima,
Yokosuka, Kanagawa, 237–0061, Japan, 5 Department of Systems Innovation, School of Engineering, The
University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113–8656, Japan, 6 Department of Zoology, University
of Oxford, South Parks Road, Oxford, OX1 3PS, United Kingdom, 7 Marine Technology and Engineering
Center (MARITEC), Japan Agency for Marine-Earth Science and Technology (JAMSTEC), 2–15
Natsushima, Yokosuka, Kanagawa, 237–0061, Japan Abstract There has been an increasing interest in seafloor exploitation such as mineral mining in
deep-sea hydrothermal fields, but the environmental impact of anthropogenic disturbance
to the seafloor is poorly known. In this study, the effect of such anthropogenic disturbance
by scientific drilling operations (IODP Expedition 331) on seabed landscape and megafau-
nal habitation was surveyed for over 3 years using remotely operated vehicle video obser-
vation in a deep-sea hydrothermal field, the Iheya North field, in the Okinawa Trough. We
focused on observations from a particular drilling site (Site C0014) where the most dynamic
change of landscape and megafaunal habitation was observed among the drilling sites of
IODP Exp. 331. No visible hydrothermal fluid discharge had been observed at the sedimen-
tary seafloor at Site C0014, where Calyptogena clam colonies were known for more than 10
years, before the drilling event. After drilling commenced, the original Calyptogena colonies
were completely buried by the drilling deposits. Several months after the drilling, diffusing
high-temperature hydrothermal fluid began to discharge from the sedimentary subseafloor
in the area of over 20 m from the drill holes, ‘artificially’ creating a new hydrothermal vent
habitat. Widespread microbial mats developed on the seafloor with the diffusing hydrother-
mal fluids and the galatheid crab Shinkaia crosnieri endemic to vents dominated the new
vent community. The previously soft, sedimentary seafloor was hardened probably due to
barite/gypsum mineralization or silicification, becoming rough and undulated with many fis-
sures after the drilling operation. Although the effects of the drilling operation on seabed Academic Editor: Sebastien Duperron, Universite
Pierre et Marie Curie, FRANCE
Received: November 14, 2014
Accepted: February 27, 2015
Published: April 22, 2015 Academic Editor: Sebastien Duperron, Universite
Pierre et Marie Curie, FRANCE
Received: November 14, 2014
Accepted: February 27, 2015
Published: April 22, 2015 Academic Editor: Sebastien Duperron, Universite
Pierre et Marie Curie, FRANCE
Received: November 14, 2014
Accepted: February 27, 2015
Published: April 22, 2015 Copyright: © 2015 Nakajima et al. This is an open
access article distributed under the terms of the Copyright: © 2015 Nakajima 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. Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited. Data Availability Statement: All relevant data are
within the paper. Funding: RN acknowledges the Environment
Research and Technology Development Fund (S9) of
the Ministry of the Environment, Japan (http://s9. conservationecology.asia). The funder 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. 1 / 22 PLOS ONE | DOI:10.1371/journal.pone.0123095
April 22, 2015 Post-Drilling Megafaunal Changes in a Deep-Sea Hydrothermal Field landscape and megafaunal composition are probably confined to an area of maximally
30 m from the drill holes, the newly established hydrothermal vent ecosystem has already
lasted 2 years and is like to continue to exist until the fluid discharge ceases and thus the
ecosystem in the area has been altered for long-term. landscape and megafaunal composition are probably confined to an area of maximally
30 m from the drill holes, the newly established hydrothermal vent ecosystem has already
lasted 2 years and is like to continue to exist until the fluid discharge ceases and thus the
ecosystem in the area has been altered for long-term. PLOS ONE | DOI:10.1371/journal.pone.0123095
April 22, 2015 Introduction In the last few decades, deep-sea hydrothermal ecosystems have been under increasing threat
from various anthropogenic activities either underway or planned [1–3]. Recent technological
developments have overcome the barrier of water depth and distance from shore, allowing the
exploitation of previously inaccessible areas [4]. This has boosted the continuous expansion of
anthropogenic activities in the hydrothermal vent ecosystems, including the exploitation of
valuable mineral resources [5]. Hydrothermal vent sites produce seafloor massive sulfide
(SMS) deposits with high-grade ores, giving them an attractive commercial prospect for mining
[1,2]. Many potential impacts on the benthic community from mining activities are predicted:
habitat loss and degradation, modification of fluid flux regimes, changes in diversity, and
change of habitat conditions [1,3,6]. Although there is no case of commercial based seafloor
mining on hydrothermal vent area so far, we can estimate the impacts or effects from mining
activities from case studies of natural and artificial disturbances, such as volcanism and drilling
[7]. Although disturbance caused by drilling may be different from proposed mining methods,
including mechanical cutting, grabbing and dredging of vent chimney and hydrothermal de-
posits, impacts from drilling have the potential to serve as supporting evidence when assessing
the risk of mining operation. At present, the impact of drilling at hydrothermal systems is poorly understood (e.g. [8]). At present, the impact of drilling at hydrothermal systems is poorly understood (e.g. [8]). Most studies on the environmental impacts associated with deep-sea exploration (or commer-
cial) drilling have been conducted in oil and gas field (e.g. [9–13]), focusing on how drilling de-
posits (cuttings and mud) affect mortality and survival rate of benthic animals (e.g. [12,11,10,14]). The drilling impact on seabed landscape and associated megabenthos in hydro-
thermal fields is likely to differ from these cases. Potential impacts that may be expected in-
clude the discharge of drilling deposits on to the seabed and subsequently high-temperature
hydrothermal fluids from subseafloor. It is conceivable that the fluid discharges will attract re-
cruits from the surrounding vent communities, creating ‘artificial’ hydrothermal vent ecosys-
tems [7]. Understanding how the benthic community responds to drill-induced disturbance
will help shape future ecosystem conservation strategy in anticipation of upcoming SMS min-
ing activities. It is therefore crucial to conduct monitoring and assessment of the effect of dril-
ling on the benthic community of deep-sea hydrothermal fields. At present, the impact of drilling at hydrothermal systems is poorly understood (e.g. [8]). Study location Situated approximately 150 km off the Okinawa Island, Japan, the Iheya North field (27°45’-
50’N; 126°53’-55’E) in the Okinawa Trough is a deep-sea hydrothermal field with a depth of ca. 1,000 m. Two decades of investigation since its discovery has revealed its geological back-
ground, fluid chemistry and microbiological characteristics [15,17–20]. Among the many ac-
tive chimney sites, the 30 m high North Big Chimney (NBC) mound is the activity center of
the field (Fig 1B, [18]). The Iheya North field is characterized by thick soft sediments offering
habitats for endobenthic invertebrates even around hydrothermal vent site, allowing both
vent-type and seep-type communities to exist in the area [21,22]. Representative species of
megabenthic vent animals in the Iheya North field are Shinkaia crosnieri galatheid crab, Alvi-
nocaris longirostris shrimp, Paralvinella polychaetes, Paralomis lithodid crab, and Bathymodio-
lus japonicus and B. platifrons mussels. Representative species of the geofluid seepage zone of
the Iheya North vent field is the endobenthic deep-sea clam, Calyptogena okutanii, which colo-
nizes the sedimentary seafloor [23]. Of the several sites drilled during the IODP Expedition 331 (Fig 1B, see also [16]), Site
C0014 is located 450 m east of the NBC mound. Around Site C0014, both active and non-active
chimneys as well as apparent hydrothermal fluid discharges were not identified through visual
observations by previous submersible surveys [21]. This site was characterized by several C. okutanii clam colonies, which had been identified for more than 10 years before the drilling
event, indicating the colonies were likely supported by seepage of hydrothermal fluid input
[16,17]. In total, seven holes were drilled at Site C0014 (i.e., Holes A-G) in a narrow area within
10 m radius, overlapping with the Calyptogena colonies (see red stars in Figs 1C and 2A). The
holes were located at 1,060 m depth (Table 1). Hole G penetrated the deepest (136.7 m below
the seafloor, mbsf), and the penetration depths of the other holes (Holes A-F) ranged from 4.2
to 44.5 mbsf (Table 1). Holes D and E were very closely located and they became one hole after
the wall between them broke down (Hole D/E hereafter). At 11 months after the drilling opera-
tion only Holes D/E, B and G were visibly recognizable while the other holes had collapsed and
filled up due to their shallow penetration (~6.5 mbsf). Ethics statement The location for this study was not privately owned or protected in any way and no specific
permits were required for the described field studies and sample collection. The field studies
did not involve any endangered or protected species. No invertebrate megafaunal specimens
were collected in this work, as it was carried out using video techniques. Introduction Most studies on the environmental impacts associated with deep-sea exploration (or commer-
cial) drilling have been conducted in oil and gas field (e.g. [9–13]), focusing on how drilling de-
posits (cuttings and mud) affect mortality and survival rate of benthic animals (e.g. In September 2010, Integrated Ocean Drilling Program (IODP) Expedition 331 was carried
out at the Iheya North hydrothermal field in the Okinawa Trough, Japan using the deep-sea
drilling vessel Chikyu to investigate active subseafloor microbial communities associated with
the physical and chemical variation of hydrothermal fluid flow [15,16]. The expedition estab-
lished several drilling-induced, ‘artificial’ hydrothermal vents [16,17]. These artificial vents
provided unique research opportunities for estimating the influence of anthropogenic drilling
to the benthic communities of deep-sea hydrothermal ecosystems. As a part of an environmen-
tal impact assessment, we surveyed the changes in the seabed landscape and habitat as well as
the abundance and composition of the megafaunal benthic community around the drill holes PLOS ONE | DOI:10.1371/journal.pone.0123095
April 22, 2015 2 / 22 Post-Drilling Megafaunal Changes in a Deep-Sea Hydrothermal Field for over 3 years. Our focuses in this study are to examine the extent and persistence of the ef-
fects of the drilling operations in the area and to elucidate how the habitat condition as well as
the benthic megabenthos communities are altered by the drilling. PLOS ONE | DOI:10.1371/journal.pone.0123095
April 22, 2015 Study location The multiple drilling operations penetrated the repeated hard layers that might have served
as cap rocks, and high-temperature hydrothermal fluid were discharged from the holes as well
as in the shallower sediments surrounding the holes [15–17]. A casing pipe was deployed down
to ca. 120 mbsf at Hole G fixed with a corrosion cap (open outlet pipe) mounted on the gim-
balled guide base [15,16]. After casing and capping, diffusing hydrothermal fluid discharged
not from the corrosion cap outlet but from the seafloor through the annulus, the space between
the wall of the hole and the casing pipe at Hole G; the temperature of the diffusing fluids was
found to be >240°C [16]. Five months after the drilling, high-temperature hydrothermal fluid
(304-311°C) discharged from the casing pipe outlet at Hole G, and the fluid discharge from the PLOS ONE | DOI:10.1371/journal.pone.0123095
April 22, 2015 3 / 22 doi:10.1371/journal.pone.0123095.g001 Post-Drilling Megafaunal Changes in a Deep-Sea Hydrothermal Field Fig 1. Maps of the study area. (a) Location of the Iheya North hydrothermal field, Okinawa Trough. (b) An event map of the Iheya North field. Red s
circles represent the IODP drilling sites (C0013, C0014 and C0016) and hydrothermal fluid venting sites, respectively. (c) Location of drill holes at Sit
(Holes A-G). doi:10.1371/journal.pone.0123095.g001 Fig 1. Maps of the study area. (a) Location of the Iheya North hydrothermal field, Okinawa Trough. (b) An event map of the Iheya North field. Red stars and
circles represent the IODP drilling sites (C0013, C0014 and C0016) and hydrothermal fluid venting sites, respectively. (c) Location of drill holes at Site C0014
(Holes A-G) doi:10.1371/journal.pone.0123095.g001 PLOS ONE | DOI:10.1371/journal.pone.0123095
April 22, 2015 4 / 22 Post-Drilling Megafaunal Changes in a Deep-Sea Hydrothermal Field Fig 2. Habitat map around Site C0014. (a) Drill holes and colonies of Calyptogena okutanii clams and Bathymodiolus mussels and assemblages
Bathymodiolus mussels and Shinkaia crosnieri galatheid crabs. (b) Pre-drilling seabed landscape near Hole D/E with C. okutanii clam colonies. (c)
example of Bathymodiolus mussels and S. crosnieri galatheid crabs assemblage. / Fig 2. Habitat map around Site C0014. (a) Drill holes and colonies of Calyptogena okutanii clams and Bathymodiolus mussels and assemblages of
Bathymodiolus mussels and Shinkaia crosnieri galatheid crabs. (b) Pre-drilling seabed landscape near Hole D/E with C. okutanii clam colonies. (c) An
example of Bathymodiolus mussels and S. crosnieri galatheid crabs assemblage. doi:10.1371/journal.pone.0123095.g002 pipe continued at 25 months after drilling [17]. Prior to the drilling (2 weeks before), a ther-
mometer and an acrylic-glass sedimentation chamber with mounting stage were placed near
Hole D/E (Fig 3A). The thermometer recorded that the bottom surface temperature near Hole
D/E had increased at 11 months and reached >50°C at 14–15 months after drilling, which was
the maximum temperature for the thermometer (the thermometer was broken at this time, see
Fig 4 in [17]). pipe continued at 25 months after drilling [17]. Prior to the drilling (2 weeks before), a ther-
mometer and an acrylic-glass sedimentation chamber with mounting stage were placed near
Hole D/E (Fig 3A). The thermometer recorded that the bottom surface temperature near Hole
D/E had increased at 11 months and reached >50°C at 14–15 months after drilling, which was
the maximum temperature for the thermometer (the thermometer was broken at this time, see
Fig 4 in [17]). Pre- and post-drilling video observations Pre- and post-drilling seafloor video observations were carried out 2 weeks before drilling and
11, 16, 25, 38 and 40 months after drilling using JAMSTEC’s remotely operating vehicles
(ROVs) either Hyper-Dolphin or Kaiko 7000 II (Table 2). Video data were recorded with colour
video cameras positioned in either vertical or oblique views by running the ROVs haphazardly
around the drilling holes. At 2 weeks before and 11 months after drilling, a forward-facing
video camera (Super HARP, Hamamatsu Photonic) recorded the seabed from the oblique view
(the vertical distance of camera to vehicle bottom was 0.9 m). At 16, 25, 38 and 40 months after
drilling, a downward-looking video camera (Handycam HDR-CX-700V, Sony) recorded the
seabed vertically below the ROVs (the vertical distance of camera to vehicle bottom was 0.4 m). The surveys were conducted at an altitude of 2.8 ± 0.3 m (the average distance of vehicle bottom
to the bottom substrate during the imaging for each dive) and vehicle speed of ca. 0.25 m s-1. The ROV angles during the video imagery were 5.1 ± 2.3° for pitch and 1.5 ± 0.6° for roll angles,
thus causing some variation in subsequent measurements of seabed feature coverage and animal
abundance. Positional data of ROVs from the Super-Short Baseline Navigation (SSBN) tran-
sponder were continuously recorded during the dives. The apparent outliers of the transponder
were excluded before estimating the positional information of the vehicle. Position aberration of
vehicles among the different survey periods were corrected based on the absolute positions of
the gimballed guide base mounted on Hole G and the sedimentation chamber base placed near
Hole D/E. During the observation at 16 months post-drilling, in situ measurement of seawater pH was
carried out using a submersible pH sensor for deep-sea (XR 420 CTD with AMP pH combined
sensor, RBR Limited) which was installed on the ROV Kaiko 7000 II. Calibration of the pH
sensor was performed pre- and post-dive operation of the ROV. In addition, hardness of the
bottom sediment was examined by testing whether a push-core sampler can be inserted to the
sediment (the concurrently collected sediment samples were used in other studies). Post-Drilling Megafaunal Changes in a Deep-Sea Hydrothermal Field Table 1. Drill hole summary at Site C0014 in the Iheya North hydrothermal field, Okinawa Trough. Hole
Latitude (N)
Longitude (E)
Water depth
Hole depth
(m)
(mbsf)
A
27°47.4140'
126°54.0487'
1059.5
6.5
B
27°47.4131'
126°54.0448'
1059.0
44.5
C
27°47.4194'
126°54.0391'
1060.0
6.5
D
27°47.4158'
126°54.0406'
1060.0
16.0
E
27°47.4158'
126°54.0406'
1060.0
35.0
F
27°47.4185'
126°54.0443'
1060.8
4.2
G
27°47.4165'
126°54.0463'
1059.8
136.7
doi:10 1371/journal pone 0123095 t001 Table 1. Drill hole summary at Site C0014 in the Iheya North hydrothermal field, Okinawa Trough. PLOS ONE | DOI:10.1371/journal.pone.0123095
April 22, 2015 5 / 22 PLOS ONE | DOI:10.1371/journal.pone.0123095
April 22, 2015 Video data analysis The oblique and vertical video images were used for quantitative analysis of seabed features
and megabenthic animals. Video clips were captured at 10-second intervals, using the software
GOM Player (Gretech) in order to provide still image frames. Overlapping and unsuitable pho-
tographs (e.g., out of focus and high sediment re-suspension) were excluded from the analysis
(excluded images constituted ~ 30% of the total generated images frames). In order to derive % coverage of disturbed sediments indicated as white-colored clay-like
substrate (drilling deposits) and microbial filamentous mats seen as either white or pink, the
image frame sets collected by the video cameras were imported in the image analysis software PLOS ONE | DOI:10.1371/journal.pone.0123095
April 22, 2015 6 / 22 Post-Drilling Megafaunal Changes in a Deep-Sea Hydrothermal Field Fig 3. Temporal sequence of landscape at/around Hole D/E. Arrows in a-f indicate the sedimentation
chamber base, which was placed pre-drilling (a). Photo g shows the edge of cuttings and photo h shows
bottom of the sedimentation chamber at 16 months post-drilling. Photos i-k are perpendicular images of Fig 3. Temporal sequence of landscape at/around Hole D/E. Arrows in a-f indicate the sedimentation
chamber base, which was placed pre-drilling (a). Photo g shows the edge of cuttings and photo h shows the
bottom of the sedimentation chamber at 16 months post-drilling. Photos i-k are perpendicular images of Hole PLOS ONE | DOI:10.1371/journal.pone.0123095
April 22, 2015 7 / 22 Post-Drilling Megafaunal Changes in a Deep-Sea Hydrothermal Field D/E at 16, 25 and 38 months post-drilling, respectively. Photo l is a perpendicular image of the bottom
substratum at 40 months post-drilling, near Hole D/E. D/E at 16, 25 and 38 months post-drilling, respectively. Photo l is a perpendicular image of the bottom
substratum at 40 months post-drilling, near Hole D/E. doi:10.1371/journal.pone.0123095.g003 CPCe (Coral Point Count with Excel extension, [24]). For each picture frame, 50–100 random
points were plotted and observed [25]. White-colored bacterial mats were impossible to distin-
guish from the white drilling deposits if the mat developed on or overlapped the deposits, and
thus the coverage of white bacterial mats may be underestimated. The presence of fluid dis-
charge observed as shimmering in each photograph was confirmed through cross-checking
with original video clips, as it was difficult to determine if there was actually shimmering fluid
from only still images. Video data analysis Distribution and coverage data of the discolored area and shimmering
were plotted in a geographic information system using the software QGIS (version 2.2.0-Val-
miera). In order to compare the seabed features (drilling deposits and microbial mats) between
different sampling periods, we used the percentage cover within 10 m radius of Hole D/E as the
hole was intensively visited by the ROVs. The megabenthic invertebrate animals appeared in each image frame were identified to the
lowest taxonomic levels possible and counted, and abundance of each animal was calculated
from each image as number of individuals per m2 (inds. m-2). The seabed area of the image was
estimated according to [26] for the perpendicular images and [27] for the oblique images using
the underwater horizontal and vertical aperture angles of the camera and the camera-to-sea-
floor distance. We considered only animals with a minimum dimension ca. >30 mm. Paralvi-
nella and polynoidea polychaetes were not counted as they were difficult to distinguish from
the bottom substrate using our video observation from some distance. The distribution and
abundance data of the megabenthic animals were plotted in geographical maps using QGIS. The abundances of observed animals between different sampling periods were compared with-
in 10 m radius of Hole D/E. The statistical differences of these values were determined by mul-
tiple comparison Steel-Dwass test. Difference with P<0.05 was considered statistically
significant. At 16 and 40 months after drilling, carapace width length (mm) of the galatheid crab Shin-
kaia crosnieri that appeared in the perpendicular images recorded around Hole D/E was mea-
sured using the image analysis software Hakarundesu v0.7.1 (Natchan). The sedimentation
chamber mount stage (base, 250 mm × 250 mm; height, 200 mm) that appeared in the images
was used for length calibration. The statistical difference in the size of S. crosnieri between the
different sampling periods was determined by two-tailed Student’s t-test. Difference with
P<0.05 was considered statistically significant. PLOS ONE | DOI:10.1371/journal.pone.0123095
April 22, 2015 PLOS ONE | DOI:10.1371/journal.pone.0123095
April 22, 2015 Seabed landscapes before and after drilling Prior to drilling, the sediment at Site C0014 was entirely dominated by fine-grained silty sedi-
ment (mud) (Figs 2A and 2B and 3A). Pre-drilling video surveys revealed that there were no
vent endemic animals within a 15 m radius of the center of the drill holes. The hydrothermal
fluid seepage community characterized by live Calyptogena clam colonies was the most promi-
nent feature in the soft sediment (Fig 2B), though visual observation confirmed that more than
90% of the Calyptogena were dead shells. Rossellid sponges and laetmogonid holothrians were
commonly observed around the clam colonies. The 15 m~ northwest of the drilling site was
covered by hard substratum consisting of exposed bedrocks and boulders (Fig 2A). Diffusing
hydrothermal fluids were discharged from the fissures of the exposed bedrocks at some 20–30
m west-northwest of the drilling site, where vent endemic animals Shinkaia crosnieri galatheid PLOS ONE | DOI:10.1371/journal.pone.0123095
April 22, 2015 8 / 22 Post-Drilling Megafaunal Changes in a Deep-Sea Hydrothermal Field PLOS ONE | DOI:10.1371/journal.pone.0123095
April 22, 2015 9 / 22 Post-Drilling Megafaunal Changes in a Deep-Sea Hydrothermal Field Fig 4. Temporal sequence of drilling deposits seen as white-color sediment as well as hydrothermal
fluid discharges seen as shimmering at Site C0014. Dotted circles indicate the area within 10 m radius
from Hole D/E. doi:10.1371/journal.pone.0123095.g004 Fig 4. Temporal sequence of drilling deposits seen as white-color sediment as well as hydrothermal
fluid discharges seen as shimmering at Site C0014. Dotted circles indicate the area within 10 m radius
from Hole D/E. doi:10.1371/journal.pone.0123095.g004 crabs and Bathymodiolus mussel beds were observed (Fig 2A and 2C). There was also a single
small assemblage of S. crosnieri and Bathymodiolus mussels ca. 12 m northwest from Hole D/E
(Fig 2A). crabs and Bathymodiolus mussel beds were observed (Fig 2A and 2C). There was also a single
small assemblage of S. crosnieri and Bathymodiolus mussels ca. 12 m northwest from Hole D/E
(Fig 2A). After drilling commenced the drilling process deposited white-colored clay-like sediments
(probably originating from drill cuttings and mud with clay mineral, barite and bentonite) on
the seabed in the vicinity of the holes (Fig 3B). This was visually confirmed at 11 months post-
drilling. The multiple drilling operations drastically altered the seafloor landscape as the Calyp-
togena clam colonies were completely buried under the white-colored sediments. Seabed landscapes before and after drilling The white
sediment extended 13–25 m from the center of drill holes at 16 months after drilling (Figs 3G
and 4). The mean coverage (%) of drilling deposits seen as clay-like white sediments within a
10 m radius of Hole D/E was 60.4 ± 31.2% at 16 months after drilling. The coverage of drill de-
posits had decreased thereafter: 36.0 ± 21.5% at 25 months, and 20.3 ± 17.6% at 38–40 months
post-drilling (Fig 4). Although it was difficult to accurately assess the thickness of the newly de-
posited sediments, dead clam shells that appeared inside of Hole D/E suggest this was at least
ca. 300 mm (Fig 3I). At 11 months after drilling, numerous tiny chimneys and drilling-induced hydrothermal
fluid discharges seen as shimmering were observed on the white seafloor (Fig 3B, see also Fig
2L in [17]). The extent of shimmering fluid discharges induced by the drilling operations at 16
months after drilling was consistent with that of the white sediment, extending up to 24 m
from the center of drill holes (Fig 4). Naturally discharged shimmering water was also recog-
nized from exposed bedrock areas at some 20 m west-northwest the drill site at this time (Fig
4). The seawater pH showed lower values around the holes compared to the surrounding sea-
floor due to diffusive hydrothermal fluid discharges from the seafloor (Fig 5). At 16 months
after drilling the seawater pH increased with increasing distance from the holes; 7.09 ± 0.13
within 10 m from Hole D/E, 7.12 ± 0.12 at 10–20 m, 7.22 ± 0.06 at 20–30 m, and 7.39 ± 0.17 at
30–40 m. Upon collection of the sedimentation chamber placed near Hole D/E at 16 months, bottom
of the chamber was partially melted due to the increased bottom temperature (Fig 3H). Consid-
ering the melting point of acrylic-glass [28], the seabed temperature likely have increased to at
least 160°C at 16 months. At 11 and 16 months after drilling, the bottom sediment around
Hole D/E was soft enough for push core-samplers to penetrate into the sediment. However, the
bottom had hardened after 25 months and the area which the core-samplers could penetrate Table 2. Timing of investigations relative to the drilling event at Site C0014 in the Iheya North hydrothermal field, Okinawa Trough. Seabed landscapes before and after drilling Date
Time elapsed
Cruise
ROV
Dive number
(mm/yy)
Sep/2010
2 weeks before
NT10-E01
Hyper-Dolphin
1178
Sep/2010
drilling
IODP Exp331
ROV-C
August/2011
11 months
NT11-15/16
Hyper-Dolphin
1312/1315
Jan/2012
16 months
KR12-02
Kaiko 7000II
537/538
Oct/2012
25 months
NT12-27
Hyper-Dolphin
1446/1447/1448/1450
Nov/2013
38 months
NT13-22
Hyper-Dolphin
1593/1595
Jan/2014
40 months
KY14-01
Hyper-Dolphin
1611
doi:10.1371/journal.pone.0123095.t002 igations relative to the drilling event at Site C0014 in the Iheya North hydrothermal field, Okinawa Trough. 2. Timing of investigations relative to the drilling event at Site C0014 in the Iheya North hydrothermal field, O Table 2. Timing of investigations relative to the drilling event at Site C0014 in the Iheya North hydrothermal field, Okinawa Trough. PLOS ONE | DOI:10.1371/journal.pone.0123095
April 22, 2015 10 / 22 Post-Drilling Megafaunal Changes in a Deep-Sea Hydrothermal Field Fig 5. Distribution of seawater pH around drill holes at Site C0014 at 16 months post-drilling. Dotted circles indicate the area within 10 m radius from
Hole D/E. drill holes at Site C0014 at 16 months post-drilling. Dotted circles indicate the area within 10 m radius from Fig 5. Distribution of seawater pH around drill holes at Site C0014 at 16 months post-drilling. Dotted circles indicate the area within 10 m radius from
Hole D/E Fig 5. Distribution of seawater pH around drill holes at Site C0014 at 16 months post-drilling. Dotted circles indicate the area within 10 m radius from
Hole D/E. Fig 5. Distribution of seawater pH around drill holes at Site C0014 at 16 months post-drilling. Dotted circles in
Hole D/E. doi:10.1371/journal.pone.0123095.g005 doi:10.1371/journal.pone.0123095.g005 into had become limited. At 38–40 months after drilling, the bottom was further hardened and
it was impossible to insert the core-sampler into the sediment around Hole D/E and the bottom
substratum was visually seen to be undulating with many fissures (Fig 3F and 3L). Unlike the drilling deposits, the coverage of bacterial mats around the holes increased over
time (Fig 6A and 6B). Two types of microbial mats, seen as white and pink colors, have devel-
oped after drilling commenced, extending 5–25 m from the center of holes. Before the drilling
commenced, white bacteria mats covered 10.3 ± 16.1% the seabed within 10 m radius from the
point of Hole D/E, probably because the area hosted hydrothermal fluid seepage which also
supported Calyptogena clam colonies. Seabed landscapes before and after drilling After drilling, the coverage of white bacterial mats had
increased to 14.1 ± 18.4% at 16 months, 34.6 ± 19.7% at 25 months, and 46.5 ± 18.9% at 38–40
months (Fig 6A). The pink-colored bacterial mats were not observed before drilling, but were
first confirmed at 16 months post-drilling, covering 1.9 ± 3.6% within 10 m radius from Hole
D/E, increasing thereafter: 7.7 ± 12.5% at 25 months and 12.5 ± 16.0% at 38–40 months post-
drilling (Fig 6B). Changes in megafaunal assemblage composition after drilling At 11 months after drilling, no benthic animals were observed 10 m radius around Hole D/E as
the original Calyptogena clam colonies were buried under the drill deposits. At this time, PLOS ONE | DOI:10.1371/journal.pone.0123095
April 22, 2015 11 / 22 PLOS ONE | DOI:10.1371/journal.pone.0123095
April 22, 2015 Post-Drilling Megafaunal Changes in a Deep-Sea Hydrothermal Field Fig 6. Temporal sequence of microbial mats at Site C0014. (a) white and (b) pink mats. Dotted circles indicate the area within 10
doi:10.1371/journal.pone.0123095.g006
PLOS ONE | DOI:10.1371/journal.pone.0123095
April 22, 2015 mats at Site C0014. (a) white and (b) pink mats. Dotted circles indicate the area within 10 m radius from Hole D/E. Fig 6. Temporal sequence of microbial mats at Site C0014. (a) white and (b) pink mats. Dotted circles indicate the area within 10 m radius from Hole D/E. doi:10.1371/journal.pone.0123095.g006 PLOS ONE | DOI:10.1371/journal.pone.0123095
April 22, 2015 12 / 22 Post-Drilling Megafaunal Changes in a Deep-Sea Hydrothermal Field Fig 7. Temporal variations in the abundance of newly colonized megabenthos around Hole D/E. Abundance is the average within 10 m radius from
Hole D/E. Fig 7 Temporal variations in the abundance of newly colonized megabenthos around Hole D/E Abundance is the average within 10 m radius from however, a very low density (~0.27 inds. m-3) of vent endemic galatheid crab S. crosnieri was
found at 7.2–9.7 m northwest of Hole D/E (Fig 7, geographical map not shown). At 16 months
post-drilling the population of S. crosnieri has become widely distributed and dominated areas
around the holes (Figs 7 and 8A). Another vent animal found at 16 months was Alvinocaris
longirostris shrimp (Fig 8B). We also confirmed the presence of other vent endemic animals
such as Paralvinella polychaetes and polynoid polychaetes at this time, although their abun-
dance was not counted. The main distribution of S. crosnieri was restricted within a 10–15 m radius from the holes
(Fig 8A). The mean abundance of S. crosnieri within a 10 m radius of Hole D/E was 2.4 ± 7.2
inds. m-3 (max, 43 inds. m-3) at 16 months after drilling (Fig 7). Since the population of S. cros-
nieri was very patchily distributed, standard deviation (SD) was higher than the average. Al-
though there were no significant differences among the different post-drilling periods (Steel-
Dwass test; all test statistics, <0.6; 5% reference point, 2.57) probably due to the patchy
Fig 7. Temporal variations in the abundance of newly colonized megabenthos around Hole D/E. Abundance is the average within 10 m radius from
Hole D/E. doi:10.1371/journal.pone.0123095.g007
PLOS ONE | DOI:10.1371/journal.pone.0123095
April 22, 2015
13 / 22 Fig 7. Temporal variations in the abundance of newly colonized megabenthos around Hole D/E. doi:10.1371/journal.pone.0123095.g007 Abundance is the average within 10 m radius from
Hole D/E doi:10.1371/journal.pone.0123095.g007 doi:10.1371/journal.pone.0123095.g007 however, a very low density (~0.27 inds. m-3) of vent endemic galatheid crab S. crosnieri was
found at 7.2–9.7 m northwest of Hole D/E (Fig 7, geographical map not shown). At 16 months
post-drilling the population of S. crosnieri has become widely distributed and dominated areas
around the holes (Figs 7 and 8A). Another vent animal found at 16 months was Alvinocaris
longirostris shrimp (Fig 8B). We also confirmed the presence of other vent endemic animals
such as Paralvinella polychaetes and polynoid polychaetes at this time, although their abun-
dance was not counted. The main distribution of S. crosnieri was restricted within a 10–15 m radius from the holes
(Fig 8A). The mean abundance of S. crosnieri within a 10 m radius of Hole D/E was 2.4 ± 7.2
inds. m-3 (max, 43 inds. m-3) at 16 months after drilling (Fig 7). Since the population of S. cros-
nieri was very patchily distributed, standard deviation (SD) was higher than the average. Al-
though there were no significant differences among the different post-drilling periods (Steel-
Dwass test; all test statistics, <0.6; 5% reference point, 2.57) probably due to the patchy PLOS ONE | DOI:10.1371/journal.pone.0123095
April 22, 2015 13 / 22 Post-Drilling Megafaunal Changes in a Deep-Sea Hydrothermal Field Fig 8. Temporal sequence in distribution and abundance of megabenthos at Site C0014. (a) Shinkaia crosnieri galatheid crabs and (b) Alvinocaris
longirostris shrimps. Dotted circles indicate the area within 10 m radius from Hole D/E. Fig 8. Temporal sequence in distribution and abundance of megabenthos at Site C0014. (a) Shinkaia crosnier
longirostris shrimps. Dotted circles indicate the area within 10 m radius from Hole D/E. Fig 8. Temporal sequence in distribution and abundance of megabenthos at Site C0014. (a) Shinkaia crosnieri galatheid crabs and (b) Alvinocaris
longirostris shrimps. Dotted circles indicate the area within 10 m radius from Hole D/E. Fig 8. Temporal sequence in distribution and abundance of megabenthos at Site C0014. (a) Shinkaia crosnieri galatheid crabs and (b) Alvinocaris
longirostris shrimps. Dotted circles indicate the area within 10 m radius from Hole D/E. doi:10.1371/journal.pone.0123095.g008 distribution, mean abundance of S. crosnieri had increased by 4.5-fold at 25 months (mean,
10.5 ± 28.7 inds. m-3; max, 110 inds. m-3) compared to those at 16 months after drilling and be-
came relatively stable thereafter at 38 months (mean, 10.4 ± 20.4 inds. m-3; max, 109 inds. m-3)
and 40 months (9.6 ± 20.1 inds. m-3; max, 114 inds. m-3). S. crosnieri was not found inside
Hole D/E at 16 months but were found inside the holes at 25 months post-drilling and thereaf-
ter (Fig 3I–3K). The mean carapace width length of S. crosnieri at 16 months (51 ± 11 mm) was significantly
(t-test, P = 0.0015×10−6) higher than those at 40 months after drilling (43 ± 13 mm) (Fig 9). At 16 months after drilling, larger individuals (60–70 mm width) dominated (35.1%), while at
40 months post-drilling smaller individuals (40–50 mm width) were the most predominant
(25.8%) (Fig 9). The proportion of individuals with 30–40 mm carapace width was only 3.0% at
16 months, but had increased to 15.6% at 40 months. There were no individuals with 20–30 mm
carapace width at 16 months, but these appeared at 40 months (1.8%). The mean abundance of A. longirostris shrimps at 16 months was 0.3 ± 0.8 inds. m-3 within
10 m radius of Hole D/E (max: 3.7 inds. m-3). The abundance of A. longirostris at 25, 38 and 40
months after drilling was 0.2 ± 1.2 inds. m-3, 1.1 ± 4.0 inds. m-3 and 0.4 ± 1.2 inds. Discussion The results of this study revealed significant changes in the seabed landscape and megafaunal
benthic community after an intensive drilling event in a deep-sea hydrothermal field. Although
benthic animals that occupied the drilling site prior to drilling (such as live Calyptogena clams
in this case) were buried under drilling deposits as reported by previous studies (e.g., [17]), the
amount of megabenthic animals lost in this drilling event was much greater than those at a
normal, non-chemosynthetic deep-sea floor. This is due to deep-sea chemosynthetic habitats
harboring a remarkably high abundance of benthic animals (e.g., 70–1,000 ind. m-2, [29]) with
a biomass of up to 500–1000 times greater [30,31], compared to the surrounding non-chemo-
synthetic sea floor (e.g., 0.2–6 inds. m-2, [13]). m-3 with
maximum abundance of 6.0, 21.6 and 10.0 inds. m-3, respectively (Figs 7 and 8B). Paralomis sp. was not consistently observed within a 10 m radius of Hole D/E at 16 months, but appeared at 25,
38 and 40 months after drilling (Figs 7 and 10A), with a mean abundance of 0.07 ± 0.4 inds. m-3
at 25 months (max: 1.0 inds. m-3), 0.004 ± 0.02 inds. m-3 at 38 months (max: 0.1 inds. m-3) and
0.09 ± 0.2 inds. m-3 at 40 months (max: 0.7 inds. m-3). However Bathymodiolus mussel, one of the
typical sessile vent animals of the Okinawa Trough vents, has never been observed around the
holes from our video investigations up to the most recent observation (i.e. 40 months post-dril-
ling) (Fig 10B). Post-Drilling Megafaunal Changes in a Deep-Sea Hydrothermal Field PLOS ONE | DOI:10.1371/journal.pone.0123095
April 22, 2015 14 / 22 doi:10.1371/journal.pone.0123095.g009 The extent of drilling impact on seabed landscape The horizontal extent of the white drilling deposits in the present study was within 25 m of the
drilling. This is similar to that reported from Orinoco Fan, Venezuela (10–25 m, [11]), while
some other studies reported physical influence of exploitation drilling extended to approxi-
mately 100 m [9,12,13]. These differences may be due to the difference in the depths of drill
holes and periods of time analyzed. Unlike any other reports though, the drilling event in the
present study has subsequently caused hydrothermal fluid discharges seen as shimmering and
lowering of seawater pH from the holes and the surrounding sediments, because Hole G PLOS ONE | DOI:10.1371/journal.pone.0123095
April 22, 2015 15 / 22 Post-Drilling Megafaunal Changes in a Deep-Sea Hydrothermal Field Fig 9. Size-frequency distribution of Shinkaia crosnieri galatheid crabs at (a) 16 months and (b) 40 months post-drilling. Each size-
on galatheid crabs taken near Hole D/E. doi:10.1371/journal.pone.0123095.g009
Post-Drilling Megafaunal Changes in a Deep-Sea H
PLOS ONE | DOI:10.1371/journal.pone.0123095
April 22, 2015 Fig 9. Size-frequency distribution of Shinkaia crosnieri galatheid crabs at (a) 16 months and (b) 40 months post-drilling. Each size-structure is Fig 9. Size-frequency distribution of Shinkaia crosnieri galatheid crabs at (a) 16 months and (b) 40 months post-drilling. Each size-structure is based
on galatheid crabs taken near Hole D/E. doi:10.1371/journal.pone.0123095.g009 PLOS ONE | DOI:10.1371/journal.pone.0123095
April 22, 2015 16 / 22 Post-Drilling Megafaunal Changes in a Deep-Sea Hydrothermal Field Fig 10. Temporal sequence in distribution and abundance of megabenthos at Site C0014. (a) Paralomis lithodid crabs and (b) Bathymodiolus mussels. Dotted circles indicate the area within 10 m radius from Hole D/E. Fig 10. Temporal sequence in distribution and abundance of megabenthos at Site C0014. (a) Paralomis lithodid crabs and (b) Bathymodiolus mussels. Dotted circles indicate the area within 10 m radius from Hole D/E. doi:10.1371/journal.pone.0123095.g010 penetrated into the subseafloor hydrothermal fluid reservoir [16]. The multiple drilling opera-
tions that disturbed the subseafloor hydrogeological structures may provide numerous vertical
and lateral hydrothermal fluid pathway networks in the shallower sediments surrounding the
holes [17]. During the study period, deposits from the drilling event have been gradually removed
probably by lateral advection during high current events and from vertical redistribution in the
sediments [12]. In contrast, bacterial mats have developed and become widespread. The newly
emerged hydrothermal fluid discharges increased hydrothermal fluid inputs such as methane
and hydrogen sulfide and increased the temperature of seafloor surface, promoting bacterial
mat growth [32]. An increase in the areal extent of microbial mats up until 40 months post-
drilling indicates a continuous supply of hydrothermal fluids from the subseafloor sources. This phenomenon differs from the microbial mat succession observed at the East Pacific Rise
volcanic eruption in 1991, which reported a marked reduction in the areal coverage of the mi-
crobial mats within one year (from >20 m2 to <5 m2) and no longer present 32 months after
the eruption due to the reduction of hydrothermal flux [7]. Considering the horizontal extent
of the shimmering fluid and white drilling deposits as well as newly developed bacterial mats,
the horizontal impact distance by the scientific drilling was probably less than 30 m. Seabed around the drill holes has been hardening throughout the post-drilling period, be-
coming rough and undulated with many fissures at 38–40 months after drilling. Unfortunately,
we did not have sediment samples to characterize the hardening process in this study. Howev-
er, the most likely process is barite/gypsum precipitation or silicification of the seafloor sedi-
ment. As mentioned earlier, high-temperature hydrothermal fluid (>160°C) discharged
through the seafloor sediment. If this hot fluid was mixed with ambient cold (4°C) seawater,
sulfate minerals such as anhydrite (CaSO4), gypsum (CaSO4•2H2O) and barite (BaSO4) could
be precipitated. The solubility of anhydrite in the ocean has a reverse relationship as a function
of the temperature [33]. Thus, most of the anhydrite would be dissolved in the water when the
seabed temperature decreased to ambient seawater temperature as previously observed at an-
other IODP 331 drilling site (Site C0013, [17]), while some may remain as gypsum. Conversely,
the solubility of barite is approximately three orders of magnitude lower than that of anhydrite
[34], leading to survival of barite crystals. Indeed, a barite-rich chimney sample has been col-
lected at the Iheya North field in the past, prior to the present study [35]. Barite also originated
from drilling mud, which is used together with bentonite to adjust the density of circulative
drilling mud water. Therefore, some fractions of barite crystal that harden the seabed may be of
an artificial origin. Such barite/gypsum precipitation or silicification of the seafloor sediment
might have sealed and plugged the hydrothermal paths within the sediment, which in turn con-
verged the subseafloor hydrothermal flow. This process is a possible factor for the numerous
fissures observed at 38–40 months, due to fluid pressure. Since bentonite is well known to pro-
duce low permeable layer that shut out the flow [36–38], artificially added bentonite in the dril-
ling mud fluid may have also contributed to converge the subseafloor hydrothermal flow. Further investigation and additional sediment sampling are necessary to elucidate the detailed
mechanism of the seabed hardening after drilling. Further investigation and additional sediment sampling are necessary to elucidate the detailed
mechanism of the seabed hardening after drilling. Post-Drilling Megafaunal Changes in a Deep-Sea Hydrothermal Field PLOS ONE | DOI:10.1371/journal.pone.0123095
April 22, 2015 PLOS ONE | DOI:10.1371/journal.pone.0123095
April 22, 2015 17 / 22 Megafaunal assemblages at the artificial hydrothermal vents Previous studies on the deep-sea drilling impacts in oil and gas fields showed that megafaunal
density and diversity recovers partially from drilling disturbance after 3 years as there was PLOS ONE | DOI:10.1371/journal.pone.0123095
April 22, 2015 18 / 22 Post-Drilling Megafaunal Changes in a Deep-Sea Hydrothermal Field significant removal of cuttings from those initially deposited [12]. Contrariwise, the disturbed
ecosystems in the present study have not recovered to the pre-disturbed condition after 3
years, due to newly emerged hydrothermal fluid discharges from the subseafloor establishing
new hydrothermal vent ecosystems. The ‘artificial’ vent ecosystem created has already lasted 2
years and is likely to be maintained as long as the hydrothermal fluid supply continues. Originally, the drilling site was characterized by a chemosynthetic community sustained by
hydrothermal fluid seepage (geofluid seep), where Calyptogena clams dominated. The clam
colonies were however completely buried under the drilling deposits, and converted to a com-
munity consisting of typical vent-associated animals after several months. The galatheid crab S. crosnieri was predominant in the ‘artificial’ vent community, and their abundance around
Hole D/E increased considerably from 11 months to 25 months, stablizing thereafter. We con-
firmed that a small number of S. crosnieri at 11 months within 10 m of Hole D/E and subse-
quent higher abundance at 16 months after drilling. This suggests the galatheid crabs started
migrating to the drill site from 11 months when the bottom surface temperature around the
holes started to increase, indicating hydrothermal fluid discharges [17]. Compared to the popu-
lation at 40 months post-drilling, most individuals of S. crosnieri were larger in size in the 16
months post-drilling population, which also lacked small individuals (<3 cm carapace width). This strongly indicates that S. crosnieri migrated on foot to the drill site from existing nearby
colonies by sensing the fluid discharge (chemistry and/or lowering of seawater pH), rather
than through planktonic larvae dispersal. Although it may be possible for planktonic larvae of
S. crosnieri to arrive at the drill site after 11 months, it is unlikely that individuals developed
from planktonic larvae to adult in this short period (5 months, from 11 to 16 months) consid-
ering the general crustacean growth rate [39]. Previous seabed observations confirmed several
colonies of S. crosnieri outside of 20–40 m radius of the drill holes, implying their ability to mi-
grate at least 20 m distance on foot. Megafaunal assemblages at the artificial hydrothermal vents Similarly, another typical vent animal Alvinocaris shrimp was confirmed at 16 months after
drilling. Paralomis lithodid crabs have appeared at 25 months after drilling. Paralomis crabs
are typical carnivores [23] and may have migrated into the drilling site following the migration
of their potential preys, possibly Shinkaia galatheid crabs. Yet, another representative vent ani-
mal in the Iheya North field, Bathymodiolus mussels, has never been observed around Hole D/
E throughout the study periods even when there are several mussel bed colonies at ca. 12–30 m
distance around the drill holes. Although the sessile Bathymodiolus mussels can move short
distances by depositing and releasing byssal threads [40,41], it is unlikely that they move such a
long distance (20~ m) on foot. Consequently they can probably only migrate to the drill site
through planktonic larvae dispersal. Assuming that the larvae had a steady growth of ca. 2 cm
year-1 [42], if they had settled after establishment of the ‘artificial’ vent community, these mus-
sels must be visually recognizable from our ROV camera observation within 40 months after
drilling. This strongly indicates that the drill site is unsuitable for mussel colonization. In the
Okinawa Trough, Shinkaia crabs, Alvinocaris shrimps and Paralvinella polychaetes have been
reported as being closely associated with active vents, while Bathymodiolus mussels occur dis-
tantly from active vents probably due to their lower tolerance levels of temperature and/or
toxic substances such as hydrogen sulfide [43]. The high bottom temperature and/or high
concentrations of hydrogen sulfide at the drill site may not provide suitable habitat for the
mussels. Acknowledgments We thank Center for Deep Earth Exploration (CDEX) for providing data for the IODP Expedi-
tion 331; the operation teams of ROVs (Hyper-Dolphin and Kaiko 7000 II) and the ship crews
of R/V Natsushima, Kairei and Kaiyo; M. Morita and K. Yagasaki for their help in image analy-
sis, and B. Thornton and A. Bodenmann (The University of Tokyo) for providing the altitude
data of ROV. Conclusions In conclusion, the intensive drilling campaign in the Iheya North hydrothermal field resulted
in the complete collapse of the original hydrothermal-fluid-seepage community visually PLOS ONE | DOI:10.1371/journal.pone.0123095
April 22, 2015 19 / 22 Post-Drilling Megafaunal Changes in a Deep-Sea Hydrothermal Field characterized by Calyptogena clams (some alive) due to drilling deposits. The subsequent ‘arti-
ficial’ hydrothermal fluid discharges from the holes and the surrounding seabed created ‘artifi-
cial’ hydrothermal vent ecosystems. The vent-endemic galatheid crab S. crosnieri quickly
migrated to the newly created habitats and dominated the new vent community. Bottom sub-
strate had hardened probably due to barite/gypsum mineralization or silicification, becoming
rough and undulated with many fissures after drilling operation. Although the impacts of the
drilling operation on seabed landscape and megafaual invertebrate composition were confined
to an area of 30 m maximum from the drill holes, the newly established ‘artificial’ vent ecosys-
tem from these drillings is likely to continue to exist until the fluid discharge ceases. Conse-
quently, ecosystem in this area has been altered for long-term. It is indefinite how violent the
disturbances to seabed will be, in the case of full-scale commercial mining. Such mining and
their prerequisite feasibility studies of SMS deposits may require a large number of subseafloor
drilling, for mineral deposit reserve assessment. Therefore, massive commercial mining antici-
pated in the near future will potentially create more ‘artificial’ hydrothermal vent ecosystems at
a much greater scale. characterized by Calyptogena clams (some alive) due to drilling deposits. The subsequent ‘arti-
ficial’ hydrothermal fluid discharges from the holes and the surrounding seabed created ‘artifi-
cial’ hydrothermal vent ecosystems. The vent-endemic galatheid crab S. crosnieri quickly Author Contributions Conceived and designed the experiments: RN HY SK KT. Performed the experiments: RN HY
SK YT TN CC KF TM KT. Analyzed the data: RN. Contributed reagents/materials/analysis
tools: RN HY TM. Wrote the paper: RN HY SK YT TN CC KF KT. Conceived and designed the experiments: RN HY SK KT. Performed the experiments: RN HY
SK YT TN CC KF TM KT. Analyzed the data: RN. Contributed reagents/materials/analysis
tools: RN HY TM. Wrote the paper: RN HY SK YT TN CC KF KT. PLOS ONE | DOI:10.1371/journal.pone.0123095
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English
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Study on the efficacy of amorphous calcium carbonate as a consolidant for calcareous matrix
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Heritage science
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© The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the
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mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Abstract The popular involvement of amorphous calcium carbonate (ACC) in organisms, usually via a complicated non-clas-
sical crystallization pathway, has aroused interest in the scientific community. In recent years, a lot of work has been
carried out for the preparation and application of ACC. In this paper, the effectiveness of ACC for the consolidation
of calcareous matrix was evaluated. ACC was synthesized through direct carbonation of calcium oxide in ethanol
and was introduced into simulated samples as consolidant. A comprehensive study for the assessment of protective
effect was carried out by testing surface strength, chromaticity, compressive strength, water vapor permeability, water
absorption and freezing–thawing resistance. The results showed that the addition of ACC could significantly improve
the surface strength, compressive strength and weatherability of treated samples, while it had only a slight influence
on color difference and water vapor permeability. It was confirmed that ACC could connect the loose calcite particles
by ‘spot welding’ through heterogeneous nucleation and growth processes. As a consolidant, it demonstrated its mer-
its in this study and could be proposed as an alternative for the protection of weathered calcareous stones. Keywords: Amorphous calcium carbonate, Amorphous to crystal transformation, Calcareous matrix, Efficacy of
consolidation ous calcium carbonate, Amorphous to crystal transformation, Calcareous matrix, Efficacy of and reinforcement. Until now, commonly used materials
for the protection of weathered historic calcareous stones
include organic consolidant [1–5], inorganic consolidant
[6–8] and the hybrid one [9]. Organic consolidant usu-
ally possesses nice workability [10], such as good perme-
ability, low viscosity, and exhibit excellent reinforcement
performance [11]. However, since the organic materi-
als are apt to be attacked by microorganisms and suffer
from thermal-aging and photo-aging, the conservation
efficiency of organic polymers has been deeply con-
cerned by the scientific community in this field. While
considering the obvious incompatibility with calcare-
ous stones, the shortage of using organic consolidant
becomes much clearer. Owing to better physicochemi-
cal stability and compatibility than organic consolidant,
inorganic material has been considered as an alternative
for the consolidation of weathered calcareous stones. Although inorganic consolidant is not easy to breed
microorganisms and has good hydrophilicity, its poor
permeability and sometimes harmful side-effect caused Wang et al. Heritage Science (2022) 10:165
https://doi.org/10.1186/s40494-022-00804-4 Open Access Introduction Limestone is a sedimentary rock made of calcium car-
bonate (CaCO3), usually in the form of calcite or arago-
nite. It is abundant in the earth’s crust and has been used
as construction and building materials from very ancient
times. Many immovable cultural relics, such as stone
carvings, monuments, niches and grotto temples, were
constructed on the base of limestone. Around the world,
especially in Europe, many famous churches were con-
structed using limestone as the main building material. However, these cultural relics have suffered from damage
after long exposure to the natural environment. To delay further deterioration of these cultural relics
bearing fruitful artistic, historic, and scientific informa-
tion, the most effective way is conducting conservation *Correspondence: sdki@shu.edu.cn
1 Institute for the Conservation of Cultural Heritage, Shanghai University,
Shanghai 200444, People’s Republic of China
Full list of author information is available at the end of the article *Correspondence: sdki@shu.edu.cn
1 Institute for the Conservation of Cultural Heritage, Shanghai University,
Shanghai 200444, People’s Republic of China
Full list of author information is available at the end of the article *Correspondence: sdki@shu.edu.cn
1 Institute for the Conservation of Cultural Heritage, Shanghai University,
Shanghai 200444, People’s Republic of China
Full list of author information is available at the end of the article [40]
presented the first direct nanoscale evidence showing
that faceted calcite crystals could grow non-classically by
a layer-by-layer process through the amorphous-to-crys-
talline transformation and fusion of amorphous calcium
carbonate with the underlying calcite substrate. The novel consolidation approach using ACC prepared
by mixing the solutions of CaCl2.2H2O and Na2CO3 was
first proposed and tested by A. Burgos Cara et al. [41] in
2017. The authors demonstrated a significant increase in
drilling resistance and durability of the treated stone by a
dispersion of ACC in isopropanol. In 2021, Pavlakou et al. [42] reported the possible use of ACC for decayed mar-
ble. They tested the possibility of treating calcite mate-
rials with suspensions of amorphous calcium carbonate
(am-CaCO3, ACC) and amorphous silica by measuring
the rate of dissolution of treated samples in slightly acidic
and undersaturated CaCO3 solutions. Samples deposited
with amorphous silica outperformed those treated with
ACC. However, the direct use of ACC as a consolidant
was still in its very early stage. There are several polymorphs of CaCO3. The repre-
sentative crystalline forms include calcite, aragonite and
vaterite [18]. Besides these crystal phases, amorphous
calcium carbonate also exists and has been found to play
a very important role in the formation of many biomin-
erals [19] and also in the initial crystallization stage of
biomimetic mineralization. The popular involvement of
amorphous calcium carbonate in biological process [19,
20] in organism, usually via a complicated non-classical
crystallization pathway [21, 22], has aroused interest in
the scientist community. In recent years, a lot of work has
been carried out for the preparation and application of
ACC. From the viewpoint of thermodynamics, ACC is a
metastable phase and tends to crystallize spontaneously. Taking advantage of this characteristic of ACC, the main
objective of this paper was to explore the possibility of
strengthening weathered calcareous matrix by ACC. In
this study, a green synthesis process was used by direct
carbonation of CaO in alcohol for the formation of ACC. High pure ACC was used as a potential consolidant and
its performance was assessed by comprehensive tests. To date, plenty of methods had been reported for the
synthesis of ACC. Wang et al. Heritage Science (2022) 10:165 Page 2 of 14 Page 2 of 14 from crystallization and expansion [12] of soluble salts
restricted its wild application. from aqueous solutions usually shows poor stability, so
far, most studies involving preparation of stable ACC
have introduced organic molecules or inorganic ions
to prevent ACC from crystallization. These additives
include poly (acrylic acid) [25], carboxyl species [34],
magnesium ions [35], phosphorus ions [36], silicate [37]
etc.. Recently, Sun et al. [29] reported that high pure
ACC could be synthesized by direct carbonation of CaO
in methanol, which provided an alternative way for the
preparation of phase pure ACC.h Limewater has been used for a long history. It is widely
known that the solubility of calcium hydroxide in water
is too low to provide enough consolidant in aqueous
phase. Meanwhile, the early stage reinforcement perfor-
mance [13] of inorganic consolidant is relatively poor. Taking limewater as an example, it needs to absorb CO2
from air to facilitate carbonation [14] and usually it is a
time-consuming process. Attempts had been carried out
through adding calcium hydroxide particles and stabiliz-
ers in limewater. The breakthrough work was reported by
Dei’s group [15] for the synthesis of stable nano-Ca(OH)2
particles in alcohols. It is a well-validated state-of-the-
art technology and the effectiveness has been proven in
numerous real life conservation cases. The consolidation
of calcareous relics by limewater or Nano-Ca (OH)2 is
usually accompanied by the formation of CaCO3 which
is the outcome of carbonation of Ca (OH)2 [16]. Rodri-
guez-Navarro et al. [17] revealed that the carbonation
of nano-lime in humid air at room temperature involves
the initial formation of amorphous calcium carbonate
(hereinafter referred to as ACC) and its transformation
to metastable vaterite (and minor aragonite) via a dis-
solution–precipitation process, followed by nonclassi-
cal nanoparticle-mediated crystal growth. Subsequently,
vaterite (and aragonite) partially dissolves and stable cal-
cite precipitates. The intriguing transformation process from ACC to
crystal state still has not been well mastered by scientists
until now [18]. In some cases, ACC transfers to crystal
state following a traditional ion-by-ion crystal growth
pathway, while the non-classical crystal growth pathway
[38] was usually reported in other studies. The end prod-
uct could be presented in multi-crystal powder or even
single crystal [39] which depended on the crystallization
conditions. In recent years, Rodriguez-Navarro et al. According to the originating of CO3
2−,
they could be categorized to the followings: gas diffusion
[23, 24], direct mixing of the aqueous solutions of cal-
cium and carbonate salts [25, 26], hydrolysis of dimethyl
carbonate [27], Kitano solution [28] and supplying gase-
ous CO2 [29, 30]. A lot of work has been carried out to
prepare stable ACC in ethanol media adopting CaCl2 as
the source of calcium [23, 24, 31]. From the view point
of conservation, the introducing of chloride ion into ACC
may cause the crystallization of soluble salts which are
harmful for historic relics [32, 33]. Since ACC prepared Characterization of amorphous calcium carbonateh p
The morphology of ACC was observed by the transmis-
sion electron microscope (TEM, JEM-2010, JEOL, Japan)
equipped with energy dispersive spectroscopy (EDS). The
attachments of high-resolution transmission electron
microscopy (HRTEM) image and selected area electron
diffraction (SAED) of JEM-2010 were used to get crystal-
lographic information. The crystal structure was inves-
tigated by X-ray diffractometry (XRD, D/MAX2200V
PCX) using Cu Kα radiation (λ = 1.5406 Å), operated
under 40 kV and 40 mA. The infrared absorption spec-
tra were determined by a Fourier Transform infrared
spectrometer (FTIR, Nicolet IS50). Thermogravimetric
(TGA) and differential scanning calorimetry (DSC) anal-
ysis were carried out using a Mettler Toledo TGA/DSC Synthesis of ACCh The synthesis of ACC was performed according to the
previously reported method [29] with some modifica-
tion by direct carbonation of CaO in ethanol. In a typi-
cal procedure, 1.25 g of CaO was added into 100 mL of
ethanol, and the slurry was grinded in a planet mill for
three hours to disperse it evenly. Then the uniform slurry
was transferred to an autoclave. When the temperature
reached 50℃, CO2 was fed into it. The pressure was kept
at 0.5 MPa and the reaction continued for 2.5 h under
constant stirring at 500 rpm. After the reaction, the pres-
surized CO2 was released from the autoclave. To remove
the small amount of over grown ACC particles, the reac-
tion mixture was centrifuged at 11,000 rpm for 10 min. An almost hyaline ACC ethanol suspension was acquired
after centrifugation. After drying the test blocks under ambient condition
for 24 h, different amounts of ACC were introduced into
simulated samples for reinforcement at room tempera-
ture. ACC dispersed in ethanol synthesized in “Synthesis
of ACC” section was infiltrated into the simulated sam-
ples by spraying. The information about the introduced
amount of ACC was shown in Table 1. The amount of
ACC introduced by each spray was controlled to be less
than 2 wt.% based on the weight of compressed block and
the interval between each spray was 24 h. After infiltrat-
ing the required amount of ACC, the simulated samples
were kept in the environment at room temperature and
relative humidity of 70% with different periods before
further investigation. Preparation and consolidation of simulated samples Preparation and consolidation of simulated samples
Simulated samples were prepared by a similar method
suggested by Yang [43] and Zeng et al. [44], which could
be considered as a proxy for heavily degraded calcare-
ous stones. Four kinds of calcium carbonate powders (10
mesh, 100 mesh, 400 mesh, and 1250 mesh) were mixed
uniformly in the weight ratio of 1:1:1:1. Ethanol was
added into the mixture in the ratio of 10 wt.% based on
the mass of calcium carbonate powder and then the mix-
ture was homogenized by stirring. The mixture of 10 g
or 30 g was weighed for each sample and pressed under
2 MPa or 5 MPa respectively by a tableting machine for
preparation of simulated samples. After that, the blocks
with the approximate size of Φ20 × 15 mm for 10 g pow-
der and Φ50 × 7 mm for 30 g powder were obtained. Materials Calcium Oxide (CaO, 98% +), ethanol (high performance
liquid chromatography grade, 99.9%) were received from Page 3 of 14 Wang et al. Heritage Science (2022) 10:165 Wang et al. Heritage Science (2022) 10:165 Wang et al. Heritage Science (2022) 10:165 Adamas-beta and used without further purification. Car-
bon dioxide (CO2, 99.995%) was purchased from Air Liq-
uide (Kunshan) Gas Technology Co., Ltd. Commercially
available calcium carbonate power with different grades
identified by their particle size (10 mesh, 100 mesh, 400
mesh, 1250 mesh) were purchased from Henan Yixiang
New Materials Co., Ltd. and used directly without any
pretreatment. 3+ (Schwerzenbach, Switzerland). The TGA/DSC curves
were recorded in the temperature range of 25 to 1000 °C
in air at a heating rate of 10 °C/min. Viscosity was meas-
ured by a rotational viscometer (Smart series, ChemTron
Co.) at 25 °C. The particle size of ACC dispersed in etha-
nol were determined by the Zeta Potentiometer (Zeta
sizer Nano). Evaluation of consolidation effectf This configuration pro-
vided the high-pressure side of partial water vapor, while
the relative humidity of the low-pressure side of partial
water vapor was kept at 0% via the absorption of water by
anhydrous CaCl2 loaded in the cup. The temperature was
maintained at 23.0 ± 0.5 °C during the test. The freezing–
thawing resistance test was conducted according to the
Chinese national standard of GB/T 50,266–2013. Before
freezing–thawing resistance test, samples were satu-
rated with water by soaking them in water at 15–20 °C
for 48 h. One freezing–thawing cycle included the freez-
ing phase (immersing sample in refrigerating fluid at absorption, and freezing–thawing resistance tests were
carried out in this study. Surface strength was deter-
mined according to ASTM D3359-09e2. Chromatic-
ity was measured by a I5 desktop spectrophotometer
(XRite, USA). The reported chromaticity was the average
of the measured values of three individual samples. The
color difference,ΔE*, was calculated by the equation of
E∗=
( L∗)2 + ( a∗)2 + ( b∗)2 (where L*, a* and
b* were the measured chromaticity parameters: L* was
brightness, a* and b* were chromaticity coordinates (a*
was the red–green parameter and b* was the blue–yel-
low parameter)). Compressive strength was measured by
a microcomputer controlled electronic universal testing
machine (CTM8050) under the loading rate of 0.5 mm/
min. Water vapor permeability was determined by a dry
test method according to the Chinese national stand-
ard of GB/T 17146–2015. A mixture of microcrystalline
wax (60 wt.%) and paraffin (40 wt.%) was used as seal-
ing material. The sample with the diameter of 50 mm
was used and it was sealed on the top of the test cup. According to that cord, the setup was put in a chamber
with the humidity about 50 ± 3%. This configuration pro-
vided the high-pressure side of partial water vapor, while
the relative humidity of the low-pressure side of partial
water vapor was kept at 0% via the absorption of water by
anhydrous CaCl2 loaded in the cup. The temperature was
maintained at 23.0 ± 0.5 °C during the test. The freezing–
thawing resistance test was conducted according to the
Chinese national standard of GB/T 50,266–2013. Before
freezing–thawing resistance test, samples were satu-
rated with water by soaking them in water at 15–20 °C
for 48 h. Characterization of ACC XRD pattern in the upper part of Fig. 1a showed the
crystallinity of the powder obtained by rapid-drying the
transparent colloidal suspension synthesized in “Syn-
thesis of ACC” section at 150 °C in a pre-heated oven. It
presented two broad humps at 30° and 45° respectively,
indicating an amorphous phase characteristic [45] of this
powder. XRD pattern for powder kept in ambient for
a week was also shown in the lower part of Fig. 1a. All
peaks could be attributed to the diffraction of calcite,
confirming the powder could change from amorphous
state to crystal one spontaneously. Samples dried at a
lower temperature increasing rate (for example, heat-
ing up from room temperature to 150 °C in an electric
oven by its full power supply) were highly unstable. XRD
patterns obtained from samples dried by this process
showed it was always the mixture of some sharp peaks
and two broad traditional humps. All these results indi-
cated that ACC could nucleate spontaneously from
amorphous to crystal state under natural conditions. The measurement of FT-IR could provide fruit-
ful structure information and it has been performed by
many researchers for the characterization of ACC. Fig-
ure 1b showed the IR absorption spectra of ACC and
calcite at room temperature. The Fourier transform infra-
red (FT-IR) spectroscopy spectrum of the rapid drying
powder showed typical characteristics of an ACC phase. It could be found from the spectra that the characteristic y
tis
n
et
n
I
a.u. Initial
10
20
30
40
50
60
70
80
2θ(°)
After a week
(a)
4000
3500
3000
2500
2000
1500
1000
500
). u
.a
(e
c
n
a
b
r
o
s
b
A
Wavenumber(cm-1)
ACC
CaCO3
712
876
1434
2510
2870
(b)
Fig. 1 a XRD patterns for powder obtained by rapid-drying process (upper part) and powder obtained by keeping it under ambient for one-week
(lower part), the bar chart in the lower part was the reference of calcite; b IR spectra of ACC and CaCO3 at room temperature 4000
3500
3000
2500
2000
1500
1000
500
). u
.a
(e
c
n
a
b
r
o
s
b
A
Wavenumber(cm-1)
ACC
CaCO3
712
876
1434
2510
2870
(b) y
tis
n
et
n
I
a.u. Evaluation of consolidation effectf To evaluate the consolidation effect of ACC on simu-
lated samples, surface strength, chromaticity, sim-
ple compression, water vapor permeability, water Table 1 specification of simulated samples and the information of infiltrated ACC content for each sample
* Errors for the mass of samples and the ACC colloid were ± 0.001 g; the solid content of ACC in ethanol dispersion was 2.91% based on the TGA analysis and
corresponding colloid was introduced based on this value. Specification of simulated sample
Infiltrated content of ACC (wt.%)
Amount of ACC colloid* (g)
Size (mm)
Mass* (g)
1wt.%
2wt.%
3wt.%
4wt.%
5wt.%
6wt.%
Φ20 × 15
10
3.436
6.873
10.309
13.746
17.182
20.619
Φ50 × 7
30
10.309
20.619
30.928
41.237
51.546
61.856 Table 1 specification of simulated samples and the information of infiltrated ACC content for each sample * Errors for the mass of samples and the ACC colloid were ± 0.001 g; the solid content of ACC in ethanol dispersion was 2.91% based on the TGA analysis and
corresponding colloid was introduced based on this value. Wang et al. Heritage Science (2022) 10:165 Wang et al. Heritage Science (2022) 10:165 Wang et al. Heritage Science (2022) 10:165 Page 4 of 14 absorption, and freezing–thawing resistance tests were
carried out in this study. Surface strength was deter-
mined according to ASTM D3359-09e2. Chromatic-
ity was measured by a I5 desktop spectrophotometer
(XRite, USA). The reported chromaticity was the average
of the measured values of three individual samples. The
color difference,ΔE*, was calculated by the equation of
E∗=
( L∗)2 + ( a∗)2 + ( b∗)2 (where L*, a* and
b* were the measured chromaticity parameters: L* was
brightness, a* and b* were chromaticity coordinates (a*
was the red–green parameter and b* was the blue–yel-
low parameter)). Compressive strength was measured by
a microcomputer controlled electronic universal testing
machine (CTM8050) under the loading rate of 0.5 mm/
min. Water vapor permeability was determined by a dry
test method according to the Chinese national stand-
ard of GB/T 17146–2015. A mixture of microcrystalline
wax (60 wt.%) and paraffin (40 wt.%) was used as seal-
ing material. The sample with the diameter of 50 mm
was used and it was sealed on the top of the test cup. According to that cord, the setup was put in a chamber
with the humidity about 50 ± 3%. Evaluation of consolidation effectf One freezing–thawing cycle included the freez-
ing phase (immersing sample in refrigerating fluid at − 20 °C ± 2 °C for four hours) and the following thaw-
ing phase (soaking sample in water at 20 °C ± 2 °C for
another four hours). Characterization of ACC Initial
10
20
30
40
50
60
70
80
2θ(°)
After a week
(a) (b) (a) (
)
Fig. 1 a XRD patterns for powder obtained by rapid-drying process (upper part) and powder obtained by keeping it under ambient for one-week
(lower part), the bar chart in the lower part was the reference of calcite; b IR spectra of ACC and CaCO3 at room temperature Page 5 of 14 Wang et al. Heritage Science (2022) 10:165 band ν4 of calcite at 712 cm−1 was replaced by two low-
intensity splitting bands at 695 and 725 cm−1, which was
a typical feature of ACC [23, 29]. The band at 695 cm−1
could be assigned to O–C–O bending (ν4) in the struc-
ture of the amorphous state of CaCO3. Bands at 864 and
1076 cm−1 could be attributed to out-of-plane bending
(ν2) and symmetric stretch (ν1) in non-centrosymmetric
structures [21]. Compared with the sharp out-of-plane
bending (ν2) at 876 cm−1 for calcite, the corresponding
one shifted to 864 cm−1 and became relatively wider for
ACC. In addition, it was worth noting that the infrared
spectra of ACC and calcite were quite different in the ν1
band at 1076 cm−1 and the ν3 band at 1434 cm−1. The
absorption peak of ACC at 1076 cm−1 was caused by the
symmetric stretching vibration of the non-centrosym-
metric structure in proto-calcite form. The relative broad
absorption peak at 1434 cm−1 which could be attributed
to the asymmetric stretch of the carbonate ions (ν3) for
calcite separated into two peaks located at 1474 cm−1
and 1416 cm−1. The peaks splitting at 712 and 1434 cm−1
for calcite were typical characteristics of ACC vibrations
[23]. The broad peaks at around 1636 cm−1 and the broad
band at around 3367 cm−1 were due to vibrations of
structural water molecules of ACC. as that high energy state ACC transformed to a lower
energy stable state of crystalline calcium carbonate and
released the energy of the system. In addition to the mass drop associated with water, a
weight loss above 600 °C could also be observed on the
curves of TGA and DTG. This mass drop was associated
with the thermal decomposition of CaCO3 into CaO. The
decomposition of CaCO3 transformed from ACC shifted
to lower temperature compared with that occurred for
the reference CaCO3 powder. Evaluation of the conservation efficiency Nice workability, one of it was penetration property,
could be considered as one major aspect for the fulfill-
ment of conservation. The measured value of viscosity for
the obtained colloid was 2.1 cP at 25 °C. Since the ACC
prepared in this study had a very small particle size and
colloidal suspension had a very low viscosity, its excellent
penetration property should be expected. Here, to test
the penetration property of ACC in simulated samples,
the instantaneous states about a droplet of ACC colloidal
suspension from the contact with the simulated sample
to completely penetrate into it was visually recorded by
high-speed photography. Figure 4 showed the scenarios
from 0–0.3 s about a droplet of ACC. It’s very clear that
a drop of ACC suspension could quickly penetrate into
the simulated sample, which took less than 0.3 s for the
whole process. It illustrated that ACC truly had a good
property about penetration which was suitable for fur-
ther reinforcement of weathered heritage relics.h All the above results indicated that ACC was success-
fully synthesized in our experiment. The electron diffrac-
tion (ED) pattern shown in Fig. 2b further confirmed the
lack of crystallinity in the sample, as only diffuse diffrac-
tion rings were observed. From the TEM image in Fig. 2a,
it could be found that ACC tended to form secondary
agglomeration particles from nanoparticles with several
or several tens’ nanometers. The histogram in Fig. 2c
unraveled that the particle size distribution of ACC was
relatively narrow and centered at 13 nm, which was con-
sistent with the results obtained from TEM observation. The Tyndall effect (Fig. 2d) could be clearly observed in
ACC ethanol suspension by an incident light beam, dem-
onstrating a stable dispersed nanoparticle system.f To understand the difference in thermodynamic trans-
formation characteristics between ACC and calcite, the
TGA/DSC measurements were carried out. The TGA and
DTG (Derivative Thermogravimetry) curves were shown
in Fig. 3a, b. The mass drops about 12.38% observed in
the TGA curve before 200 ℃ were related to physical
absorbed and structural water. Considering the prepara-
tion conditions and drying process of ACC, the lost water
here was the free water absorbed from air during the dry-
ing process of liquid samples. Characterization of ACC The decrease of the decom-
position temperature of CaCO3 could be attributed to
the nano-size effect. From the weight loss quantity in the
high temperature section, it could be calculated that the
CaCO3 content in ACC was more than 96.9 wt.% which
showed high purity of ACC. Based on the experimental
dosage, the solid content of ACC in the colloid after full
carbonization of CaO could reach 3.00wt%. The calcu-
lated value of the solid content of ACC in ethanol disper-
sion was 2.91% based on the TGA analysis. The decrease
of that value was attributed to the removal of a small
amount of over grown ACC particles. Evaluation of the conservation efficiency In addition, combined with
the analysis of TGA and DSC curves, there was an obvi-
ous exothermic peak in DSC curve where there was no
weight loss at 326 ℃ in TGA curve. It indicated that ACC
underwent a crystal transformation around this tempera-
ture. The exothermic peak could be reasonably explained The surface strength test after the addition of variable
amount of ACC to the simulated samples was carried out
by the ‘Scotch Tape Test’ method. The surface strength
was evaluated by the calculation of removed mass from
the surface of the simulated sample by the scotch tape. The smaller the mass of the removed material from ele-
mental area, the greater the surface strength of the sim-
ulated sample. It could be found from Fig. 5a that the
surface strength of the simulated sample was significantly
improved with the increase of infiltrated ACC content
and the effectiveness became tender when the infiltrated
amount of ACC exceeded 3 wt.%. The sample treated by Wang et al. Heritage Science (2022) 10:165 Page 6 of 14
)
%
(
y
tis
n
et
n
I
Size(nm)
13nm
(c)
(a)
(b)
(d)
Fig. 2 a TEM images of ACC, b electron diffraction (ED) pattern taken from particles within the red circle in a; c Particle size distribution of ACC; d
optical photos for ACC colloid and its Tyndall light scattering shown in the lower part (b) (a)
(b) (a) (b) (a)
)
%
(
y
tis
n
et
n
I
Size(nm)
13nm
(c)
(d)
Fig. 2 a TEM images of ACC, b electron diffraction (ED) pattern taken from particles within the red circle in a; c Particle size distribution of ACC; d
optical photos for ACC colloid and its Tyndall light scattering shown in the lower part
)
%
(
y
tis
n
et
n
I
Size(nm)
13nm
(c)
(d) (c) (d) Fig. Evaluation of the conservation efficiency 2 a TEM images of ACC, b electron diffraction (ED) pattern taken from particles within the red circle in a; c Particle size distribution of ACC; d
optical photos for ACC colloid and its Tyndall light scattering shown in the lower part the addition of 6 wt.% ACC showed that the strength was
about 13 times higher than that of the blank sample (0
wt.% ACC).hf conservation, since such small variation will not be easily
detected by naked eyes. In our experiments, the addition
of ACC resulted a tiny color change while the addition
of 2 wt.% GPTMS made the variation of E* value exceed
this criterion. However, it should be noticed that this
result might be different from the consolidation of natu-
rally decayed limestone since the simulated samples were
made up of white calcite powder in this study. The appearance of samples with different amounts of
ACC (1 ~ 6 wt.%) were similar with the blank one with-
out the addition of ACC. Here, only chromatic properties
of the blank and the samples with the addition of 2 wt.%
ACC were listed in Table 2. As comparison, samples with
the addition of 2 wt.% GPTMS were also measured. Gen-
erally, ΔE* < 5 is considered to be acceptable for practical To assess the compressive strength of the treated sim-
ulated samples, a simple uniaxial compressive test was Wang et al. Heritage Science (2022) 10:165 Page 7 of 14 200
400
600
800
1000
50
60
70
80
90
100
)
%
(ss
ol
t
h
gie
W
T(℃)
CaCO3
ACC
H2O
CO2
CO2
(a)
87.62%
54.91%
50.28%
200
400
600
800
1000
-0.8
-0.6
-0.4
-0.2
0.0
0.2
)
℃
/
%
(
G
T
D
CaCO3
ACC
(b)
684℃
696℃
150
300
450
600
750
900
-8
-4
0
4
8
12
)
W
m
(t
d
/
H
d
CaCO3
ACC
(c)
326℃
T(℃)
T(℃)
Fig. Evaluation of the conservation efficiency Heritage Science (2022) 10:165 Page 8 of 14
Mass per unit area(g/m2)
Content (wt%)
(a)
0
1
2
3
4
5
6
0.5
1.0
1.5
2.0
2.5
3.0
Compress strength(MPa)
(b)
Content (wt%)
Fig. 5 aThe surface strength of the simulated sample with the penetration of ACC; b Scatter diagram (purple) and its fitting curve (red dotted line)
of compressive strength of treated simulated samples by the addition of ACC
Mass per unit area(g/m2)
Content (wt%)
(a) 0
1
2
3
4
5
6
0.5
1.0
1.5
2.0
2.5
3.0
Compress strength(MPa)
(b)
Content (wt%) Fig. 5 aThe surface strength of the simulated sample with the penetration of ACC; b Scatter diagram (purple) and its fitting curve (red dotted line)
of compressive strength of treated simulated samples by the addition of ACC Table 2 Chromaticity parameters for specific samples
GPTMS 3-Glycidoxypropyltrimethoxysilane
Blank
2wt% ACC
2wt% GPTMS
L*
92.29
91.73
87.76
a*
0.84
0.94
1.64
b*
3.48
4.22
6.98
ΔE*
–
0.93
5.79 value of 2.80880 kPa in this experiment; RH1 represented
the relative humidity (50%) on the high water vapor pres-
sure side; RH2 represented the relative humidity (0%) on
the low water vapor pressure side.h The results shown in Fig. 6a revealed that the water
vapor permeability coefficient did not decrease sig-
nificantly after adding ACC to the simulated sample. As
comparison, the water vapor permeability for sample
with the addition of 2 wt.% 3-Glycidoxypropyltrimeth-
oxysilane as a consolidant was also tested. Introduc-
ing 2 wt.% of ACC into simulated sample caused a 13%
decrease of water vapor permeability, while that value
was 33% for sample introduced the same amount of
3-Glycidoxypropyltrimethoxysilane. Since silane had
been wildly used as consolidant to reinforce weath-
ered stone. Our results indicated that ACC adopted as
an additive for the reinforcement of weathered stone
showed better performance than silicone-based mate-
rial [46] in terms of water vapor permeability. And it also
suggested that ACC could be considered as a suitable
consolidant, since it did not dramatically alter the water
vapor permeability. Nowadays, retaining the pathway for
water and gas exchange after the addition of conserva-
tive materials to heritage relics has aroused deep concern
during conservation practice. Evaluation of the conservation efficiency 3 a The TGA curves; b the DTG curves and c the DSC curves of ACC and calcium carbonate 200
400
600
800
1000
-0.8
-0.6
-0.4
-0.2
0.0
0.2
)
℃
/
%
(
G
T
D
CaCO3
ACC
(b)
684℃
696℃
T(℃) 200
400
600
800
1000
50
60
70
80
90
100
)
%
(ss
ol
t
h
gie
W
T(℃)
CaCO3
ACC
H2O
CO2
CO2
(a)
87.62%
54.91%
50.28% (b) (a) (
)
150
300
450
600
750
900
-8
-4
0
4
8
12
)
W
m
(t
d
/
H
d
CaCO3
ACC
(c)
326℃
(
)
T(℃) (c) 150
300
450
600
750
900
-8
-4
0
4
8
12
)
W
m
(t
d
/
H
d
CaCO3
ACC
(c)
326℃
T(℃) ig. 3 a The TGA curves; b the DTG curves and c the DSC curves of ACC and calcium carbonate performed. It could be speculated from Fig. 5b that as
the mass of the infiltrated ACC increased, the compres-
sive strength of the simulated samples increased linearly. The fitting formula was y = 0.5767 + 0.3964x, where, “y”
represented the compressive strength (MPa) of the simu-
lated sample, and “x” represented the mass ratio of ACC
penetrated into the simulated sample. Fig. 4 From a–d, the pictures showed the instantaneous state of the
surface of simulated sample recording the penetration process about
a drop of ACC colloidal dispersion Fig. 4 From a–d, the pictures showed the instantaneous state of the
surface of simulated sample recording the penetration process about
a drop of ACC colloidal dispersion It could be concluded from our experiments that ACC
could be considered as an effective candidate for the rein-
forcement of weathered calcareous matrix. The introduc-
ing of 1wt. % ACC to the simulated sample could double
the compressive strength. And the compressive strength
had been increased to about 5 folds after introducing
ACC with a mass ratio about 6 wt. %. Similar tests were
conducted by Yang et al. [43] where nano-Ca(OH)2 dis-
persed in Isopropyl alcohol was used as consolidant for
comparable simulated samples in our study. In Yang’s
work, the introduced nano-Ca(OH)2 particles trans-
formed into loose rhombohedral crystalline calcite grains Fig. 4 From a–d, the pictures showed the instantaneous state of the
surface of simulated sample recording the penetration process about
a drop of ACC colloidal dispersion Wang et al. Evaluation of the conservation efficiency The purple bar corresponding to 2Si in a expressed the water vapor permeability coefficient of simulated sample
containing 3-Glycidoxypropyltrimethoxysilane with 2wt% of its own mass; c Picture of the blank sample after soaking in water for 1 min; d Picture of
the simulated weathered sample with 1wt% ACC after soaking in water for 24 h 0
1
2
3
4
5
6
2Si
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
Content(wt%)
(a)
kg 10-11/s m Pa
Absorption(%)
Content(%)
(b) 0
1
2
3
4
5
6
2Si
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
Content(wt%)
Absorption(%)
Content(%)
(b)
(a)
kg 10-11/s m Pa (b) (c)
(d)
Fig.6 The variation trend of a water vapor permeability coefficient of simulated sample and b water absorption rate of simulated sample with
the increase of ACC content. The purple bar corresponding to 2Si in a expressed the water vapor permeability coefficient of simulated sample
containing 3-Glycidoxypropyltrimethoxysilane with 2wt% of its own mass; c Picture of the blank sample after soaking in water for 1 min; d Picture of
the simulated weathered sample with 1wt% ACC after soaking in water for 24 h (d) Fig.6 The variation trend of a water vapor permeability coefficient of simulated sample and b water absorption rate of simulated sample with
the increase of ACC content. The purple bar corresponding to 2Si in a expressed the water vapor permeability coefficient of simulated sample
containing 3-Glycidoxypropyltrimethoxysilane with 2wt% of its own mass; c Picture of the blank sample after soaking in water for 1 min; d Picture o
the simulated weathered sample with 1wt% ACC after soaking in water for 24 h disintegration resistance performance also confirmed
the consolidation efficiency of ACC. It could be found
from Fig. 6b that with the increase of the amount of ACC
introduced to the samples the water absorption of the
simulated sample decreased to some acceptable extent,
which was consistent with the results for water vapor
permeability. The decrease of water vapor permeability
and water absorption were the outcome of the reduc-
tion of the volume of pores in the tested samples. Evaluation of the conservation efficiency It has a profound effect on
improving the durability of conserved heritage relics,
since blocking the pathway for the transportation of salt
and water could cause severe damage by the addition of
conservation materials [30]. after two weeks’ incubation in air. There was no obvi-
ous improvement in superficial strength and compres-
sive strength for the simulated weathered stone samples
treated with the nano-Ca (OH)2 dispersion. However, it
was very clear that the infiltration of ACC could improve
the surface strength and compressive strength of the
simulated samples effectively at the same time in our
experiments.h The water vapor permeability of samples was tested
through the dry method by sealing the simulated sample
on the top of the test cup with anhydrous CaCl2 in it. The
water vapor permeability coefficient δ (kg/s·m·Pa) of the
measured sample was calculated according to formula
(1): (1)
δ =
G
A×Ps×(RH1−RH2) (1) where ‘G’ represented the wet flow in the unit of kilo-
gram per second (kg/s) through sample, which could be
deduced by drawing the curve of the mass increase of
the test system with time; ‘A’ was the exposed area of the
tested sample in square meters (m2); Ps was the saturated
vapor pressure at the test temperature (23 °C) with the Since water vapor permeability has a close relationship
with porosity [47] of the tested sample, the water absorp-
tion property of the simulated sample with or without the
addition of ACC was tested by recording the mass change Wang et al. Heritage Science (2022) 10:165 Page 9 of 14 0
1
2
3
4
5
6
2Si
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
Content(wt%)
Absorption(%)
Content(%)
(b)
(a)
kg 10-11/s m Pa
(c)
(d)
Fig.6 The variation trend of a water vapor permeability coefficient of simulated sample and b water absorption rate of simulated sample with
the increase of ACC content. Evaluation of the conservation efficiency It could be found that the number of
anti-freezing–thawing cycles of the simulated sample
increased significantly with the increase of the amount
of introduced ACC. The number of anti-freezing–thaw-
ing cycles increased from 2 to 20 as the content of infil-
trated ACC increased from 1 wt.% to 6 wt.%. When 4
wt.% ACC was introduced into the simulated sample,
the number of freezing–thawing cycles of the test sam-
ple reached 19. Therefore, combined with the results for
the study of mechanical strength, it showed that in the
process of infiltration reinforcement of simulated sample,
the infiltration of 4 wt.% could achieve satisfactory rein-
forcement effect. At the same time, with the increase of
freezing–thawing cycles, the mass of absorbed water in
the tested sample increased gradually. It indicated that
the freezing–thawing process gradually caused damage Combining the above experimental results, it could
be deducted that the penetration of ACC for the rein-
forcement of simulated samples was not resulted from a
simple particle filling effect in the pores but a cementi-
tious effect on the particles of the tested simulated sam-
ple through its own epitaxial growth by nucleation and
crystallization or direct transformation by a non-classical
pathway. The mechanism will be discussed in detail in the
later section. The utility of ACC as a consolidant could
not only reinforce the weathered sample effectively but
also maintain the water vapor transportation function. Evaluation of the conservation efficiency It was
also an acceptable and reasonable side-effect for the rein-
forcement of weathered stone by the addition of consoli-
dant especially when the decrease was not severe. of the simulated sample after 48 h immersing in pure
water at room temperature. The value of water absorption
for untreated sample could not be obtained since it col-
lapsed completely after immersion in water for only a few
minutes. This situation was shown in Fig. 6c. Since there
was no cementitious material in the untreated simulated
sample, disintegration became inevitable when it was
immersed in water. Contrast sharply with the untreated
sample destroyed by water rapidly, sample reinforced by
the addition of 1wt.% of ACC could maintain intact in
water after a long period of immersion. Observation for
samples reinforced by the addition of 1 ~ 6 wt.% of ACC
had lasted for 30 days after being immersed in water and
there was no obvious change for these samples. Nice Freezing–thawing resistance experiments were carried
out to assess weather resistance of the tested simulated Wang et al. Heritage Science (2022) 10:165 Page 10 of 14 to the tested sample by losing the cementitious material
transformed from ACC. samples treated by ACC. The number of freezing–thaw-
ing cycles for specific simulated sample was counted
until obvious cracks appeared in it. The results were
shown in Fig. 7. It could be found that the number of
anti-freezing–thawing cycles of the simulated sample
increased significantly with the increase of the amount
of introduced ACC. The number of anti-freezing–thaw-
ing cycles increased from 2 to 20 as the content of infil-
trated ACC increased from 1 wt.% to 6 wt.%. When 4
wt.% ACC was introduced into the simulated sample,
the number of freezing–thawing cycles of the test sam-
ple reached 19. Therefore, combined with the results for
the study of mechanical strength, it showed that in the
process of infiltration reinforcement of simulated sample,
the infiltration of 4 wt.% could achieve satisfactory rein-
forcement effect. At the same time, with the increase of
freezing–thawing cycles, the mass of absorbed water in
the tested sample increased gradually. It indicated that
the freezing–thawing process gradually caused damage samples treated by ACC. The number of freezing–thaw-
ing cycles for specific simulated sample was counted
until obvious cracks appeared in it. The results were
shown in Fig. 7. Microstructure analysis and the protective mechanism
of ACC for simulated samplef After that, the calcium carbon-
ate continuously precipitated in the high-energy surface
of the newly formed crystals to reduce the energy of the
system and make the nucleation and growth much easier.i spontaneous transformation of metastable ACC to sta-
ble calcite under nature conditions had been elucidated
by many researchers and it was also confirmed in this
study. In some cases, the single crystal could also be pre-
pared by this transformation process, when it underwent
in well controlled conditions. For example, in the work
reported by Tang et al. [30], centimeter-sized mono-
lithic CaCO3 signal crystal could be obtained by the
crosslinking of CaCO3 oligomers using different thermal
treatment protocols with careful control of temperature
between 300 to 325 ℃, or the damaged single-crystalline
calcite (Geological Iceland spars was used in their work)
could be perfectly repaired using CaCO3 oligomers. Combined with the results in our study, the specific
consolidation mechanism of ACC for calcareous matrix
was assumed and schematically shown in Fig. 9c. When
ACC dispersed in ethanol penetrated into the simulated
sample, ACC nanoparticles in colloid quickly adhered to
the surface of calcite powder and then the transformation
was followed by heterogeneous nucleation and growth. It was assumed that the growth pathway of ACC was
similar with that revealed by Rodriguez-Navarro et al. [40] through a nonclassical crystal growth mechanism
via the attachment of ACC nanoparticle. The process
involved surface diffusion and ultimate incorporation
coincided with dehydration and restructuring at macro-
steps, deriving from previously attached ACC particles
that form 2D islands or macro-spirals on the calcite sur-
face. Since nucleation and growth of calcite from ACC
was involved, the kinetics of this process deserved to be
further studied. Relative works had been done for the
carbonation process of nanolime and the beneficial for
future study for ACC system could be forecasted. For
example, the ‘Boundary Nucleation and Growth’ model
was adopted by Camerini et al. [49] for the first time to
consider the effect of surface area of Ca(OH)2 nanoparti-
cles on the carbonation kinetics. In their study, the effect
of temperature on the carbonation kinetics and on the
CaCO3 polymorphs formation was evaluated. However, the laboratory-controlled condition was far
from that we met when the weathered stone was con-
served under natural condition which might cover a
wide range of temperature and relative humidity. Microstructure analysis and the protective mechanism
of ACC for simulated samplef Positive effects for the utility of ACC as a consolidant for
calcareous matrix had been revealed clearly in this study. The exhibition of nice performance for the conservation
of simulated samples by the addition of ACC should have
an inherent relationship with the change of microstruc-
ture. To understand this, SEM images corresponding to
samples before and after the filtration of ACC were com-
pared. It could be found from the image in Fig. 8a that
the edges and surfaces of particles were regular and the
boundaries between particles were clear in the untreated
sample. When it was reinforced by ACC, the edges and
surfaces of particles became irregular and the boundaries
between particles became obscure. Some representative
spots were highlighted in the red circles in Fig. 8b.h Fig. 7 The number of freezing–thawing cycle and water absorption
quality for tested samples with different content of ACC The general impression obtained from the analysis of
these SEM images was that the enhancement of mechan-
ical properties for simulated samples was aroused by
the fusing of the contacted particles and the filling of
powders transformed from ACC. This result could be
reasonably explained by heterogeneous nucleation and
growth of ACC on the surface of calcareous matrix. The Fig. 7 The number of freezing–thawing cycle and water absorption
quality for tested samples with different content of ACC Fig. 7 The number of freezing–thawing cycle and water absorption
quality for tested samples with different content of ACC Fig. 8 The scanning microscopy image of unreinforced simulated stone a and simulated stone reinforced by the addition of 4wt% ACC (b). the red
circles highlighted the heterogeneous nucleation sites for ACC Fig. 8 The scanning microscopy image of unreinforced simulated stone a and simulated stone reinforced by the addition of 4wt% ACC (b). the red
circles highlighted the heterogeneous nucleation sites for ACC Wang et al. Heritage Science (2022) 10:165 Wang et al. Heritage Science (2022) 10:165 Page 11 of 14 Page 11 of 14 Wang et al. Heritage Science (2022) 10:165 formation and growth in crystallography. When the cal-
cite powder was used as substrate, ACC nucleated and
transformed into the calcium carbonate crystal and grew
on the substrate firstly. Microstructure analysis and the protective mechanism
of ACC for simulated samplef This
study was carried out in a relatively standard condition,
which was 25℃ and 70% RH. It had been revealed that
relative humidity and temperature could result to differ-
ent consolidation effects when nano-Ca(OH)2 was used
for calcite matrix. For instance, P. López-Arce et al. [48]
consolidated dolostone samples used in historical build-
ings by exposure slaked lime (Ca(OH)2) nanoparticles at
33 and 75% relative humidity (RH). They found that 75%
RH favors the consolidation process by a fast transforma-
tion of nano-Ca(OH)2 into vaterite, monohydrocalcite
and calcite and significantly improve the physical and
hydric properties of the stones, but do not favor calcite
re-crystallization as it occurs at 33% RH. To explore ACC
as a consolidant, the impact of temperature and humidity
on the evolution of carbonate phases from ACC should
be carefully studied in the future. In our experiments, the synthesized ACC colloid
showed low viscosity and high penetration capability. With the volatilization of ethanol, ACC nanoparticles
could deposit on the surface of calcite powder. In the pro-
cess of reinforcement, it was found that a small amount
of ACC, such as 4 wt.%, could result in a very good rein-
forcement effect. Since the calcite transformed from
ACC could not be clearly distinguished from the calcar-
eous matrix. The growing habit of calcite spontaneously
transformed from metastable ACC colloid was observed
on the surface of glass or on the surface of calcite. The
morphologies of the resulted calcite were shown in Fig. 9. The calcite functioned as a substrate and it would influ-
ence the crystallizing habit of ACC. Under natural condi-
tions without a calcite substrate, it could be found that
ACC grew into spherical or short rod-like crystalline
CaCO3 as shown in Fig. 9a. When it grew on the surface
of calcite in a confined state, the crystals became very
fine and compacted in a closely connected state. These
phenomena accorded with the theory of crystal nucleus It should be noticed that the nucleation and growth
might happen randomly and continue to grow until the
tips of the macro-spirals or islands contacted and fused
with the counterpart growing from the adjacent calcite
powder. So, the simulated sample was strengthened by
‘spot welding’ through heterogeneous nucleation and
growth pathway of ACC.. Obviously, in our experiments
we could not assure that all ACC particles were con-
sumed in this effective way. Microstructure analysis and the protective mechanism
of ACC for simulated samplef So, to effectively use ACC as
consolidant, the method of application and the pretreat-
ment of the substrate deserved to be carefully studied in
the future. Conclusion Colloid ACC in ethanol was successfully synthesized
by direct carbonation of CaO and it was adopted as
a consolidant for calcareous matrix taking advantage
of its spontaneous transformation from high energy
amorphous state to low energy crystal state confined
on the surface of calcite powder. The surface strength, Wang et al. Heritage Science (2022) 10:165 Page 12 of 14 Fig. 9 Morphologies of particles transformed from ACC in natural conditions: on the surface of glass (a); with influence of calcite powder as
substrate (b). and the proposed mechanism for the reinforcement of simulated sample by ACC (c) Fig. 9 Morphologies of particles transformed from ACC in natural conditions: on the surface of glass (a); with influence of calcite powder as
substrate (b). and the proposed mechanism for the reinforcement of simulated sample by ACC (c) Fig. 9 Morphologies of particles transformed from ACC in natural conditions: on the surface of glass (a); with influence of calcite powder as
substrate (b). and the proposed mechanism for the reinforcement of simulated sample by ACC (c) about the assessment of conservation efficiency indicated
high potential of ACC for the application of the protec-
tion for stone cultural relics with calcareous matrix. compressive strength and freezing–thawing resistance
of simulated sample could be greatly improved by intro-
ducing ACC. Compared with the untreated sample, the
compressive strength increased by 5 times and the sur-
face strength increased by 12 times after the addition of
6wt% ACC into the simulated samples. The addition of
ACC as consolidant did not change the pore structure
significantly, which was reflected by the tender decrease
of water vapor permeability coefficient and water absorp-
tion rate. The nice conservation performance of ACC
could be explained by ‘spot welding’ through heteroge-
neous nucleation and growth pathway of ACC. Results Abbreviations ACC: Amorphous calcium carbonate; TEM: Transmission electron microscope;
EDS: Energy dispersive spectroscopy; HRTEM: High-resolution transmission
electron microscopy; SAED: Selected area electron diffraction; XRD: X-ray dif-
fractometry; FTIR: Fourier transform infrared spectrometer; TGA: Thermogravi-
metric; DSC: Differential scanning calorimetry; ED: Electron diffraction; DTG:
Derivative thermogravimetry. ACC: Amorphous calcium carbonate; TEM: Transmission electron microscope;
EDS: Energy dispersive spectroscopy; HRTEM: High-resolution transmission
electron microscopy; SAED: Selected area electron diffraction; XRD: X-ray dif-
fractometry; FTIR: Fourier transform infrared spectrometer; TGA: Thermogravi-
metric; DSC: Differential scanning calorimetry; ED: Electron diffraction; DTG:
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historical sites. Constr Build Mater. 2020;230:117055. 35. Shaked H, Polishchuk I, Nagel A, Bekenstein Y, Pokroy B. Long-term stabi-
lized amorphous calcium carbonate-an ink for bio-inspired 3D printing. Mater Today Bio. Declarations 20. Politi Y, Arad T, Klein E, Steve Weiner S, Addadi L. Sea urchin spine calcite
forms via a transient amorphous calcium carbonate phase. Science. 2004;306(5699):1161–4. Acknowledgements
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Not applicable. Page 13 of 14 Wang et al. Heritage Science (2022) 10:165 Wang et al. Heritage Science (2022) 10:165 Wang et al. Heritage Science (2022) 10:165 Availability of data and materials 18. Rimer JD. Inorganic ions regulate amorphous-to-crystal shape preserva-
tion in biomineralization. Proc Natl Acad Sci U S A. 2020;117(7):3360–2. All data generated or analyzed during this study are included in this
manuscript. tion in biomineralization. Proc Natl Acad Sci U S A. 2020;117(7):3360 19. Beniash E, Aizenberg J, Addadi L, Weiner S. Amorphous calcium carbon-
ate transforms into calcite during sea urchin larval spicule growth. Proc R
Soc Lond B Biol Sci. 1997;264(1380):461–5. Funding 16. Silva BA, Ferreira Pinto AP, Gomes A, Candeias A. Effects of natural and
accelerated carbonation on the properties of lime-based materials. J CO2
Util. 2021;49:101552. This study was supported by the National Natural Science Foundation of
China (NSFC, No. 52172297) and the National Key R&D Program of China
(Project No. 2021YFC1523400; Subject No. 2021YFC1523403). 17. Rodriguez-Navarro C, Elert K, Ševčík R. Amorphous and crystalline cal-
cium carbonate phases during carbonation of nanolimes: implications in
heritage conservation. CrystEngComm. 2016;18(35):6594–607. Author details
1 1 Institute for the Conservation of Cultural Heritage, Shanghai University,
Shanghai 200444, People’s Republic of China. 2 School of Material Science
and Engineering, Shaanxi University of Science & Technology, Shang-
hai 710021, People’s Republic of China. 22. Jeon T, Na YE, Jang D, Kim IW. Stabilized amorphous calcium carbonate as
a precursor of microcoating on calcite. Materials. 2020;13(17):3762. 23. Chen SF, Colfen H, Antonietti M, Yu SH. Ethanol assisted synthesis of pure
and stable amorphous calcium carbonate nanoparticles. Chem Com-
mun. 2013;49(83):9564–6. Received: 29 July 2022 Accepted: 6 October 2022 Received: 29 July 2022 Accepted: 6 October 2022 24. Rao C, Guo X, Li M, Sun X, Lian X, Wang H, Gao X, Niu B, Li W. In vitro
preparation and characterization of amorphous calcium carbon-
ate nanoparticles for applications in curcumin delivery. J Mater Sci. 2019;54(16):11243–53. Competing interests The authors declare no conflict of interest. 21. Addadi L, Raz S, Weiner S. Taking advantage of disorder: amorphous
calcium carbonate and its roles in biomineralization. Adv Mater. 2003;15(12):959–70. Author contributions 14. Wei G, Zhang H, Wang H, Fang S, Zhang B, Yang F. An experimental study
on application of sticky rice–lime mortar in conservation of the stone
tower in the Xiangji Temple. Constr Build Mater. 2012;28(1):624–32. Conceptualization; SJI. Methodology and Investigation; WW, SW, QL, XW. Writ-
ing—original draft preparation; WW. Writing—review and editing; WW, SW,
QL, XW, JZ, HL, SJ. All authors read and approved the final manuscript. 15. Salvadori B, Dei L. Synthesis of Ca(OH)2 nanoparticles from diols. Lang-
muir. 2001;17:2371–4. References 2021;11: 100120. 36. Zou Z, Xie J, Macías-Sánchez E, Fu Z. Nonclassical crystallization of
amorphous calcium carbonate in the presence of phosphate ions. Cryst
Growth Des. 2020;21(1):414–23. Page 14 of 14 Wang et al. Heritage Science (2022) 10:165 Wang et al. Heritage Science (2022) 10:165 Wang et al. Heritage Science (2022) 10:165 37. Gal A, Weiner S, Addadi L. The stabilizing effect of silicate on bio-
genic and synthetic amorphous calcium carbonate. J Am Chem Soc. 2010;132(38):13208–11. 37. Gal A, Weiner S, Addadi L. The stabilizing effect of silicate on bi 38. Bruno M. A two-step nucleation model based on diffuse interface theory
(DIT) to explain the non-classical view of calcium carbonate polymorph
formation. CrystEngComm. 2019;21(33):4918–24. 39. Walker JM, Marzec B, Nudelman F. Solid-state transformation of amor-
phous calcium carbonate to aragonite captured by cryoTEM. Angew
Chem Int Ed Engl. 2017;56(39):11740–3. 40. Rodriguez-Navarro C, Burgos Cara A, Elert K, Putnis CV, Ruiz-Agudo E. Direct nanoscale imaging reveals the growth of calcite crystals via amor-
phous nanoparticles. Cryst Growth Des. 2016;16(4):1850–60. y
41. Burgos-Cara, A. Biomimetic strategies for the consolidation and protec-
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42. Pavlakou EI, Agrafiotis AG, Tsolaki TG, Lemonia C, Zouvani E, Paraskeva CA,
Koutsoukos PG. The protection of building materials of historical monu-
ments with nanoparticle suspensions. Heritage. 2021;4(4):3970–86. 43. Yang F, Zhang B, Liu Y, Wei G, Zhang H, Chen W, Xu Z. Biomimic
conservation of weathered calcareous stones by apatite. New J Chem. 2011;35(4):887–92. 44. Zeng Y, Zhang B, Liang X. A case study and mechanism investigation of
typical mortars used on ancient architecture in China. Thermochim Acta. 2008;473:1–6. 45. Gebauer D, Gunawidjaja PN, Ko JY, Bacsik Z, Aziz B, Liu L, Hu Y, Berg-
strom L, Tai CW, Sham TK, Eden M, Hedin N. Proto-calcite and proto-
vaterite in amorphous calcium carbonates. Angew Chem Int Ed Engl. 2010;49(47):8889–91. 46. Rodrigues A, da Fonseca BS, Pinto AF, Piçarra S, Montemor MF. Tailoring
alkoxysilanes with poly(ethylene glycol) as potential consolidants for
carbonate stones. Constr Build Mater. 2021;289:123048. 47. Ban M, Mascha E, Weber J, Rohatsch A, Rodrigues JD. Efficiency and
compatibility of selected alkoxysilanes on porous carbonate and silicate
stones. Materials. 2019;12(1):156. 48. López-Arce P, Gomez-Villalba LS, Pinho L, Fernández-Valle ME, de Buergo
MÁ, Fort R. References Influence of porosity and relative humidity on consolidation
of dolostone with calcium hydroxide nanoparticles: effectiveness assess-
ment with non-destructive techniques. Mater Charact. 2010;61(2):168–84. 49. Camerini R, Poggi G, Chelazzi D, Ridi F, Giorgi R, Baglioni P. The carbona-
tion kinetics of calcium hydroxide nanoparticles: A Boundary Nucleation
and Growth description. J Colloid Interface Sci. 2019;547:370–81. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub-
lished maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in pub-
lished maps and institutional affiliations.
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The Changing Nature of Elections in Africa: Impact on Peacebuilding
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CHAPTER 10 The Changing Nature of Elections in Africa:
Impact on Peacebuilding Franklin Oduro F. Oduro (B)
Ghana Center for Democratic Development (CDD-Ghana), Accra, Ghana
e-mail: f.oduro@cddgh.org © The Author(s) 2021
T. McNamee and M. Muyangwa (eds.),
The State of Peacebuilding in Africa,
https://doi.org/10.1007/978-3-030-46636-7_10 e-mail: f.oduro@cddgh.org
© The Author(s) 2021
T. McNamee and M. Muyangwa (eds.),
The State of Peacebuilding in Africa,
https://doi.org/10.1007/978-3-030-46636-7_10
163 © The Author(s) 2021
T. McNamee and M. Muyangwa (eds.),
The State of Peacebuilding in Africa,
https://doi.org/10.1007/978-3-030-46636-7_10 Introduction Elections remain central to the development of democratic and inclusive
societies in Africa. Not only do elections provide means to economic
growth and development since citizens can elect leaders based on their
campaign promises and policies on socio-economic development, regular
elections also contribute to peacebuilding by conferring authority on
leaders, facilitating peaceful transfers of power, and promoting citi-
zens’ participation and inclusion in governance. Indeed, regular and
credible elections have been recognized by various global, regional, sub-
regional, and national entities and infrastructures as critical for sustainable
peacebuilding. In Africa, through the African Union (AU) Charter on
Democracy, Elections and Governance (ACDEG), the African Peace and
Security Architecture (APSA), and the African Governance Architecture
(AGA), among others, member countries have been provided guidelines 163 F. ODURO 164 to ensure that the conduct of elections promotes good governance, and
ensures peace and stability.1 Notwithstanding the general recognition of the centrality of elections
to sustainable peacebuilding efforts, many elections have resulted in less
peace and more destabilization. The continent has recorded several cases
of pre- and post-election violence that have threatened stability in some
countries, and generated prolonged civil wars, conflict, and violence in
others such as Ethiopia (2005), Togo (2005), Kenya (2007 and 2017),
Zimbabwe (2008), and Nigeria (2007 and 2011),2 to cite only a few
examples. Even in countries, such as Ghana, Zambia, and Senegal where
peaceful election outcomes have been recorded, the (flawed) conduct of
elections has stoked fears of instability and violence, which could flare
up at any time. Except for a few countries characterized by single party
and/or candidate dominance, such as Rwanda, there remain uncertain-
ties about how sustainable “peaceful outcomes of elections” are in many
African countries. This chapter provides an overview of elections and their contributions
to peacebuilding in Africa. The argument is made that while elections
should be viewed as a central part of peacebuilding, their impact is depen-
dent on the regularity, inclusiveness, integrity, and credibility of election
processes and outcomes. Drawing on key lessons and international best
practices, this chapter highlights challenges and emerging threats to the
conduct of peaceful elections in Africa. They include the increasing cost of
financing elections, abuse of presidential term limits, and the increasing
use of technology in the administration of elections. The final section
provides four recommendations for addressing the key drivers of election-
related violence and instability. Introduction These recommendations, informed by
three decades of election experiences in Africa, are advanced in order that
elections may contribute positively to peace on the continent. Elections and Peacebuilding The role of elections in peacebuilding is recognized globally. Rather than
viewing them as ends in themselves, they are viewed as one (impor-
tant) tool in broader efforts to promote peace and stability. Elections are
part of democratic political transitions and are critical during the imple-
mentation of peace agreements. As argued by Matanock, “Post-conflict
elections can greatly enhance the durability of peace agreements…specif-
ically, [peace] agreements that enable rebel and government parties to 10
THE CHANGING NATURE OF ELECTIONS IN AFRICA … 165 participate in elections.”3 Accordingly, international assistance for peace-
building programs, including deployments by the United Nations (UN)
and its affiliated agencies, invariably have an election component. This is
particularly true of post-conflict and/or transitional settings, which are
common in Africa.4 Post-conflict or transitional elections create the opportunity for all
stakeholders to contribute to building legal and constitutional frame-
works, as well as democratic institutions that advance a sustainable
peacebuilding agenda. In addition, because election periods provide a
path—for many citizens, the only path—to political participation, they
create the feeling of belonging, thereby fostering inclusion and unity
which, in turn, promotes peace. In other words, the electoral impera-
tive, the opportunity for citizens to periodically participate in leadership
change through a credible and competitive process, offers the promise of
peace and stability in societies.5 Fundamentally, the link between elections
and peacebuilding is premised on the belief that regular and credible elec-
tions offer the most promising instrument for addressing and managing
societal tensions and conflict without the use of violence.6 Three Decades of Elections in Africa: What Do
We Know About Their Impact on Peacebuilding? Whereas the nexus between elections and peacebuilding is widely under-
stood in its positive manifestations—e.g., allowing citizens to choose their
political leaders freely and, thus, allocate power peacefully—there is also
clear evidence to suggest that elections have often triggered violence
and undermined peacebuilding on the continent. Mostly, this is due to
the way elections are conducted. A poorly conducted election can ignite
the underlying tensions in societies, already amplified by the very high-
stakes—e.g., “winner-takes-all”—associated with electoral competition in
Africa. Studies on electoral violence in Sub-Saharan Africa highlight a
number of factors and threats to electoral peace.7 For example, Straus
and Taylor, examining data from 1990 to 2007, suggest a typology of
election violence that ranges from low intensity to violent harassment and
large-scale violence, which results in death and instability.8 Election-related violence on the African continent has been variously
influenced by: 166
F. ODURO 166 i. Intense competition—“winner-takes-all” mentality—among polit-
ical elites for state power and resources9; i. Intense competition—“winner-takes-all” mentality—among polit-
ical elites for state power and resources9; i. Intense competition—“winner-takes-all” mentality—among polit-
ical elites for state power and resources9; ii. weak election management bodies (EMBs); iii. flawed electoral registers; and iv. a lack of transparency and/or inadequate pre-election preparation These and other factors10 contribute greatly toward a deficit in trust in
elections among citizens and electoral opponents. The story is not all negative, however. Regular elections have increas-
ingly become the norm in Africa. Acceptance by citizens11 and political
elites that the ballot box is the only legitimate means for electing leaders
is now widespread. This is a far cry from the past when leaders ruled
through the barrel of a gun in many parts of Africa. Not anymore. The compliance of ACDEG protocols regarding regularity of elections in
member countries is growing. A recent study assessing compliance of six
AU-member countries (Ghana, Ethiopia, Rwanda, South Africa, Zambia,
and Nigeria) on regular elections confirmed as much, although capacity
to conduct elections varied across the studied countries.12 While regu-
larity of elections is improving, their quality remains a serious concern. Even with this concern, the fact that political elites and citizens prefer
elections, rather than other methods, for choosing leaders and allocating
power is beneficial to peacebuilding efforts in Africa. p
p
g
Gradually,
election
management
bodies
are
getting
better
and
becoming more professionalized. Three Decades of Elections in Africa: What Do
We Know About Their Impact on Peacebuilding? This has helped to drive even more
demand for transparency in election preparations and management. The
application of technology is also helping EMBs to address other concerns
and build societal trust in electoral processes. EMBs in Africa have been
the test-bed for biometric technology in registering potential voters. Where it has been introduced effectively, it has improved the integrity
of voter registers and enhanced transparency in the collation and declara-
tion of results. All of this has helped to make elections in some countries
more credible and thus minimize their potential for triggering violence
and instability. As a result of the improvement in election administration and
increasing professionalism of EMBs, the continent is also experiencing
increasing (even if sometimes difficult) acceptance of election results by
losing candidates, and thereby effecting peaceful election outcomes.13
The accompanying leadership transitions recorded in Benin, Senegal,
Nigeria, Ghana, Sierra Leone, and Liberia are significant success stories 10
THE CHANGING NATURE OF ELECTIONS IN AFRICA … 167 that are contributing to reducing tensions surrounding the conduct of
elections. Finally, the contributions of elections to peacebuilding processes in
Africa cannot be discussed without acknowledging the role of election
monitors and observers, both domestic and international. Despite some
limitations, the short- and long-term observation activities of election
observers, are contributing to and supporting peaceful election outcomes. The AU’s electoral assistance division within the department of political
affairs has played a critical role and continues to provide critical support
and observation missions (both long-term and short-term) to African
countries in compliance with ACDEG. The AU’s presence and role in
elections in Nigeria (2015 and 2019), Ghana (2016), Kenya (2017),
Liberia (2017), and Sierra Leone (2018) are clear cases for reference,
where their role in promoting peace before, during, and after elections
was critical. Winner-Takes-All Politics Winner-Takes-All Politics The “winner-takes-all” nature of African politics can be especially insid-
ious come election time, owing to the high stakes involved. The desire
of African political elites to capture state power, and by extension state
resources for themselves and their cronies, has fueled the quest to secure
election victory at any cost. As Atta-Asamoah notes, “Such [winner-takes-
all] politics, if left unchecked in the context of the complex cocktail of
development and security challenges in Africa, could derail the sustain-
ability of democratic gains, development strides, and the maintenance of
peace and security in many fragile states.”18 p
y
y
g
To curtail this trend, reforms should be made to key governance insti-
tutions—such as the legislature as a countervailing force to the executive
branch of government; constitutions to foster broad-based inclusive poli-
tics—and to electoral systems (e.g., move away from “first past the post”
and toward “proportional representation” and “mixed plurality”).19 With
particular reference to electoral systems, and as argued elsewhere with
respect to the Southern African Development Cooperation (SADC)
region, Khabele Matlosa, for example, recommends that “SADC states
must make deliberate efforts to address election-related conflicts and war
by, among other things, reforming their electoral systems.”20 Observa-
tions seem to suggest that the “first past the post” systems tends to
amplify the “winner-takes-all” approach. This, in turn, raises the potential
for election-related violence. Three Decades of Elections in Africa: What Do
We Know About Their Impact on Peacebuilding? As Lappin argues, while admitting limitations, “…interna-
tional election observation missions (IEOMs) remain essential elements
to peacebuilding…”14 Those limitations were most starkly evident during
the 2019 elections in Malawi, where the African Union Election Obser-
vationer Mission reported that “…the elections took place in a peaceful,
transparent and orderly manner, and thus met national, regional, conti-
nental and international standards for democratic elections…”15 For
their part, the European Union’s Election Observation Mission, which
covered 27 of the 28 voting districts of Malawi in its preliminary report,
concluded that despite an unlevel playing field, the election was “Well-
managed, inclusive, transparent and competitive.”16 However, Malawi’s
own Constitutional Court concluded in 2020 that this assessment was
flawed, thus confirming allegations of widespread irregularities. It ordered
new elections, which resulted in the defeat of the winner of the annulled
2019 vote.17 Both positive and negative voting experiences have served to heighten
Africa’s focus on developing best practices and institutional reforms to
ensure elections that better serve the interests of peace. Among many
lessons learned, two stand out: first, the negative consequences of the
persistent “winner-takes-all” mentality; and second, that the timing and
sequencing of elections are crucial, particularly in transitional periods. 168
F. ODURO 168
F. ODURO 168 Sequencing and Timing A badly timed election can, sometimes, be worse than having no elec-
tion at all. Promoters of democracy and good governance, whether
foreign or local, tend to push for elections at the first opportunity during
transitional settings. This is not always good for building sustainable
peace.21 Where peace is still fragile and trust is lacking, EMBs are usually
poorly resourced; basic legal and institutional reforms have not been
implemented; new and opposition political parties have not been given
sufficient time to prepare; and elections are often hurriedly organized. All
of these factors can impede democratic progress. As Fath-Lihic and Brancati argue, the ability of national and inter-
national policy actors to understand and assess the complex political,
legal, technical, operational, participatory, and security-related challenges 10
THE CHANGING NATURE OF ELECTIONS IN AFRICA … 169 in determining the ideal timing for the conduct of transitional elections is
critical for peacebuilding processes. A “one-size-fits-all” election solution,
they add, does not exist. Every transition is different. Much depends on
historical context, the nature of the conflict, and what the elections are
designed to achieve, which is not always as straightforward as it seems.22 “Elections are often the final stage of a peace process, if not the ulti-
mate objective,” observe McNamee et al. “The hope is that elections can
have a stabilizing effect on a fractured society. Done well and timeously,
they can. But conducted too early, before security problems have been
converted into political problems, they can have the opposite effect: exac-
erbate divisions and foment violence.”23 Fostering local ownership and
inclusivity, building trust and promoting broad-based popular support
for electoral processes are vital. The benefits to peace of encouraging a
national tone of mutual respect and tolerance during the electoral phases
(pre, during, and post) are also not to be underestimated. Challenges and Emerging Threats
to Election Peace in Africa In addition to the failings highlighted above, elections during the past
decade have revealed new and emerging problems which can serve to
undermine peace and stability. These emerging issues, including failures
in the (increasing) application of technology during elections, mounting
legal challenges to election results by contenders, incumbents ignoring
term limits, and the rising cost of elections, merit closer scrutiny. Technology and Elections In some cases, the use of biometric technology by EMBs in the regis-
tration and authentication of voters has helped to counter voter fraud
and enhance the credibility of electoral registers. Recently, EMBs have
also been scaling up efforts—notably in Ghana, Kenya, and Nigeria—
to use information technology to increase transparency in election results
transmission.24 In other instances, however, the introduction of new tech-
nologies has led to myriad problems, with negative consequences for
stability and peace. Questions about the procurement and management
of the equipment, equipment breakdowns, unreliable devices, and the
suspect integrity and poor handling of these devices by election officials F. ODURO 170 have been common.25 Failures in the collation and transmission of elec-
tion results in Kenya resulted in a period of grave uncertainty, as the
“defeated” opposition successfully disputed the results of the election,
then withdrew from the subsequent re-scheduled vote. Many feared that
the political turmoil sparked by the 2017 election saga would trigger
a repeat of the 2007/2008 election violence. Fortunately, this did not
happen. pp
All the evidence suggests that, despite these and other problems such
as weak transparency and inclusion in procurement practices, Africa’s
EMBs are pushing for more information technology in election manage-
ment. But that push, in itself, can be detrimental to situations where
elections become a tool for promoting peacebuilding. Recent electoral
processes and outcomes have exposed weaknesses in the methods and
conduct of election observers. Their ability, for example, in the era of
tech-driven elections to detect potential manipulation at results collation
centers seems limited. As Judd Devermont points out, “Most observers
do not have the technological expertise to counter… [and] circumvent
government internet controls, safeguard election results from hacking, or
detect digital tampering.”26 Hacking and other digital attacks can effec-
tively undermine many of the key ingredients of election integrity: voter
registration, vote casting, and vote tabulation. As many analysts have also
observed, the timing and context in which election-related information,
communication, and technology are introduced often do not allow for
adequate time for familiarization by opposition parties.27 The risks of
politicization become high at a very early stage, meaning that many elec-
tion results have already been effectively rejected by the opposition well
before the election itself is actually held. Litigating Election Results Recent elections in Nigeria (2011 and 2019), Ghana (2012 and 2016),
Kenya (2017), Liberia (2017), Sierra Leone (2018) and Malawi (2019)28
gave rise to new dynamics in election dispute management and potential
threats to stability. The resort to judicial processes to contest elec-
tion results in these cases is, obviously, a progressive step forward from
the extra-judicial means used in the past to contest and change elec-
toral outcomes. That said, this trend presents its own challenges to
peacebuilding. 10
THE CHANGING NATURE OF ELECTIONS IN AFRICA … 171 When the judiciary is put at the center of deciding election outcomes,
it also ends up pronouncing on them. Though the difference in language
is subtle, the implications can be significant. Judicial pronouncements on
election outcomes will be based on interpretations of the law, technical-
ities, procedures, and legal principles which may contradict vote tallies. Citizens who feel that their votes have been overturned by seemingly
abstract judicial rulings may be more susceptible to joining violent forms
of resistance and mass mobilization. They may even be given succor by
alternative swearing-in ceremonies (as recently witnessed in Kenya) where
the opposition candidate claims “victor status” despite official results not
going his or her way.29 Civic education in Africa has not sensitized people
to the idea that one’s chosen candidate can “win” the election at the
ballot box but lose it in court. The length of judicial litigations on election outcome disputes could
also serve as potential triggers for election violence. The decision by the
Kenyan Supreme Court after the 2017 election is a case in point. The
relative speed (within a month) with which the court concluded on the
petition brought before it by the opposition parties raised concerns that
the court did not spend adequate time examining the case and competing
arguments before deciding to annul the election results. In Ghana, the
opposite occurred: the post-2012 election petition to the Supreme Court
on the results of the presidential election took too long. The eight-month
delay created a wellspring of anxiety across society, which could have
boiled-over in a country where the declared winner was in office but not
yet confirmed as legitimate. On the face of it, there is nothing wrong with the judiciary playing a
greater role in ensuring elections are sound. Litigating Election Results The Malawi Constitutional
Court’s decision to order a re-run of the 2019 election has generally
been viewed as a boost for Malawi’s journey toward democracy. But as
more disgruntled politicians and parties try to exploit potential loop-
holes in electoral laws in African countries, weak election administration
infrastructures are exposed. Judiciaries cannot be expected to fill this
gap—balancing interpretations of rules and technicalities and legal prin-
ciples against the will of the people, however that might be determined. A recent study illustrated some of the dangers: in the case of Nigeria, it
argues that its judiciary has been complicit in perpetrating electoral fraud
in Nigerian elections.30 The authors conclude that, while the Nigerian
political class sees the judiciary as another platform for resolving election
disputes, the manipulation of the judiciary and its associated process to 172
F. ODURO F. ODURO 172 perpetrate electoral fraud has resulted in a high level of public mistrust of
the judiciary.31 Resolving this electoral conundrum will be critical to the
future of peacebuilding in Africa. Abusing Presidential Term Limits Another emerging threat to election peace relates to attempts by ruling
elites to extend their stay in office through removal of constitutional
term limits, mostly against the wishes of their populations.32 In the post-
Cold War era, approximately 30 African leaders have attempted to change
constitutional impositions to extend their rule—in perpetuity, in some
cases—and almost half of these attempts have succeeded.33 The idea of
“third termism” can sometimes be described as lawful, due to the consti-
tutional procedures adopted to legitimize the changes in the law, but the
popular opposition to such moves stores up considerable potential for
violence and instability. Moreover, attempts by African political leaders to
extend their stay in power typically precludes opportunities for multi-party
electoral engagements. The absurd corollary of the “president for life” trend—that oppo-
sition parties aren’t allowed to compete for office—foments myriad
extra-legal/judicial means of seeking power. As Taylor et al. argue,
election-related violence is more likely in situations where incumbents
have unfettered right to contest elections; conversely, where incumbents
don’t run, violence becomes less likely.34 Increasing Cost of Financing Elections and Elective Politics Increasing Cost of Financing Elections and Elective Politics The increasing cost of financing African elections is another potential
threat to peacebuilding.35 Due to limited resources, African countries
have turned more and more to donor partners, especially Western part-
ners, to fund their elections. This trend raises stark questions about
resilience and sustainability: what happens if traditional funding streams
suddenly dry up? Will governments be more inclined to suspend or delay
elections, or indeed not have them at all? And what will be the conse-
quent impacts on peace and stability? Through logistics and (putative)
funding-related delays in holding scheduled elections over more than two
years, from 2016–2018, the government of the Democratic Republic of
the Congo (DRC)36 was perhaps lucky to have escaped with only minor
eruptions of violence by discontented Congolese. 10
THE CHANGING NATURE OF ELECTIONS IN AFRICA … 173 It is not just the cost of the elections themselves that is problematic;
campaign financing can also seed security problems. The increasing cost of
electoral campaigns makes it difficult for opposition parties to compete on
a level playing field. Ruling parties and candidates often raid state coffers
to fund their campaigns; opposition parties might not be able to secure
private funding, owing to fears of the private sector, especially compa-
nies, that opportunities for business with the state will be closed if they
are found to be funding the opposition. Significantly, electoral politics in
much of Africa has become the sole preserve of the rich. A study in Ghana
revealed that around US $86,000 was spent on average by members of
parliament in their election campaigns.37 This is completely out of reach
for the vast majority of Africans, adding to concerns of inclusivity. If
money alienates ordinary Africans from electoral politics, this is bad for
peacebuilding in the long-term. Africa needs to take a hard look at its electoral systems and decide
whether to encourage peace or promote division. The “winner-takes-all”
politics common across Africa are a significant trigger of election-related
conflict.38 Any system that encourages political exclusion, Gyampoh
argues, “can, potentially, jeopardize the fragile election peace.”39 Alter-
natives such as proportional representation have their own shortcomings,
but to the extent that proportional representation lessens the marginal-
ization of losers and spreads the benefits of victory more widely, it can
reduce the likelihood of elections undermining peace and peacebuilding. Key Recommendations Strengthening the integrity and quality of electoral processes in Africa
will contribute positively to peacebuilding. This requires a multifaceted
response on the part of local, national, and international stakeholders,
including organizations specializing in elections and democratic assistance
and consolidation. As part of this holistic response, four specific policy
recommendations are offered below. 1. Re-examine electoral laws in African Union-member countries. There is a need to re-examine electoral laws in order to ensure that
these laws are in compliance with provisions in the African Union
Charter on Democracy, Elections and Governance (ACDEG). The
recurring challenge in deepening democratic governance in Africa 174
F. ODURO F. ODURO 174
F 174 has, generally, been the failure of African Union member coun-
tries to domesticate various protocols and charters to which they
are signatories. After three decades of operating transitional elec-
toral laws, there has yet to be an adequate sifting of the policies
and protocols which have worked from those that have not. The
reforms should address electoral processes as well as rules and legisla-
tion that promote a level playing field, reducing the unfair advantage
held by incumbents. Such a review must take into account gaps and
non-compliance that result in electoral disputes and litigations, as
well as a lack of transparency and inclusiveness. Given the regu-
larity of elections and their changing dynamics, Africa needs new
or updated mechanisms to address election-related threats to peace
and stability. Pro-democracy organizations working to strengthen
election management bodies should be at the forefront in engaging
with these growing threats. g
g
2. Confront the challenges related to the growing use of technology in
the administration of elections. There is a critical need to stimu-
late global and continental discussions on emerging challenges to
election observers’ work in an era of technologically driven elec-
tion administration. Independent and non-partisan citizen election
observation groups, who are already incorporating information tech-
nology in their observation as well as international election observer
missions, must begin to find ways of including verification proto-
cols in electronic transmittal and collation of election results in their
observation programs. With increasing sophistication in election
administration, election observer groups must also align their proto-
cols to the emerging threats to election peace. Key Recommendations “…like modern-day
police chasing cyber-thieves instead of masked bank robbers, inter-
national observers must adapt to new technologies.”40 Accordingly,
it may be prudent to review the Declaration of Global Principles
for Non-Partisan Election Observation and Monitoring by Citizen
Organizations, and Code of Conduct for Non-Partisan Citizen Elec-
tion Observers and Monitors to take into account protocols for
addressing gaps in the more obscure aspects of election technology
processes, i.e., the back-end. By addressing this gap, the reports of
election observer groups will be more reliable and better received by
host countries. Creating spaces for observer groups to engage elec-
tion management bodies in this area of election administration is
critical for enhanced transparency, trust-building, and inclusiveness. 10
THE CHANGING NATURE OF ELECTIONS IN AFRICA … 175 3. Assure sustainable financing of elections. Stakeholders, especially
international democracy promoters, must engage on the issues of
financing. In order to forestall opportunities for leaders to use a
lack of resources as an excuse to suspend elections, it is impera-
tive to develop ways for instituting sustainable financing of elections. Such mechanisms could include the establishment of national elec-
tion funds designed in a manner that the ruling government does
not have a say in its management. The policy response should also
include instituting predictable national and international sources
of funding, and accountable mechanisms to ensure transparent
and judicious use of resources for election administration. In this
instance, groups such as the International Foundation for Electoral
Systems (IFES), regional economic communities (RECs) in Africa,
the African Union Department of Political Affairs, and other global
democracy promoters must lead in advocating for such a fund. Exploring ways to address sustainable financing of elections must
be accompanied by campaign finance reform to create a more level
playing field, and to assure inclusiveness and integrity of elections. 4. Counter the trend to eliminate presidential term limits. A conti-
nental and sub-regional response is required to address the trend
toward serving beyond constitutionally mandated presidential term
limits. This will require sustained, long-term advocacy from all
corners, but perhaps especially the African Union and the regional
economic communities. Key Recommendations For instance, the Economic Community of
West Africa (ECOWAS) was close to instituting such a protocol for
its members but failed when two members, Togo and The Gambia,
objected to the decision.41 With the exit of president Yahya Jammeh
in The Gambia and the reforms that have occurred in Togo, there is
a window of opportunity for civil society actors to begin a campaign
for ECOWAS to revisit the subject. 4. Counter the trend to eliminate presidential term limits. A conti-
nental and sub-regional response is required to address the trend
toward serving beyond constitutionally mandated presidential term
limits. This will require sustained, long-term advocacy from all
corners, but perhaps especially the African Union and the regional
economic communities. For instance, the Economic Community of
West Africa (ECOWAS) was close to instituting such a protocol for
its members but failed when two members, Togo and The Gambia,
objected to the decision.41 With the exit of president Yahya Jammeh
in The Gambia and the reforms that have occurred in Togo, there is
a window of opportunity for civil society actors to begin a campaign
for ECOWAS to revisit the subject. 2. Dorina A. Bekoe (ed.), Voting in Fear: Electoral Violence in Sub-Saharan
Africa (Washington, DC: United States Institute of Peace Press, 2012). Notes 1. The African Charter for Democracy, Elections and Governance (ACDEG),
adopted in 2007, articulates the universal values of democracy and respect
for human rights that are founded on supremacy of the constitution and
the holding of democratic and credible elections. 2. Dorina A. Bekoe (ed.), Voting in Fear: Electoral Violence in Sub-Saharan
Africa (Washington, DC: United States Institute of Peace Press, 2012). 176
F. ODURO 3. Aila M. Matanock, “How Elections Can Lead to Peace: Making Negoti-
ated Settlements Last,” Foreign Affairs, April 25, 2018. 4. Annette M. Fath-Lihic and Dawn Brancati, Elections and Peacebuilding:
Why Timing and Sequencing of Transitional Elections Matter (Geneva:
Electoral Integrity Initiative Policy Brief no. 4, KofiAnnan Foundation,
2017). 5. Adelaja Odukoya, “Democracy, Elections, Election Monitoring and Peace-
Building in West Africa,” African Journal of International Affairs 10, no. 1–2 (2007): 147–160. 6. Richard Lappin, “Why Observe Elections? Reassessing the Importance
of Credible Elections to Post-conflict Peacebuilding,” Peace Research:
Canadian Journal of Peace and Conflict Studies 41, no. 2 (2009):
86–117. 7. Dorina
Bekoe,
“Africa’s
Electoral
Landscape:
Concerning
Signals,
Reassuring Trends,” African Center for Strategic Studies, May 16,
2016, https://africacenter.org/spotlight/sub-saharan-africas-electoral-lan
dscape-concerning-signals-reassuring-trends/. 8. Scott Straus and Charlie Taylor, “Democratization and Electoral Violence
in Sub-Saharan Africa, 1990–2008,” in Voting in Fear: Electoral Violence
in Sub-Saharan Africa (Washington, DC: United States Institute of Peace
Press, 2012). )
9. For
detailed
discussions
on
the
implications
of
“winner-takes-all”
phenomenon on inclusive governance and peacebuilding, see Ransford
Gyampoh, “Winner-Takes-All Politics in Ghana: The Case for Effec-
tive Council of State,” Journal of Politics and Governance 4, no. 1–4
(December 2015): 17–24; and Andrews Atta-Asamoah, “Winner-Takes-
All Politics and Africa’s Future,” Institute for Security Studies, https://iss
africa.org/amp/iss-today/winner-takes-all-politics-and-africas-future. 10. Such as unfair electoral rules and unfair playing field, technical incompe-
tence of members of EMBs, and appearance of incumbent control and
influence of EMB. 11. Interviews conducted by the Afrobarometer Research Network between
2016 and 2018 reported that 75 percent of Africans (across 34 countries)
preferred to use regular, open, and honest elections to choose country
leaders. See “Africans Want High-Quality Elections-Especially If They
Bring Change, Afrobarometer Surveys Show,” Afrobarometer, June 26,
2019,
https://afrobarometer.org/sites/default/files/pressrelease//ab_
r7_pr_africans_want_high_quality_elections_especially_if_they_bring_cha
nge.pdf. g p
12. Anne McLennan, “Democratic Governance,” in Civil Society Perspectives
on African Union Member States Commitments to Democratic Governance
(Johannesburg: Wits School of Governance, University of Witwatersrand,
2017). 10
THE CHANGING NATURE OF ELECTIONS IN AFRICA … 177 13. Notes Judd Devermont notes that since 2015, 13 opposition parties have won
elections and defeated incumbent parties, which is contrary to experiences
in the past two decades. See Judd Devermont, The Game Has Changed:
Rethinking the U.S. Role in Supporting Elections in Sub-Saharan Africa
(Washington, DC: Center for Strategic and International Studies, 2019). 14 L
i
“Wh
Ob
El
ti
?” 87 14. Lappin, “Why Observe Elections?” 87. 15. See https://au.int/sites/default/files/documents/38117-doc-report_of_
the_african_union_election_observation_mission_to_the_21_may_2019_t
ripartite_elections_in_the_republic_of_malawi.pdf, 9. 16. See
https://eeas.europa.eu/election-observation-missions/eom-malawi-
2019_en/63055/EU%20EOM%20Malawi%20presents%20its%20Prelimi
nary%20Statement. 17. See
https://www.theguardian.com/world/2020/jun/27/opposition-
wins-rerun-of-malawis-presidential-election-in-historic-first. p
18. Atta-Asamoah, “Winner-Takes-All Politics and Africa’s Future.” 19. Gyampoh,
“Winner-Takes-All
Politics
in
Ghana,”
17–24;
Ransford
Edward Van Gyampoh, “Dealing with Ghana’s Winner-Takes-All Poli-
tics: The Case of an Independent Parliament,” African Review 42,
no. 2 (2015): 63–75; Ransford Edward Van Gyampoh, “Dealing with
Ghana’s Winner-Takes-All Politics: A Case for Proportional Representa-
tion?” The Journal of Social Sciences Research 1, no. 4 (2015): 41–46;
Nic Cheeseman, Karuti Kanyinga, Gabrielle Lynch, Mutuma Ruteere,
and Justin Willis, “Kenya’s 2017 Elections: Winner-Takes-All Politics as
Usual?” Journal of Eastern African Studies 13, no. 2 (2019): 215–234;
Khabele Matlosa, “Review of Electoral Systems and Democratisation in
Southern Africa” (Paper, International Roundtable on the South African
Electoral System, Cape Town, South Africa, September 9–10, 2002);
and David Thomas, “End ‘Winner Takes All’ Politics—Osinbajo,” New
African, April 21, 2015, https://newafricanmagazine.com/10741/. 20. Matlosa, “Review of Electoral Systems and Democratisation in Southern
Africa.” 21. Fath-Lihic and Brancati, Elections and Peacebuilding; Sead Alihodzic,
Nicholas Matatu, Oliver Joseph, and Katrin Lewis, Timing and Sequencing
of Transitional Elections (Policy Paper No. 18, International IDEA,
2019); and Terence McNamee, Nchimunya Hamukoma, and Chipokoa-
Mayamba Mwanawasa, Elections in Africa: Preparing a Democratic
Playbook (Johannesburg: The Brenthurst Foundation, 2017). 22. Fath-Lihic and Brancati, Elections and Peacebuilding, 9. 23. McNamee, Hamukoma, and Mwanawasa, Elections in Africa, 17. f
24. “2019: Election Results Will Be Transmitted Electronically from Polling
Units—INEC,” Vanguard, December 5, 2017, https://www.vangua
rdngr.com/2017/12/2019-election-results-will-transmitted-electroni
cally-polling-units-inec/; Lois Ugbede, “2019: INEC Seeks NCC’s Help 178
F. ODURO 178 to Electronically Transmit, Collate Results,” Premium Times, January 31,
2018,
https://www.premiumtimesng.com/news/more-news/257151-
2019-inec-seeks-nccs-help-electronically-transmit-collate-results.html. p
y
25. Rhoda Osei-Afful, “Solutions or Problems? The Increasing Role of Tech-
nology in African Elections,” African Up Close (blog), Wilson Center,
December 11, 2017, https://africaupclose.wilsoncenter.org/solutions-or-
problems-the-increasing-role-of-technology-in-african-elections/. 26. Devermont, The Game has Changed, 6. 26. Devermont, The Game has Changed, 6. 27. McNamee, Hamukoma, and Mwanawasa, Elections in Africa, 9–12. 28. 36. Jason Burke, “DRC Minister Says Country ‘Can’t Afford’ to Hold
Election This Year,” The Guardian, February 16, 2017, https://www. Notes Recent elections conducted in these countries witnessed judicial litigations
in various forms in the pre-election and post-election phases, including
litigation on results collation, transmission and declaration. 29. Devermont, The Game Has Changed, 5. k
d
f
k k
d k
h
h
h 29. Devermont, The Game Has Changed, 5. 30. Hakeem Onapajo and Ufo Okeke Uzoduke, “Rigging Through the
Courts: The Judiciary and Electoral Fraud in Nigeria,” Journal of African
Elections 13, no. 2 (2014): 137–168. 31. Onapajo and Uzoduke, “Rigging through the Courts,” 161. 32. Boniface Dulani, “African Publics Strongly Support Term Limits, Resist
Leaders’ Efforts to Extend Their Tenure,” Afrobarometer, May 25,
2015, http://afrobarometer.org/sites/default/files/publications/Dispat
ches/ab_r6_dispatchno30.pdf. 33. McNamee, Hamukoma, and Mwanawasa, Elections in Africa; Ibraheem
Bukunle Sanusi and Rizzan Nassuna, Emerging Trends in Africa’s Election
Processes (Cape Town: Policy Brief No. 158, South African Institute of
International Affairs, 2017). 34. Charles Fernandes Taylor, John C. W. Pevehouse, and Scott Straus, “Perils
of Pluralism: Electoral Violence and Incumbency in Sub-Saharan Africa,”
Journal of Peace Research 54, no. 3 (2017): 397–411. 35. In Ghana, it is reported that the recent 2016 elections cost 35 times
more than the cost of 2004 elections. See “This Is the Cost of Elections
in Ghana,” Pulse, November 17, 2016, http://www.pulse.com.gh/
news/politics/election-2016-this-is-the-cost-of-elections-in-ghana-id5
772116.html. In Kenya, the 2017 elections were projected to be the
most expensive in Africa. See “Kenya Campaign Projects Now in Doubt
as Polls Cost Shoots to $500 m,” The East African, July 16, 2017,
http://www.theeastafrican.co.ke/news/Kenya-most-expensive-elections-
in-the-world-/2558-4016484-5dw5jhz/index.html;
Abdi
Latif
Dahir,
“Kenya Is Set to Hold One of the Most Expensive Elections in Africa,”
Quartz Africa, July 18, 2017, https://qz.com/1030958/kenyas-electi
ons-will-cost-1-billion-in-government-and-campaign-spend/;
McNamee,
Hamukoma, and Mwanawasa, Elections in Africa, 13–14. 36. Jason Burke, “DRC Minister Says Country ‘Can’t Afford’ to Hold
Election This Year,” The Guardian, February 16, 2017, https://www. 10
THE CHANGING NATURE OF ELECTIONS IN AFRICA … 179 theguardian.com/world/2017/feb/16/delayed-drc-elections-could-be-
put-back-further-by-cash-shortage; Vince Chadwick, “Donors Fall Short
of Targets on Funding Pledges for DRC, But See Progress,” Devex, April
16,
2018,
https://www.devex.com/news/donors-fall-short-of-targets-
on-funding-pledges-for-drc-but-see-progress-92543; David Pilling, “Con-
golese Opposition Calls for Foreign Cash to Fund Election,” Financial
Times, March 27, 2017, https://www.ft.com/content/fd05134c-04e7-
11e7-ace0-1ce02ef0def9. 37. Even this estimated figure has been described as very conservative and
too low by Ghanaian Members of Parliament. “MPs Spend US $86,000
to Get Elected—Study,” MyJoyOnline, February 4, 2018, https://www. Notes myjoyonline.com/politics/2018/February-4th/mps-spend-us86000-to-
get-elected-study.php; “Each MP Spends GHC390,000 on Election
Campaign—Report,” Citi 97.3 FM, February 5, 2018, http://citifm
online.com/2018/02/05/mp-spends-ghc390000-election-campaign-rep
ort/; “#GhanaElections 2016—Facts and Figures,” Ghana Election 2016,
December 6, 2016, http://www.africanelections.org/ghana/news_detail. php?nws=7480&t=. 38. Gyampoh, “Dealing with Ghana’s Winner-Takes-All Politics: A Case for
Proportional Representation?” 39. Ibid., 42. 40. Nic Cheeseman, Todd Moss, and Jeffrey Smith, “IT’S time for Inter-
national Election Monitors to Start Doing Their Job,” DemocracyPost,
The Washington Post, November 15, 2017, https://www.washingto
npost.com/news/democracy-post/wp/2017/11/15/its-time-for-intern
ational-election-monitors-to-start-doing-their-job/. 41. “W. African Leaders Drop Term Limit Idea After Gambia, Togo Oppose,”
Reuters, May 19, 2015, https://www.reuters.com/article/westafrica-
democracy-limits/w-african-leaders-drop-term-limit-idea-after-gambia-
togo-oppose-idUSL5N0YA52820150519; “Gambia and Togo Opposte
Presidential Term Limits,” CGTN Africa, May 20, 2015, https://africa. cgtn.com/2015/05/20/gambia-and-togo-oppose-presidential-term-lim
its/. 180
F. ODURO F. ODURO 180 Open Access This chapter is licensed under the terms of the Creative Commons
Attribution 4.0 International License (http://creativecommons.org/licenses/
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to the material. If material is not included in the chapter’s Creative Commons
license and your intended use is not permitted by statutory regulation or exceeds
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Pengaruh Penggunaan Media Video Pembelajaran Interaktif terhadap Hasil Belajar Fisika Siswa SMAN 1 Musi Rawas
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which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Pengaruh Penggunaan Media Video Pembelajaran Interaktif Terhadap Hasil Belajar Fisika Siswa
SMAN 1 Musi Rawas ngaruh Penggunaan Media Video Pembelajaran Interaktif Terhadap Hasil Belajar Fisika Siswa
MAN 1 M
i R Pengaruh Penggunaan Media Video Pembelajaran Interaktif Terhadap Hasil Belajar Fisika Sis
SMAN 1 Musi Rawas Pengaruh Penggunaan Media Video Pembelajaran Interaktif Terhadap Hasil Belajar Fisika Siswa
SMAN 1 Musi Rawas Ahmad Fahrudin, Eka Maryam Ahmad Fahrudin, Eka Maryam This is an Open Access article distributed under the terms of the Creative Common Attribution License (http://creativecommons.org/license/by/4.0/), Pengaruh Penggunaan Media Video Pembelajaran Interaktif Terhadap Hasil Belajar Fisika Siswa
SMAN 1 Musi Rawas Pengaruh Penggunaan Media Video Pembelajaran Interaktif Terhadap Hasil Belajar Fisika Siswa
SMAN 1 Musi Rawas © 2021 The Author(s). Published by Medan Resource Center ABSTRACT This research aims to find out the influence of the use of
interactive learning video media on the learning outcomes of
SMAN1 Musi Rawas Physics. This research uses the Quasi-
Experimental Design method with nonequivalent control group
design research design. The population of this study was the
entire class X SMAN 1 Musi Rawas, with a sample of class XIC as
the experimental class and XIA as the control class. Sampling
techniques are done randomly. Data collection techniques using
tests. Data analysis techniques with t-test. The results showed
that, the results of the post-test study physics experimental class
the average value was obtained 80.14, the control class was
70.86 and the tcount value was 4.915 > ttable. This means that
there is a significant difference between the results of post-test
experimental classes that use interactive learning video media
and control classes. Fahrudin, Ahmad., & Maryam, Eka. (2021). Pengaruh Penggunaan
Media Vido Pembelajaran Interaktif Terhadap Hasil Belajar Fisika
Siswa SMAN1 Musi Rawas. Pedagogika: Jurnal Ilmu-ilmu
Kependidikan. Volume 1 (1), Page. 60 – 64 fahrudinahmad63@gmail.com © 2021 The Author(s). Published by Medan Resource Center
hi i
O
A
i l di
ib
d
d
h
f h C
i
C
A
ib i
Li
(h
//
i
/li
/b /4 0/) Pengaruh Penggunaan Media Video Pembelajaran Interaktif Terhadap Hasil Belajar Fisika Siswa
SMAN 1 Musi Rawas I 61 Menurut (Hadir, dkk 2020) Pengaruh penggunaan media pembelajaran yang tepat dapat membangkitkan minat
siswa dalam belajar. Media pembelajaran sangat menentukan keberhasilan proses belajar mengajar (Lukman Hakim,
2020). Media pembelajaran dapat membantu meningkatkan keinginan belajar siswa sehinga konsentrasi terhadap
pembelajaran lebih terfokus. Video pembelajaran fisika telah banyak dikembangkan pada materi-materi fisika yang
disusun sesuai denan kebutuhan pembelajaran, contohnya sebagai media eksperimen atau praktikum (Hafizah, 2020). Contoh media pembelajaran berbasis video ini salah satunya adalah media pembelajaran berupa Videoscribe yang
merupakan multimedia berbasis komputer, berupa audiovisual dalam bentuk animasi dengan gambar dan teks
bernarasi atau lebih dikenal dengan witheboard animation (Sakti, 2019). Keuntungan penggunaan Sparkol Video Scribe dalam pembelajaran diantaranya dapat memberikan kemudahan
untuk menjelaskan pelajaran, karena software ini membantu menjelaskan materi pembelajaran dengan bantuan
animasi computer (Yudha, Asrul, & Kamus, 2016). Sedangkan menurut (Jannah, Harijanto, &
Yushardi, 2019)
aplikasi media pembelajaran fisika berbasis Sparkol Videosribe mampu meningkatkan hasil belajar siswa, media
pembelaajaran berbasis videoscribe cukup efektif dan mendapatkan respon dari siswa yang baik. Penelitian lain juga
dilakukan oleh (Pratiwi, dkk 2020) menyatakan bahwa adanya media pembelajaran berbasis video dapat
meningkatkan kemampuan siswa dalam memahami materi pembelajaran dan layak atau dapat digunakan untuk
proses pembelajaran. Berdasarkan latar belakang yang diuraikan diatas maka penelitian ini bertujuan untuk
mengetahui Pengaruh Penggunaan Media Vido Pembelajaran Interaktif Terhadap Hasil Belajar Fisika Siswa SMAN1
Musi Rawas. PENDAHULUAN Dilarangnya kegiatan pembelajaran tatap muka yang disebabkan karena adanya wabah yang melanda dunia termasuk
juga negara Indonesia. Sehingga dengan kondisi ini pemerintah mengeluarkan kebijakan masyarakat untuk bekerja dari
rumah, belajar dari rumah, serta beribadah di rumah. Kebijakan tersebut menyebabkan banyak sekolah-sekolah
menerapkan pembelajaran daring, kantorkantor menerapkan work from home (WFH) bagi karyawannya dan beraktifitas di
rumah (setyoningrum, dkk 201). Sehingga kebijakan tersebut berdampak pada sistem pembelajaran yang tadinya tatap
muka menjadi daring atau online (Febrianto, 2020). Terkait belajar dari rumah Kemendikbud menekankan bahwa
pembelajaran dalam jaringan dilaksanakan untuk memberikan pengalaman belajar yang bermakna tanpa membebani. Banyak sekali platform belajar daring yang bisa diakses seperti Google Classroom, Zoom, dan lain-lain. Penggunaaan media
vido pembelajaran interaktif merupakan salah satu solusi untuk siswa mampu mempelajari materi pelajaran dengan baik
(Sulastri, dkk 2021). Materi yang diberikan dalam bentuk powerpoint, video singkat maupun soal latihan yang diberikan
melalui Google Classroom. Media pembelajaran merupakan alat perantara pesan dalam hal ini materi pelajaran kepada siswa (Setyahandayani,
dkk 2018). Dengan adanya media vido pembelajaran diharapkan membantu mempermudah guru dan siswa dalam
melaksanakan kegiatan belajar mengajar secara online, Selain itu dalam memahami materi tersebut siswa tidak mudah
jenuh menyimak materi pelajaran yang disampaikan oleh guru melalui media vidio pembelajaran interaktif yang digunakan
karena
siswa
terbiasa
berlama-lama
dengan
handphone
mereka
(Basri
&
Khatimah,
2019). 60 Pengaruh Penggunaan Media Video Pembelajaran Interaktif Terhadap Hasil Belajar Fisika Siswa
SMAN 1 M
i R
I61 Pengaruh Penggunaan Media Video Pembelajaran Interaktif Terhadap Hasil Belajar Fisika Siswa
SMAN 1 Musi Rawas I 61 PEMBAHASAN Hasil data distribusi frekuensi post-test pada kelas eksperimen dan kontrol
Kriteria
Kelas Eksperimen
Kelas Kontrol
Frekuensi
Frekuensi
Sangat Baik ( 85-100)
8
2
Baik ( 70-84)
14
10
Cukup (55-69)
5
11
Kurang (40-54)
1
4
Sangat Kurang ( 0-39)
0
1
Total
28
28
Rata-Rata
80,14
70,86
Nilai Tertinggi
90
88
Nilai Terendah
48
36 Kriteria
Kelas Eksperimen
Kelas Kontrol
Frekuensi
Frekuensi
Sangat Baik ( 85-100)
8
2
Baik ( 70-84)
14
10
Cukup (55-69)
5
11
Kurang (40-54)
1
4
Sangat Kurang ( 0-39)
0
1
Total
28
28
Rata-Rata
80,14
70,86
Nilai Tertinggi
90
88
Nilai Terendah
48
36 Berdasarkan data pada tabel 3, diketahui bahwa post-test hasil belajar fisika kelas eksperimen diperoleh nilai
rata-rata sebesar 80,14 dengan kriteria baik. Nilai tertinggi 95 dan nilai terendah 64. Sedangkan kelas kontrol kontrol
diperoleh nilai rata-rata secara sebesar 70,86 dengan kriteria baik. Nilai tertinggi 90 dan nilai terendah 52. Selama
proses pembelajaran menggunakan media video pembelajaran, siswa terlihat lebih mudah memahami materi yang
disampaikan, siswa lebih antusias dan lebih senang selama pembelajaran. PEMBAHASAN Sebelum diberikan perlakuan pada kelas eksperimen dan kontrol terlebih dahulu dilakukan tes (pre-test). Setelah diadakan pre-test data yang diperoleh kemudian diolah menggunakan bantuan software SPSS 16 for Windows,
untuk mengetahui data distribusi frekuensi pre-test pada kelas eksperimen dan kontrol. Rincian data distribusi
frekuensi dapat dilihat dalam tabel berikut. Tabel 1. Hasil data distribusi frekuensi pre-test pada kelas eksperimen dan kontrol
Kriteria
Kelas Eksperimen
Kelas Kontrol
Frekuensi
Frekuensi
Sangat Baik ( 85-100)
0
0
Baik ( 70-84)
0
0
Cukup (55-69)
12
15
Kurang (40-54)
10
10
Sangat Kurang ( 0-39)
6
3
Total
28
28
Rata-Rata
58,86
60,71
Nilai Tertinggi
76
68
Nilai Terendah
24
20 Tabel 1. Hasil data distribusi frekuensi pre-test pada kelas eksperimen dan kontrol Tabel 1. Hasil data distribusi frekuensi pre-test pada kelas eksperimen dan kontrol
Kriteria
Kelas Eksperimen
Kelas Kontrol
Frekuensi
Frekuensi
Sangat Baik ( 85-100)
0
0
Baik ( 70-84)
0
0
Cukup (55-69)
12
15
Kurang (40-54)
10
10
Sangat Kurang ( 0-39)
6
3
Total
28
28
Rata-Rata
58,86
60,71
Nilai Tertinggi
76
68
Nilai Terendah
24
20 Berdasarkan data pada tabel 3, diketahui bahwa rata-rata hasil pre-test belajar fisika kelas control dan
eksperimen tidak memenuhi kreteria ketuntasan minimal (KKM), nilai rata-rata kelas kontrol yaitu sebesar 60,71 dan
nilai rat-rata kelas eksperimen yaitu 58,86 dengan kriteria kurang. Dari hasil tersebut sehingga perlu dilakukan
tindakan untuk memperbaiki hasil belajar siswa yaitu dengan memberikan treadment atau perlakauan berupa sistem
belajar menggunakan media vidio pembelajaran untuk kelas eksperimen dan sistem pembelajaran seperti biasanya 62 | Ahmad Fahrudin, Eka Maryam pada kelas kontrol. Setelah diberikan treadment atau perlakauan berupa sistem belajar menggunakan media vidio
pembelajaran untuk kelas eksperimen dan sistem pembelajaran seperti biasanya pada kelas kontrol. Selanjutnya
diberikan kembali tes (post-test). Adapun Rincian data post-test dapat dilihat dalam tabel berikut. Tabel 2 Hasil data distribusi frekuensi post-test pada kelas eksperimen dan kontrol pada kelas kontrol. Setelah diberikan treadment atau perlakauan berupa sistem belajar menggunakan media vidio
pembelajaran untuk kelas eksperimen dan sistem pembelajaran seperti biasanya pada kelas kontrol. Selanjutnya
diberikan kembali tes (post-test). Adapun Rincian data post-test dapat dilihat dalam tabel berikut. Tabel 2. Hasil data distribusi frekuensi post-test pada kelas eksperimen dan kontrol Tabel 2. A. Uji Prasyarat Sebelum dilakukan uji-t, terlebih dahulu dilakukan uji prasyarat yaitu uji normalitas dan uji homogenitas
sebagai syarat agar bisa dilakukan ujin t (Usmadi, 2020). Uji normalitas dilakukan untuk mengetahui apakah sebaran
data pada kelas eksperimen dan kelas kontrol berdistribusi normal atau tidak. Pengolahan uji normalitas
menggunakan bantuan software SPSS 16. Uji normalitas pada penelitian ini menggunakan Kolmogrov-Smirnov. Data
dikatakan berdistribusi normal apabila harga koefisien Asymptotic Sig pada output Kolmogorov- Smirnov tes lebih
besar daripada nilai alpha yang ditentukan, yaitu 5% (0,05). Rangkuman data hasil uji normalitas pre-test dan post-
test pada kelas eksperimen dan kelas kontrol dapat dilihat pada tabel berikut. Tabel 3. Rangkuman Hasil Uji Normalitas Pre-test dan Post-test Hasil Belajar Fisika
Data
Kolmogorov- Smirnov
Asymp Sig
(2-tailed)
Hasil
Kesimpulan
Eksperimen
Pre-test Hasil Belajar Fisika
0,813
0,523
Berdistribusi Normal
Post-test Hasil Belajar Fisika
0,789
0,562
Berdistribusi Normal
Kontrol
Pre-test Hasil Belajar Fisika
0,581
0,889
Berdistribusi Normal
Post-test Hasil Belajar Fisika
0,545
0,928
Berdistribusi Normal
Berdasarkan pada tabel 5 , pada kelas eksperimen diperoleh nilai Asymp Sig Kolmogorov-Smirnov pre-test Tabel 3. Rangkuman Hasil Uji Normalitas Pre-test dan Post-test Hasil Belajar Fisika hasil belajar fisika sebesar 0,523 > 0,05 dan post-test hasil belajar fisika 0,562 > 0,05, Pada kelas kontrol diperoleh
nilai Asymp Sig Kolmogorov-Smirnov pada pre-test hasil belajar fisika sebesar 0,889 > 0.05 dan post-test hasil belajar
fisika sebesar 0,928 > 0,05. Hasil tersebut menunjukkan data pre-test dan post-test kelas eksperimen dan kelas
kontrol berdistribusi normal. Setelah data berdistribusi normal kemudian dilakuakan uji homogenitas. hasil belajar fisika sebesar 0,523 > 0,05 dan post-test hasil belajar fisika 0,562 > 0,05, Pada kelas kontrol diperoleh
nilai Asymp Sig Kolmogorov-Smirnov pada pre-test hasil belajar fisika sebesar 0,889 > 0.05 dan post-test hasil belajar
fisika sebesar 0,928 > 0,05. Hasil tersebut menunjukkan data pre-test dan post-test kelas eksperimen dan kelas
kontrol berdistribusi normal. Setelah data berdistribusi normal kemudian dilakuakan uji homogenitas. Uji homogenitas dilakukan untuk mengetahui apakah kelompok data berasal dari populasi yang homogen atau
tidak. Pengolahan uji homogenitas menggunakan bantuan software SPSS 16. Uji homogenitas pada penelitian ini
menggunakan Uji Levene. Data dikatakan homogen apabila harga koefisien Sig. pada output Levene Statistic lebih Pengaruh Penggunaan Media Video Pembelajaran Interaktif Terhadap Hasil Belajar Fisika Siswa
SMAN 1 Musi Rawas | 63 |
besar daripada nilai alpha yang ditentukan, yaitu 5% (0,05). Rangkuman data hasil uji homogenitas pre-test dan post-
test pada kelas eksperimen dan kelas kontrol dapat dilihat pada tabel berikut. B. Uji Hipotesis (uji t) Uji-t ini dilakukan dengan tujuan untuk mengetahui ada tidaknya perbedaan yang signifikan antara pre-test dan
post-test hasil belajar fisika kelas eksperimen dengan kelas kontrol. Hipotesis yang akan diuji dalam penelitian ini
adalah: Uji-t ini dilakukan dengan tujuan untuk mengetahui ada tidaknya perbedaan yang signifikan antara pre-test dan
post-test hasil belajar fisika kelas eksperimen dengan kelas kontrol. Hipotesis yang akan diuji dalam penelitian ini
adalah: Ho : tidak ada perbedaan yang signifikan antara hasil belajar fisika kelas eksperimen dan kelas kontrol. Ha : ada perbedaan yang signifikan antara hasil belajar fisika kelas eksperimen dan kelas kontrol. Ha : ada perbedaan yang signifikan antara hasil belajar fisika kelas eksperimen dan kelas kontrol. Kriteria yang digunakan untuk mengambil kesimpulan hipotesis dengan taraf signifikansi 5% (0.05) yaitu
apabila nilai thitung < ttabel atau sig > 0.05 maka Ho diterima dan Ha ditolak, yang artinya tidak ada perbedaan yang
signifikan antara pre-test maupun post-test hasil belajar fisika kelas eksperimen dan kelas kontrol. Sebaliknya, apabila
nilai thitung > ttabel atau sig < 0.05 maka Ho ditolak dan Ha diterima, yang artinya ada perbedaan yang signifikan antara
pre-test hasil belajar fisika kelas eksperimen dan kelas kontrol. Hasil uji-t pre test dan post-test dapat dilihat pada tabel
berikut. Tabel 7 Hasil Uji t Data Pre-test dan Post-test
Data
Kelas
t
df
Sig. (2-tailed)
Kesimpulan
Pre test
Eksperimen- Kontrol
0,726
54
0,634
Tidak ada perbedaan yang signifikan
Pos test
Eksperimen- Kontrol
4,915
54
0,000
Ada perbedaan yang signifikan
Berdasarkan tabel 7, diperoleh data uji-t pre-test hasil belajar fisika kelas eksperimen dan kelas kontrol dengan
ilai t sebesar 0, 726 < ttabel 2,004 dan nilai sig. (2- tailed) 0,634 > dari 0,05. Sehingga dapat disimpulkan bahwa Ho
iterima dan Ha ditolak yang artinya tidak ada perbedaan yang signifikan antara pre-test hasil belajar fisika kelas
ksperimen dan kelas kontrol. Sedangkan pada hasil post-test didapatkan nilai t sebesar 4,915 > ttabel 2,004 dan nilai
ig. (2- tailed) 0,000 < dari 0,05. Sehingga dapat disimpulkan bahwa Ho ditolak dan Ha diterima yang artinya ada
erbedaan yang signifikan antara post-test hasil belajar fisika kelas eksperimen dan kelas kontrol. IMPULAN Tabel 7 Hasil Uji t Data Pre-test dan Post-test
Data
Kelas
t
df
Sig. B. Uji Hipotesis (uji t) (2-tailed)
Kesimpulan
Pre test
Eksperimen- Kontrol
0,726
54
0,634
Tidak ada perbedaan yang signifikan
Pos test
Eksperimen- Kontrol
4,915
54
0,000
Ada perbedaan yang signifikan
Berdasarkan tabel 7 diperoleh data uji-t pre-test hasil belajar fisika kelas eksperimen dan kelas kontrol dengan ,
p
j
p
j
p
g
nilai t sebesar 0, 726 < ttabel 2,004 dan nilai sig. (2- tailed) 0,634 > dari 0,05. Sehingga dapat disimpulkan bahwa Ho
diterima dan Ha ditolak yang artinya tidak ada perbedaan yang signifikan antara pre-test hasil belajar fisika kelas
eksperimen dan kelas kontrol. Sedangkan pada hasil post-test didapatkan nilai t sebesar 4,915 > ttabel 2,004 dan nilai
sig. (2- tailed) 0,000 < dari 0,05. Sehingga dapat disimpulkan bahwa Ho ditolak dan Ha diterima yang artinya ada
perbedaan yang signifikan antara post-test hasil belajar fisika kelas eksperimen dan kelas kontrol. SIMPULAN A. Uji Prasyarat Tabel 4 Hasil Uji Homogenitas Pre-test dan Post-test
Data Kelas Eksperimen & Kontrol
Levene
Statistic
Sig. Keterangan
Kesimpulan
Pre-test Hasil Belajar Fisika
0,464
0,599
Sig. > 0,05
Homogen
Post-test Hasil Belajar Fisika
2,654
0,209
Sig. > 0,05
Homogen
Berdasarkan pada tabel 6. diperoleh nilai Sig. pada Levene Statistic pre-test hasil belajar fisika kelas
eksperimen-kontrol 0,599 > 0,05 dan post-test hasil belajar fisika kelas eksperimen-kontrol 0,209 > 0,05. Dari hasil
tersebut dapat disimpulkan bahwa masing-masing data bersifat homogen (sama). Tabel 4 Hasil Uji Homogenitas Pre-test dan Post-test
Data Kelas Eksperimen & Kontrol
Levene
Statistic
Sig. Keterangan
Kesimpulan
Pre-test Hasil Belajar Fisika
0,464
0,599
Sig. > 0,05
Homogen
Post-test Hasil Belajar Fisika
2,654
0,209
Sig. > 0,05
Homogen
Berdasarkan pada tabel 6. diperoleh nilai Sig. pada Levene Statistic pre-test hasil belajar fisika kelas Post-test Hasil Belajar Fisika
2,654
0,209
Sig. > 0,05
Homogen
Berdasarkan pada tabel 6. diperoleh nilai Sig. pada Levene Statistic pre-test hasil belajar fisika kelas
eksperimen-kontrol 0,599 > 0,05 dan post-test hasil belajar fisika kelas eksperimen-kontrol 0,209 > 0,05. Dari hasil
tersebut dapat disimpulkan bahwa masing-masing data bersifat homogen (sama). Berdasarkan pada tabel 6. diperoleh nilai Sig. pada Levene Statistic pre-test hasil belajar fisika kelas
eksperimen-kontrol 0,599 > 0,05 dan post-test hasil belajar fisika kelas eksperimen-kontrol 0,209 > 0,05. Dari hasil
tersebut dapat disimpulkan bahwa masing-masing data bersifat homogen (sama). Berdasarkan pada tabel 6. diperoleh nilai Sig. pada Levene Statistic pre-test hasil belajar fisika kelas
eksperimen-kontrol 0,599 > 0,05 dan post-test hasil belajar fisika kelas eksperimen-kontrol 0,209 > 0,05. Dari hasil
tersebut dapat disimpulkan bahwa masing-masing data bersifat homogen (sama). SIMPULAN Berdasarkan hasil penelitian dan pembahasan, maka dapat diambil kesimpulan sebagai berikut. Terdapat
pengaruh penggunaan media video pembelajaran terhadap hasil belajar fisika siswa kelas XI SMAN1 Musi Rawas. Hal
ini ditunjukkan pada perbedaan nilai rata-rata post-test hasil belajar fisika siswa pada kelas eksperimen 80,14 lebih
besar dari nilai rata-rata kelas kontrol yaitu 70,86. 64 | Ahmad Fahrudin, Eka Maryam
h
h
(
)
f k
d
b l
k l
d
b
h d
l 64 | Ahmad Fahrudin, Eka Maryam 64 | Ahmad Fahrudin, Eka Maryam
Basri, S., & Khatimah, H. (2019). Efektivitas Penggunaan Media Pembelajaran Sparkol Videoscribe terhadap Hasil
Belajar Fisika Peserta Didik. Jurnal. as , S , &
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( 0 9)
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Belajar Fisika Peserta Didik. Jurnal. Febrianto, P. T., Mas’udah, S., & Megasari, L. A. (2020). Implementation of Online Learning During the Covid-19
Pandemic on Madura Island, Indonesia. International Journal Of Learning, Teaching And Educational
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Pembelajaran Fisika. Jurnal Pendidikan Fisika (JPF). Vol 9 No 1. Pp 81-89. Hafizah, S. (2020). Penggunaan dan Pengembangan Video dalam Pembelajaran Fisika. Jurnal Pendidikan Fisika, 8(2),
225-240. Hakim, Lukman. (2020) . Pemilihan Platform Media Pembelajaran Online pada Masa New Normal. Justek : Jurnal
Sains Dan Teknologi Vol 3 No 2 Hal 27 36 Hakim, Lukman. (2020) . Pemilihan Platform Media Pembelajaran Online pada Masa New Normal. Justek : Jurnal
Sains Dan Teknologi. Vol. 3, No. 2, Hal. 27-36. Jannah, M., Harijanto, A., & Yushardi, Y. (2019). Aplikasi Media Pembelajaran Fisika Berbasis Sparkol Videoscribe
Pada Pokok Bahasan Suhu dan Kalor terhadap Hasil Belajar Siswa SMK. Jurnal Pembelajaran Fisika, 8(2),
6572. Masidjo. (1995). Penilaian Pencapaian Hasil Belajar Siswa di Sekolah. Yogyakarta: Kanisius. Oemar Hamalik. (1982). Media Pendidikan. Bandung: Alumni. Oemar Hamalik. (1982). Media Pendidikan. Bandung: Alumni. Pratiwi, D., Latifah, S & Mustari. M. (2019) . Pengembangan Media Pembelajaran Fisika Menggunakan Sparkol
Videoscribe. Indonesian Journal Of Science And Mathematics Education. Vol 2 No 3. Pp 303-309. Sakti, I. (2019). Pengaruh Penggunaan Media Pembelajaran Berbasis Videoscribe terhadap Pemahaman Konsep
Fisika Siswa SMP Ittihad Makassar. PHYDAGOGIC Jurnal Fisika dan Pembelajarannya, 1(2), 4954. Sakti, I. (2019). Pengaruh Penggunaan Media Pembelajaran Berbasis Videoscribe terhadap Pemahaman Konsep
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Videoscribe untuk Pembelajaran Fisika Siswa Kelas X SMA. Pillar of Physics Education, 8(1). SIMPULAN PHYDAGOGIC Jurnal Fisika dan Pembelajarannya, 1(2), 4954. Setyahandayani, U., Darmawan, H., & Matsun. (2018) . Pengembangan Media Pembelajaran Fisika Berbasis Android
pada Materi Gelombang Cahaya di Kelas XI SMA Negeri 2 Ketapang. Jurnal Pendidikan Sains Dan
Aplikasinya (Jpsa). Vol 1 No 1. Pp 32-38. Setyoningrum, A., Rahmansari, F., Zulfinanda, U., & Safitri. P.T. (2021) . Pengaruh Media Pembelajaran Online
terhadap Hasil Belajar Siswa pada Masa Pandemi Covid-19. Jurnal Gammath, Volume 6 No 1. Pp 40-46
Sugiyono. (2010). Metode Penelitian Pendidikan Pendekatan Kuantitatif, Kualitatif, R&D. Bandung: Alfabeta. Sulastri, D., Maula, Lutfi, H., & Uswatun, Dian, A. (2021) . Pemanfaatan Platform Digital dalam Pembelajaran Online
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A lightweight high-resolution Human pose estimation method with an integrated attention mechanism
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A lightweight high-resolution Human pose
estimation method with an integrated attention
mechanism Fuchen Liu
Wenzhou University
Sijia Zhou ( zhousj@wzu.edu.cn )
Wenzhou University
Xiaocui Wang
Wenzhou University
Dezhou Zhang
Wenzhou University Sijia Zhou ( zhousj@wzu.edu.cn )
Wenzhou University
Xiaocui Wang
Wenzhou University
Dezhou Zhang
Wenzhou University Research Article Keywords: Human pose estimation, High-resolution network, Attention mechanism, Feature map
visualization Posted Date: May 22nd, 2023 A lightweight high-resolution Human pose estimation method with an integrated
attention mechanism Fuchen Liu, Sijia Zhou , Xiaocui Wang , Dezhou Zhang College of Mechanical and Electrical Engineering, Wenzhou University, Zhejiang Wenzhou 325035,China College of Mechanical and Electrical Engineering, Wenzhou University, Zhejiang Wenzhou 325035,China ABSTRACT Deep learning has significant advantages in the field of human posture estimation. However, traditional methods often increase
the parameter size and complexity of posture estimation networks by widening and deepening them to improve performance. To
address this issue, this paper proposes a lightweight human posture estimation network, ADNet, based on the high-resolution
network(HRNet). ADNet replaces Basic Blocks and downsampling in the high-resolution network with GABlocks and GDBlocks
that incorporate lightweight convolution and attention mechanisms to reduce the number of parameters and computational
complexity in the network. Additionally, ADNet solves the problem of feature redundancy by fusing relationships between
different channels to make effective feature channels fully utilized. Experimental results show that ADNet achieves high-precision
detection of human keypoint information even with fewer parameters and less computational complexity than commonly used
human posture estimation networks, such as Cascaded Pyramid Network (CPN), Stacked Hourglass Network(Hourglass), and
HRNet. Results also indicate that, compared to HRNet, ADNet reduces the model parameter size by 69.1% and computational
complexity by 58.9% under the same image resolution and environment configuration. Furthermore, in the visualization of feature
maps, the network using ADNet exhibits stronger feature extraction performance and less feature redundancy. Keywords: Human pose estimation, High-resolution network·, Attention mechanism, Feature map visualization In 2014, Toshev and his team effectively improved the
accuracy of the pose estimation network by fusing human pose
estimation and keypoint regression in DeepPose[[13]]. In 2016,
Newell et al. proposed the Stacked Hourglass Network
(SHN)[[14]], which uses a sampling method of descending and
ascending order, combined with feature maps of different
resolutions to achieve high-precision prediction of keypoints. In
2018, Chen et al. proposed the Cascaded Pyramid Network (CPN)
[[10]]to address the issue of information loss in SHN. This
network employs a layered prediction approach from easy to
difficult using GlobalNet and RefineNet to improve prediction
accuracy. In 2019, Sunet al. proposed the High-Resolution
Network (HRNet)[[12]], which uses multi-subnet chaining, multi-
resolution parallelism, and fusion of information among different
branches to achieve precise localization of semantic information. These human pose estimation networks have greatly increased the
prediction accuracy while significantly increasing the number of
parameters and computation complexity. DOI: https://doi.org/10.21203/rs.3.rs-2946377/v1 License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Additional Declarations: No competing interests reported. Additional Declarations: No competing interests reported. 1. Introduction Human pose estimation [[1],[2]] is a computer vision task that
involves joint localization of individuals in images or videos. Human pose estimation has achieved significant success in
applications such as human-computer interaction, motion capture,
sports and fitness posture correction. Due to the stronger feature
extraction performance and higher accuracy of convolutional
neural networks[3-5], most human pose estimation networks
currently use them. However, the large number of parameters and
floating-point operations in convolutional neural networks make it
difficult to deploy the models on resource-limited devices such as
robots and smartphones. Therefore, the current urgent problem in
human pose estimation is how to maintain good network
prediction performance while reducing the model parameter count
and computational complexity. There are two mainstream prediction methods for human
pose estimation, namely Top-down and Bottom-up. Top-
down[[6]-[8]] first detects the human body boundary box through
a human body detection network, then crops the human body
information, and finally predicts the human body keypoints using
a human pose estimation network. The success of keypoint
prediction depends heavily on the human detection network. Bottom-up[[9]-[12]], on the other hand, directly predicts the
position of each keypoint in the human body in the image, and then
obtains the final human pose estimation information through
splicing. This end-to-end method is faster for prediction, but it is
prone to connection errors when the keypoint information of the
human body is close to each other. In 2015, Jaderberg et al. proposed the Spatial Transformer
Network (STN)[[15]], which extracts useful information from
feature maps to reduce background interference and improve
network performance. In 2017, Hu et al. proposed the Squeeze-
and-Excitation Network (SENET)[[16]], which significantly
improves network performance by adding a small number of
parameters and computation complexity. SENET suppresses
useless feature channels and enhances the full utilization of feature
information by learning the importance of each channel for the
network. In 2018, Woo et al. 1. Introduction proposed CBAM[[17]], the first
fusion of spatial attention mechanism and channel attention E-mail address: 21461440045@stu.wzu.edu.cn( FC Liu）
E-mail address: xcwang@wzu.edu.cn ( XC Wang）
E-mail address: 20200590@wzu.edu.cn ( DZ Zhang）
Corresponding author E-mail address: zhousj@wzu.edu.cn（SJ Zhou） E-mail address: 21461440045@stu.wzu.edu.cn( FC Liu）
E-mail address: xcwang@wzu.edu.cn ( XC Wang）
E-mail address: 20200590@wzu.edu.cn ( DZ Zhang）
Corresponding author E-mail address: zhousj@wzu.edu.cn（SJ Zhou） complexity, this paper proposes two basic modules for building
the network, GABlock and GDBlock, which replace BasicBlock
and downsampling in HRNet, which account for the largest
proportion of computational and parameter complexity. By
improving the Ghost module to generate more effective feature
maps, the model's accuracy is improved. In order to improve the
performance of the module, we also studied the receptive field. The small receptive field in GhostModel makes it difficult for the
model to capture global features in the image. We increased the
receptive field, which greatly improved the model's generalization
ability and performance. The GABlock process is as follows:
standard convolution is used at the front end, which generates
intrinsic feature maps with more feature information and can fully
utilize all data to retain original detail information. Then, attention
mechanism optimization is applied to the feature information in
the intrinsic feature map to obtain an attention feature map through
compression and activation. Next, a cheap operation is performed
on the attention feature map to obtain a Ghost feature map. Finally,
to reduce feature loss, the initial intrinsic feature map and the final
Ghost feature map are stacked to obtain a Ga feature map. Fig. 2
shows the structure of GABlock. mechanism to improve network performance. In 2020, Li et al. proposed SKNet[[18]], which enhances network robustness by
fusing channel information extracted by convolutional kernels of
different sizes. However, the use of multiple sizes of convolutional
kernels and multi-directional feature channel information
extraction in these two attention mechanisms increases the number
of network parameters and computation complexity. The development of lightweight networks has always been a
research focus, achieved through changing the depth and width of
convolutional neural networks to reduce the number of parameters. In 2012, to enable multi-GPU training, AlexNet [[19]] proposed
Grouped Convolution. In 2017, Howard et al. introduced
depthwise separable convolution in MobileNets[[20]], which
separates convolution into depthwise convolution (Dwise) and
pointwise convolution (Pwise), greatly reducing the number of
parameters, but also resulting in decreased model accuracy. In
2020, Han et al. 1. Introduction proposed GhostModule in GhostNet [[21]], which
improves conventional convolution by performing linear
operations on intrinsic feature maps, reducing feature redundancy,
parameter count, and computational complexity, while also
improving network performance, but still with a significant
amount of feature redundancy. Fig. 1 shows there is a presence of
feature redundancy in the GhostNet model. GABlock reduces the number of parameters and feature
redundancy, while also minimizing computational cost. The joint
attention mechanism enhances the feature extraction performance. Here, "input" refers to the input feature map, "Conv" denotes
regular convolution, and "Output" represents the output feature
map. "𝑈𝑐" refers to the intrinsic feature map generated by regular
convolution, "i " represents linear operations, "G" is the ghost
feature map generated, and "Identity" is the identity mapping. "Fsq"
denotes the squeeze operation, "Fex" denotes the excitation
operation, and "Fscale" distributes weights channel-wise and
generates inexpensive phantom channels through linear operations
on the weighted channels. Finally, the regular convolution and the
features generated by attention and phantom operations are
concatenated to obtain the Ga feature map.The parameter quantity
of the ghost operation is given in Eq. 1： Fig.1 The feature maps with redundancy still exist in
hostNet Fig.1 The feature maps with redundancy still exist in
GhostNet Fig.1 The feature maps with redundancy still exist in
GhostNet 𝐺𝑝=
𝐶𝑖𝑛×𝐶𝑜𝑢𝑡
𝑆
× k × k +
𝐶𝑜𝑢𝑡
𝑆× (S −1) × d × d （1） （1） Where “Cin”and “Cout”are the number of input and output
channels of the feature maps, “k” is the kernel size of the regular
convolution, “d” is the kernel size of the inexpensive operation,
d “S” i th
b
f h
l i th i
i
l ti Where “Cin”and “Cout”are the number of input and output
channels of the feature maps, “k” is the kernel size of the regular
convolution, “d” is the kernel size of the inexpensive operation,
and “S” is the number of channels in the inexpensive convolution. “Fsq(·)”represents the Squeeze operation, which performs
dimensionality reduction with a reduction factor of 16, The
dimensionality reduction operation is given in Eq. 2 In 2021, YU et al. proposed a lightweight unit for human pose
estimation network called Lite-HRNet[[22]], but it resulted in a
significant drop in accuracy. Building upon the research
mentioned earlier, this paper introduces a human pose estimation
network named Attentional down-sampling network (ADNet),
based on the high-resolution network HRNet, incorporating Ghost
modules and SE modules. 1. Introduction ADNet greatly reduces the number of
parameters and computational complexity while ensuring a certain
level of prediction accuracy. To achieve this goal, we drew upon
the design of the Ghost and SE modules and innovatively
developed GABlock and GDBlock. We use these two modules to
replace the BasicBlock and downsampling blocks in HRNet,
which occupy a large number of parameters and computational
complexity. In this way, ADNet minimizes the network's
parameter count and computational complexity while maintaining
prediction accuracy. and S is the number of channels in the inexpensive convolution. “Fsq(·)”represents the Squeeze operation, which performs
dimensionality reduction with a reduction factor of 16, The
dimensionality reduction operation is given in Eq. 2 𝑍𝑐= 𝐹𝑠𝑞(𝑈𝑐) =
1
𝐻×𝑊∑
∑
𝑈𝑐（𝑖, 𝑗）
𝑊
𝑗=1
𝐻
𝑖=1
（2） （2） H and W are the height and width of the convolutional output
feature. Fex(·W) is the excitation operation which performs
dimensionality expansion with a factor of 16. The up-dimension
operation is given in Eq.3 p
g
q
𝑆𝑐= 𝐹𝑒𝑥(𝑧, 𝑊) = 𝜎(𝑔(𝑧, 𝑊)) = 𝜎(𝑊2𝛿(𝑊1𝑧)) （3） 𝑆𝑐= 𝐹𝑒𝑥(𝑧, 𝑊) = 𝜎(𝑔(𝑧, 𝑊)) = 𝜎(𝑊2𝛿(𝑊1𝑧)) （3） （3） In which z represents the result after the Squeeze operation,
W1 and W2 are the weights of the fully connected layers, δ denotes
the ReLu activation function, and σ denotes the sigmoid activation
function. 2 Methodology Fscale(·) is channel-wise multiplication. The channel-wise
multiplication operation is given in Eq. 4 2.1GABlock （ 4） p
p
g
q
𝑋̃ = 𝐹𝑠𝑐𝑎𝑙𝑒(𝑈𝑐, 𝑆𝑐) = 𝑈𝑐· 𝑆𝑐 （ 4） 𝑋̃ = 𝐹𝑠𝑐𝑎𝑙𝑒(𝑈𝑐, 𝑆𝑐) = 𝑈𝑐· 𝑆𝑐 In order to improve the accuracy of high-resolution image
processing while reducing network parameters and computational Conv
Input
Fsq(·)
Fscale(·)
Φ1
Φ2
Φk
Identity
Output
Fex(·W) Output Input Identity Fig.2 Structure flowchart of GABlock reduce feature loss and maintain performance, the input feature
map is subjected to parallel operations - the first operation is a
regular convolution to generate the intrinsic feature map, while the
second operation is a low-cost process to generate the Ghost
feature map. The Ghost feature map is then compressed and
subjected to an excitation operation to obtain the attention feature
map. Finally, the attention feature map and intrinsic feature map
are stacked to obtain the Gd feature map. The intrinsic feature map
contains more feature information, while the Ghost feature map
can reduce feature redundancy and decrease network parameters
and computation, achieving the goal of lightweight networks. Fig.3 shows the structure of GDBlock. reduce feature loss and maintain performance, the input feature
map is subjected to parallel operations - the first operation is a
regular convolution to generate the intrinsic feature map, while the
second operation is a low-cost process to generate the Ghost
feature map. The Ghost feature map is then compressed and
subjected to an excitation operation to obtain the attention feature
map. Finally, the attention feature map and intrinsic feature map
are stacked to obtain the Gd feature map. The intrinsic feature map
contains more feature information, while the Ghost feature map
can reduce feature redundancy and decrease network parameters
and computation, achieving the goal of lightweight networks. Fig.3 shows the structure of GDBlock. 2.3The network architecture and parameter quantity 2.3The network architecture and parameter quantity Using HRNet as the Backbone can provide higher resolution
feature representations, allowing the model to better capture local Using HRNet as the Backbone can provide higher resolution
feature representations, allowing the model to better capture local
illustrated in Fig.4. 2×
1×
4×
Output
Input
Image
preprocessing
Conv
feature
maps
GaBlock
GdBlock
up
samp
Fig.4 Structure flowchart of ADNet
The backbone network of ADNet first preprocesses the image
i
t
t
d d 3 3
l ti
t
d
th
l ti
f
3 Experimental Results and Analysis Input feature
maps Image
preprocessing Image
preprocessing GdBlock Conv Fig.4 Structure flowchart of ADNet Fig.4 Structure flowchart of ADNet
sses the image
3 Experimental Results and Analysis Fig.4 Structure flowchart of ADNet
esses the image
3 Experimental Results and Analysis Fig.4 Structure flowchart of ADNet
sses the image
3 Experimental Results and Analysis g
The backbone network of ADNet first preprocesses the image
using two standard 3x3 convolutions to reduce the resolution of
the image to 1/4 of the original, which serves as the first stage of
feature extraction. Then, feature extraction is performed using
GABlock and down-sampling is done using GDBlock. The
network uses parallel connections, greatly reducing the risk of
feature loss. GABlock and GDBlock use lightweight and attention
mechanisms to reduce computation while increasing accuracy. Stage 1 of the model includes StemNet and three Bottlenecks to
extract features and increase channels. Stage 2, Stage 3, and Stage
4 use GABlock to extract features, and GDBlock is used for
downsampling between the different stages to reduce parameters
and enhance model performance. The parameter, computational
complexity, and compression ratio of GABlock and GDBlock are
denoted by GP, GG, and Rs, respectively, as shown in Eq.5, Eq.6,
and Eq.7. 2.2GDBlock Due to the extensive downsampling present in HRNet, which
accounts for a significant portion of the parameter count, feature
loss can easily occur. To address this issue, the GDBlock was
proposed to reduce the parameters involved in downsampling
while maintaining accuracy. The GDBlock is able to obtain
channel relationships and original feature information with
minimal parameters, thereby reducing the feature loss incurred
during downsampling and improving performance without
sacrificing accuracy. The process of the GDBlock is as follows.To g
y
p
Conv
Input
Fsq(·)
Fscale(·)
Φ1
Φ2
Φk
Identity
Output
Fex(·W)
Fig.3 Structure flowchart of GDBlock Fex(·W) Output Fig.3 Structure flowchart of GDBlock represents the squeeze operation, Fex represents the excitation
operation. Through Fscale, the weights are allocated to the
channels, and then the channels with allocated weights undergo
linear operations to generate low-cost phantom channels. Finally,
the normal convolution and the features generated by attention and GDBlock utilizes the feature map output from the upper
layers as the intrinsic feature map. Since downsampling can easily
result in feature loss, using feature maps from upper layers can
reduce feature loss and enhance network performance. Here, input
represents the input feature map, Conv represents the normal
convolution operation, Φi represents the linear operation, Fsq phantom operations are concatenated to obtain the final Gd feature
map. details. The fusion of multi-scale features can improve the
robustness of the model, while keeping computational costs low. By using both upsampling and downsampling, the model can fully
extract and utilize information, enabling it to better capture the
shape and details of human pose, as well as important features such
as small objects and object boundaries. The structure of ADNet is
illustrated in Fig.4. 3.3Implementation details of experiment and training 3.3Implementation details of experiment and training The experimental environment in this study was configured
as follows: a Windows 10 64-bit system with a single GeForce Conv
Input
Φ1
Φ2
Φk
Identity
Fscale(·)
Fsq(·)
Fex(·W)
Output
G 3.1Dataset Introduction The specific implementation
method is shown in Eq. 8. RTX 3080 12G graphics card, and the Pytorch 1.13.0 deep
learning framework was utilized. During the training process on
the COCO dataset, the images were scaled and cropped to a fixed
size of 256×192. The AdamW optimizer was employed for
network training with an initial learning rate of 1e-3, which was
decayed to 1e-4 at the 170th epoch. The entire network was trained
for 260 epochs with a batch size of 48. To augment the dataset,
random flipping and rotation were applied during the training
process. 𝑂𝐾𝑆𝑝=
∑exp (−𝑑𝑝𝑖
2/2𝑠𝑝2𝜎𝑖2)𝛿(𝑣𝑝𝑖>0)
𝑖
∑𝛿(𝑣𝑝𝑖>0)
𝑖
(8) (8) In the above formula, p represents a person in GroundTruth,
pi represents the keypoints of the person with id p, dpi represents
the Euclidean distance between the detected keypoints of the
person with current id p and the annotated keypoints in the dataset, In the above formula, p represents a person in GroundTruth,
pi represents the keypoints of the person with id p, dpi represents
the Euclidean distance between the detected keypoints of the
person with current id p and the annotated keypoints in the dataset,
Sp represents the scale factor of person with id p, which is the
square root of the area of the pedestrian detection box, σi
represents the keypoint normalization factor of the keypoint with
id i, which is the standard deviation between the predicted
keypoint and the annotated keypoint in the dataset, and Vpi is the
visibility information of the keypoint. OKS ranges between 0 and
1, where OKS=0 means the predicted value does not match the
ground truth keypoint, and OKS=1 means a perfect prediction of
the ground truth keypoint. The closer the OKS is to 1, the higher
the matching degree. 3.1Dataset Introduction The COCO dataset is primarily used in computer vision
research. The COCO training set contains 118,287 images, the
validation set contains 5,000 images, and the test set contains
2,000 images. The dataset includes annotations for 17 keypoints,
as shown in Table 1, which are used for tasks such as human pose
estimation. Table 1 Annotation Information for COCO Keypoints Table 1 Annotation Information for COCO Keypoints
Body Part
Index
Body Part
Index
Nose
0
Left Wrist
9
Left Eye
1
Right Wrist
10
Right Eye
2
Left Hip
11
Left Ear
3
Right Hip
12
Right Ear
4
Left Knee
13
Left Shoulder
5
Right Knee
14
Right Shoulder
6
Left Ankle
15
Left Elbow
7
Right Ankle
16
Right Elbow
8
-
-
3.2Evaluation Metrics 𝐺𝑃=
𝐶𝑜𝑢𝑡
𝑠× 𝐶𝑖𝑛× 𝑘× 𝑘+ (𝑠−1) ×
𝐶𝑜𝑢𝑡
𝑠× 𝑑× 𝑑+ 2
(
𝐶𝑖𝑛
𝑠)2
𝑟
（5 ）
𝐺𝐺= 𝐻× 𝑊× (
𝐶𝑜𝑢𝑡
𝑠× 𝐶1 × 𝑘× 𝑘+ (𝑠−1) ×
𝐶𝑜𝑢𝑡
𝑠
× 𝐻× 𝑊×
𝑑× 𝑑+ 2
(
𝐶𝑖𝑛
𝑠)
2
𝑟
) (6）
𝑅𝑆=
𝐶𝑜𝑢𝑡×𝐻×𝑊×𝐶𝑖𝑛×𝑘×𝑘
𝐶𝑜𝑢𝑡
𝑠
×𝐻×𝑊×𝐶𝑖𝑛×𝑘×𝑘+(𝑠−1)×𝐶𝑜𝑢𝑡
𝑠
×𝐻×𝑊×𝑑×𝑑+
2(𝑐𝑖𝑛
𝑠)2
𝑟
=
𝐶𝑖𝑛×𝑘×𝑘
1
𝑠×𝐶𝑖𝑛×𝑘×𝑘+(𝑠−1)×1
𝑠×𝑑×𝑑≈
𝑆×𝐶𝑖𝑛
𝐶𝑖𝑛+𝑆−1 ≈𝑠 (7) 3.2Evaluation Metrics (7) In this study, experiments were conducted on the COCO
dataset and validated on the COCO validation set. The evaluation
metric used is Object Keypoint Similarity (OKS), where AP50
represents the accuracy of detecting keypoints with OKS=0.5,
AP75 represents the accuracy with OKS=0.75, mAP represents the
average accuracy of predicting keypoints at ten threshold values of
OKS between 0.50 and 0.95 with a step size of 0.05, APM
represents the detection accuracy of keypoints for medium-sized
objects, APL represents the detection accuracy of keypoints for
large-sized objects, and AR represents the average recall of Among these, Cin represents the number of channels in the
input feature map, Cout represents the number of channels in the
output feature map, H and W denote the height and width of the
feature map, s represents the number of squeeze channels, k is the
size of the conventional convolution kernel, d is the size of the
depth-wise convolution kernel, and r is the channel compression
ratio, which is typically set to 16. The parameter increment and
computational complexity in the SEBlock are negligible relative
to the overall network. predicting keypoints at ten threshold values of OKS between 0.50
and 0.95 with a step size of 0.05. 3.4 Experimental results and analysis The experimental results on the COCO dataset for pose
estimation analysis are shown below in the table. It can be
observed that ADNet, the proposed model in this paper, exhibits
certain advantages compared to other existing pose estimation
models. When the baseline model is HRNet32, using the ADNet
framework leads to a small decrease in accuracy but a significant
decrease in parameter count by 224% and floating-point
operations by 125%, while achieving a 2% increase in AP50. Compared to CNP and SimpleBaseline, ADNet demonstrates
improved accuracy while substantially reducing parameter count
and computational complexity. The performance comparison on
the COCO validation set is presented in Table 2. 3.5Ablation experiment 3.5Ablation experiment In order to evaluate the effectiveness of GABlock and
GDBlock in incorporating attention mechanisms in human pose
estimation models, a series of ablation experiments were
conducted in this section. Output Identity The input image size was 256×192, AdamW was used as the
optimizer for network training with an initial learning rate of 1e-3,
which was decayed to 1e-4 at 170 epochs. The network was trained
for a total of 260 epochs with a batch size of 48, and random
flipping and rotation were used for data augmentation during
training. Fig.5 GhostBlock integrates SE attention mechanism The present study conducted ablation experiments on the
COCO dataset to validate the effectiveness of GABlock and
GDBlock in network models by performing separate experiments
using GhostBlock and GhostBlock with the attention mechanism
SEBlock replacing the BasicBlock. The experimental results, as
shown in Table 3, demonstrate the efficacy of GABlock and
GDBlock. GhostBlock was used to replace BasicBlock in HRNet, and
SEBlock was inserted afterwards to reduce the number of network
parameters while improving performance. The module model can
be seen in Fig.5. g
Table 2 Performance comparison of COCO validation set
Table 3 Results of ablation experiments with different models
Methos
Backbone
Inputsize
#Params/107
GFLOPs
MAP
AP50
AP75
APM
APL
AR
Hrnetw32
HRNet
256×192
28.5M
7.69G
74.4
90.5
81.9
70.8
80.1
78.9
Hrnetw48
HRNet
256×192
63.6M
14.6G
75.1
90.8
82.9
72.3
83.4
81.2
Hrnetw32
Ghost Block
256×192
20.8M
5.84G
64.8
89.4
72.6
62.0
69.1
68.4
Hrnetw32
GhostBlock+SEBlock
256×192
20.9M
5.84G
66.7
90.0
74.0
64.0
70.6
70.0
Methos
Input size
#Params/107
GFLOPs
mAP
AP50
AP75
APM
APL
AR
Hourglass
256×192
25.1
14.3
66.9
-
-
-
-
-
CPN
256×192
27.0
6.2
68.6
-
-
-
-
-
SimpleBaseline
256×192
34.0
8.9
70.4
88.6
78.3
67.1
77.2
76.3
HRNetw32
256×192
28.5
7.1
74.4
90.5
81.9
70.8
80.1
79.8
HRNetw48
256×192
63.6
14.6
75.1
90.6
82.2
71.5
81.8
80.4
ADNetw32（our）
256×192
8.8
3.16
72.1
92.5
79.4
69.0
76.3
76.4
ADNetw48（our）
256×192
19.2
5.54
74.0
92.6
81.6
71.2
78.2
76.9 5.54G
74.1
92.6
81.6
71.2
78.2
76.9
(a) Single person unshielded (b) Multi-person unshielded
(c) Single person shielded (d) Multi-person shielded
Fig.7 Prediction of key points of human body
We have compared the processing of fine-grained
information between HRNet and ADNet. Compared to HRNet,
ADNet is able to extract more information, as shown in Fig.11. 4. Visualization research and analysis 4. Visualization research and analysis 4. Visualization research and analysis
This paper analyzes the feature map visualization and compares
the feature maps in the same location in ADNet and HRNet
networks. See Fig.6 for the feature map visualization and
comparison of the feature maps in the same location in ADNet and
HRNet . (c) Single person shielded (d) Multi-person shielded
Fig.7 Prediction of key points of human body
We have compared the processing of fine-grained
information between HRNet and ADNet. Compared to HRNet,
ADNet is able to extract more information, as shown in Fig.11. （a）HRNet (b)GhostBlock+SEBlock Fig.8 Visual comparison between HRNet and ADNet
5.Conclusion
H
ti
ti
i
f
d
t l t
k i
th fi ld
f 3.5Ablation experiment (b) Multi-person unshielded From the experimental results, it can be observed that the use
of GhostBlock led to a significant decrease in network accuracy
despite a reduction in model parameters. However, the
incorporation of SEBlock attention mechanism into GhostBlock
resulted in a 2% increase in accuracy, indicating that attention
mechanisms can greatly enhance the network's feature extraction
capabilities. Following the use of ADNet, traditional convolutions
in downsampling were replaced with lightweight convolutions,
resulting in a significant improvement in model performance and
a lighter network. Compared to GhostNet, the use of ADNet
reduced parameter count by 136% and computational complexity
by 84%, while also increasing mAP by 7.3%. These experiments
demonstrate that Ghost modules and attention mechanisms can
ensure a certain level of accuracy while maintaining a lightweight
design, highlighting the importance of initial features in model
performance. Furthermore, ADNet outperformed GhostNet in
terms of both lightweight design and accuracy. (b) Multi-person unshielded (d) Multi-person shielded
ts of human body
processing of fine-grained
d ADNet. Compared to HRNet,
ormation as shown in Fig 11 reference： [18] Li X, Wang W, Hu X, Yang, J(2019)Selective Kernel
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Acknowledgements [16] Hu J, Shen L, Albanie S, Sun G, Wu E(2017)Squeeze-and-
Excitation Networks. IEEE Transactions on Pattern Analysis
and Machine Intelligence, 42:2011–2023. This work was supported in part by the following projects： NaturalScience Foundation of Zhejiang Province
(LO22E050012) NaturalScience Foundation of Zhejiang Province [17] Woo S, Park J, Lee J, Kweon I(2018)CBAM: Convolutional
Block Attention Module. arXiv preprint arXiv:1807.06521. (c)ADNet
Fig.6 Feature map visualization (c)ADNet
Fig.6 Feature map visualization ( )
Fig.6 Feature map visualization In the illustration above, there exist a significant amount of
feature redundancy in both the HRNet feature maps and the
GhostBlock with SE attention mechanism, particularly between
adjacent feature maps. With the use of ADNet, however, feature
redundancy is greatly reduced, as the attention mechanism in
ADNet assigns channel weights to each feature map, resulting in
visually evident darkening of feature maps with lower weights,
while highlighting key points and enhancing the network's feature
extraction capabilities. This study conducted experiments on the
COCO 2017 validation dataset, with results for non-occluded
single and multiple person detection, as well as occluded single
and multiple person detection, shown in Fig.7. Fig.8 Visual comparison between HRNet and ADNet
5.Conclusion Human pose estimation is a fundamental task in the field of
artificial intelligence, as it serves as the foundation for future AI
applications aimed at serving and understanding humans. Lightweight network models represent the most cost-effective
solution for deploying algorithms on low-performance hardware
while reducing power consumption. This study proposes a
lightweight human pose estimation network, ADNet, based on
HRNet, with the aim of reducing network parameterization and
computational complexity. ADNet achieves a high prediction
accuracy
with
low
parameterization
and
computational
complexity, meeting the objective of model lightweighting. Experiments conducted on the COCO dataset demonstrate that IEEE/CVF Conference on Computer Vision and Pattern
Recognition (CVPR), pp5686–5696. IEEE/CVF Conference on Computer Vision and Pattern
Recognition (CVPR), pp5686–5696. ADNet outperforms networks based on GhostModel and
GhostModel+SEModel, and achieves better feature redundancy
handling compared to GhostNet. In light of the convenience of
mobile devices, future research will focus on model migration to
mobile devices, with the goal of achieving real-time monitoring on
mobile platforms while improving model performance. [13] Toshev A, Szegedy C (2013)DeepPose: Human Pose
Estimation via Deep Neural Networks. 2014 IEEE
Conference on Computer Vision and Pattern
Recognition(CVPR), pp1653–1660. Recognition(CVPR), pp1653–1660. [14] Newell A, Yang K, Deng J(2016)Stacked Hourglass
Networks for Human Pose Estimation. arXiv preprint
arXiv:1603.06937. Declaration of Competing Interest We declare that we have no financial and personal
relationships with other people or organizations that can
inappropriately influence our work, there is no professional or
other personal interest of any nature or kind in any product, service
and /or company that could be construed as influencing the
position presented in, or the review of,the manuscript entitled. [15] Jaderberg M, Simonyan K, Zisserman A, Kavukcuoglu
K(2015)Spatial Transformer Networks. Conference and
Workshop on Neural Information Processing Systems
(NIPS). pp665–673. reference： [11] Xiao B, Wu H, Wei Y(2018)Simple Baselines for Human
Pose Estimation and Tracking. In proceedings of the
european conference on computer vision (ECCV).pp466–
481. [12] Sun K, Xiao B, Liu D, Wang J(2019)Deep High-Resolution
Representation Learning for Human Pose Estimation. 2019
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https://link.springer.com/content/pdf/10.1007/s44196-021-00022-z.pdf
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English
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Evolutionary Social Poisson Factorizationfor Temporal Recommendation
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The International journal of computational intelligence systems/International journal of computational intelligence systems
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cc-by
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(2021) 14:185 (2021) 14:185 International Journal of Computational Intelligence Systems (2021) 14:185
https://doi.org/10.1007/s44196-021-00022-z RESEARCH ARTICLE Abstract Preference-based recommendation systems analyze user-item interactions to reveal latent factors that explain our latent
preferences for items and form personalized recommendations based on the behavior of others with similar tastes. Most
of the works in the recommendation systems literature have been developed under the assumption that user preference is a
static pattern, although user preferences and item attributes may be changed through time. To achieve this goal, we develop
an Evolutionary Social Poisson Factorization (EPF_Social) model, a new Bayesian factorization model that can effectively
model the smoothly drifting latent factors using Conjugate Gamma–Markov chains. Otherwise, EPF_Social can obtain the
impact of friends on social network for user’ latent preferences. We studied our models with two large real-world datasets,
and demonstrated that our model gives better predictive performance than state-of-the-art static factorization models. Keywords Poisson factorization · Recommendation system · Gamma–Markov chains Evolutionary Social Poisson Factorizationfor Temporal
Recommendation ChunYan Yin1 · YongHeng Chen2 · Wanli Zuo3 Received: 29 May 2020 / Accepted: 9 September 2021
© The Author(s) 2021 1 Introduction with a Poisson likelihood and non-negative representations. So we can also view PF as a type of Bayesian non-negative
matrix factorization. A Bayesian treatment of the Poisson
model, with Gamma conjugate priors on the latent factors, laid
the foundation for the more recent hierarchical Poisson fac-
torization. Poisson factorization demonstrates more efficient
inference and better recommendations than both traditional
matrix factorization and its variants that adjust for sparse data. The conjugate gamma Poisson structure of Poisson Factoriza-
tion model ensures computationally tractable approaches for
high-dimensional data using variational inference. However,
traditional recommendation systems assume that all the users
are independent and identically distributed. This assumption
ignores the social interactions or connections among users [3]. Through incorporating social network information into the
traditional factorization model, Social Poisson Factorization
(SPF) enriched the preference-based recommendations [4]. But the time evolving of users’ action is not considered in SPF
model, the rate of adopting an item by a user will be increased
by any time after her friend adopted this item. Otherwise, most
widely-used PF models for collaborative filtering assume that
user preferences and item attributes are static over time, while
users’ continuously time-varying preferences remain largely
under explored [5, 6]. However, in many real-world scenarios,
items’ popularity and reception continuously evolve as new
items and categories emerging, and users’ preferences and The web provides amazing access to products and knowledge,
but these choices increasingly overwhelm its consumers. Recommendation systems can reduce this problem, broadly
speaking, which use his or her historical data of purchased or
ratings to infer uses’ preferences, and then use the inferred
preferences to predict which items a user tends to like [25, 26]. One of the most important classes of recommendation meth-
ods is collaborative filtering. * YongHeng Chen
cyh771@163.com
ChunYan Yin
chuny_yin@163.com
Wanli Zuo
wanli@jlu.edu.cn
1
Business School, Lingnan Normal University,
Zhanjiang 524048, Guangzhou, China
2
School of Information Engineering, Lingnan Normal
University, Zhanjiang 524048, Guangzhou, China
3
College of Computer Science and Technology, Jilin
University, Changchun 130012, Jilin, China 2.1 Conjugate Gamma Markov Chain In order to implement the evolutionary Poisson Factoriza-
tion, we preserved the gamma-Poisson conjugate structure
by placing GMC (Gamma Markov Chain) as prior on each
latent factor. The conjugacy makes it possible to design fast
inference algorithms for the factors in a flexible and closed
form. while the finite items that users consume, most of
items that a small number of tail users consume [16, 24] So
we place long tail gamma priors on the latent attributes and
latent preferences, which controls the average size of the rep-
resentations of the users and items. In addition, it enables us
to capture the diversity of users, some users tend to consume
more than others, and the diversity of items, some users are
more popular than others. A straightforward model of GMC as shown in Fig. 1
would generate the ith component of the user’s latent factor
at current time slice t by considering previous time slice
t − 1 as follows: y
p
In this paper, we focus firstly on addressing the problem of
evolving user and item factors within the context of Poisson
factorization. Evolutionary Social Poisson Factorization (EPF_
Social) model, which models the time evolving latent user and
item factors by incorporating multiplicative Gamma–Markov
chains, is proposed. EPF_Social has the capacity to eliminate
the defects of previously mentioned Gaussian state-space
model and assumes the actions of user are either triggered by
her intrinsic interests or by previous pleasant experiences of
her friends, which can jointly model the dynamic interests of
users and popularity of items over time and time-aware peer
influence among users in social network. In summary, we have
the following main contributions: (1)
휃t
n,i|휃t−1
n,i ∼Gamma(휃a, 휃b휃t−1
n,i )] (1) where 휃t
n,i is the ith component of the user n’s latent factor at
time t, and Gamma distribution has a fixed shape parameter
휃a and a rate parameter 휃b휃t−1
n,i .i ,
This way defines a discrete state Markov chain where
each state encodes a certain regime. However, in this case
the full conditional distribution p
(
휃t
n,i
|||휃t−1
n,i , 휃t+1
n,i
)
turns out
to be non-conjugate since it has 휃i , 1∕휃i and log 휃i , making
inference harder. 1 Introduction In collaborative filtering, Pois-
son factorization (PF) [1] is a recent variant of probabilistic
matrix factorization [2] for recommendation, which replaces
the usual Gaussian likelihood and real-valued representations * YongHeng Chen
cyh771@163.com
ChunYan Yin
chuny_yin@163.com
Wanli Zuo
wanli@jlu.edu.cn
1
Business School, Lingnan Normal University,
Zhanjiang 524048, Guangzhou, China
2
School of Information Engineering, Lingnan Normal
University, Zhanjiang 524048, Guangzhou, China
3
College of Computer Science and Technology, Jilin
University, Changchun 130012, Jilin, China * YongHeng Chen
cyh771@163.com
ChunYan Yin
chuny_yin@163.com
Wanli Zuo
wanli@jlu.edu.cn 1
Business School, Lingnan Normal University,
Zhanjiang 524048, Guangzhou, China 2
School of Information Engineering, Lingnan Normal
University, Zhanjiang 524048, Guangzhou, China 3
College of Computer Science and Technology, Jilin
University, Changchun 130012, Jilin, China (0121 3456789)
3 3456789)
3 International Journal of Computational Intelligence Systems (2021) 14:185 Page 2 of 10 185 needs drift over time, leading them to interact with items in a
time-varying way [7, 8, 27]. We denote the behaviors in these
scenarios as dynamic selection. Giving the dynamic selection
data, a significant and attractive problem appears: using the
historical behavior about what a user clicks as selecting items,
how can the items that will be selected by the potential user in
the next time period be predicted? (i.e., slowly and smoothly evolving in time), which has
the capacity to eliminate the defects of Gaussian state-
space model and allows us to leverage the long-tailed
gamma prior’s better fit to user and item factors in sparse
matrices. In addition this conjugate chain construction
guarantees a straightforward scalable inference. (i.e., slowly and smoothly evolving in time), which has
the capacity to eliminate the defects of Gaussian state-
space model and allows us to leverage the long-tailed
gamma prior’s better fit to user and item factors in sparse
matrices. In addition this conjugate chain construction
guarantees a straightforward scalable inference. 2. 2. We develop an Evolutionary Social Poisson Factoriza-
tion (EPF_Social) model, which can incorporate the peer
influence and the network of users and obtain the impact
of friends on social network for user’s latent preferences. 2. We develop an Evolutionary Social Poisson Factoriza-
tion (EPF_Social) model, which can incorporate the peer
influence and the network of users and obtain the impact
of friends on social network for user’s latent preferences. There is a wealth of research to track the aforementioned
problem in the context of collaborative filtering frameworks. 1 Introduction The first attempt to model time-evolving preference patterns
in the context of CF systems can be traced back to the time
SVD++ model [9, 10]. This model can effectively capture local
changes of user preference, but ignore the drifting nature of
item attributes. Similarly, based on imposition of a dynamic
hierarchical Dirichlet process [11], dynamic Bayesian probabil-
istic matrix factorization (dBPMF) is proposed, which focuses
mainly on modeling dynamic memberships of users [12]. Nevertheless, the temporal dynamics of latent factors are not
modeled across time intervals in dBPMF. In addition to only
considering the local effects captured by user group latent fac-
tors, item attractiveness is assumed to be slowly changed and
item latent factors are thus assumed to be static and capture
global effects [13, 21]. Dynamic Poisson Factorization (DPF)
captures the time evolving latent factors with a Kalman filter,
while modeling the temporal structure using Gaussian tech-
niques under a Poisson likelihood [14]. However, DPF assumes
a simple Gaussian state-space model, which lacks the expres-
sive transition structure of the linear dynamical system [15, 22]. In practice, DPF replaces the gamma priors with a Gaussian
state space model. However, this model damages the conjuga-
tion of the model and leads to the intractability of computation,
or in the accurate numerical approximation of posterior infer-
ence. In fact, the lack of conjugacy prevents simple closed-form
updates and consequently prevents convenient extensions to the
model. Furthermore, Gaussian priors at each time step fail to
capture the empirical response distribution and the long-tailed
Gamma distributions, as demonstrated by Gopalan et al. [1].i 3. We conduct several experiments on two real datasets to
demonstrate the performance of our model, where EPF_
Social model achieves a higher predictive accuracy than
state-of-the-art static and dynamic factorization models. 3. We conduct several experiments on two real datasets to
demonstrate the performance of our model, where EPF_
Social model achieves a higher predictive accuracy than
state-of-the-art static and dynamic factorization models. 2.1 Conjugate Gamma Markov Chain Dividing 휈 by five means that we can
interpret it as the number of time intervals after which the
user will have little influence for other users’ action. For
example, in Fig. 2, users intrinsic interest contribute partly
the adoption of items. The interests of user and attributions
of item can be evolved by our conjugate gamma markov
chain. Otherwise, the adoption of item1 by user2 and user3
cause also adoption of item1 by user1. where N(u) is the set of indices of other users connected to
u, 휆u,v represents the per-neighbor non-negative latent influ-
ence of user v on user u and rt′
v,i is the number of times user
v clicked on item i at time t′ . The trust factor 휆u,휐 is equal
to zero for all users that are not connected in the social net-
work. In order to ensure the effect of interaction decreas-
ing over time, we uses exponential decay function f(t, t ) to
realize this purpose. Dividing 휈 by five means that we can
interpret it as the number of time intervals after which the
user will have little influence for other users’ action. For
example, in Fig. 2, users intrinsic interest contribute partly
the adoption of items. The interests of user and attributions
of item can be evolved by our conjugate gamma markov
chain. Otherwise, the adoption of item1 by user2 and user3
cause also adoption of item1 by user1. Fig. 1 The dynamic of uses’ preferences and items’ attributes with
Gamma Markov Chain (2)
p1
n,k ∼Gamma(훿P, bP
u)
휃1
u,k|p1
u,k ∼Gamma(휂P, 휌Pp1
uk)
pt
u,k|휃t−1
u,k ∼Gamma(훿P, oP휃t−1
u,k ) (2) where p is latent auxiliary variables, the parameter bP
u , used
for the initialization of each chain when t = 1, is separated
from the transition or chaining parameters 휌P and oP. Through inserting auxiliary variables into GMC, we
exploit the transition kernel of Markov Chain. A notable
feature here is that we consider separately the parameter bp
휇
to initialize the GMC at first time slice, which enables us to
capture the static property of latent factor. In order to fol-
low the track of latent factor along with time, we model the
temporal corrections by decoupling these parameters. The
strength of the correlation is managed by the absolute value
of 휂 and 훿 , and ratio 휂휌∕훿o manages the skewness. 3 Model Description In this section we will describe Social Evolutionary Poisson
Factorization model for recommendation. Data about users,
items and neighbors of users are given, where each user u as a
vector 휃 of K latent preferences and each item i as a vector 훽 of
K latent attributes. We introduce a set of auxiliary latent varia-
bles p and l to preserve the positive correlation for the chains of
휃 and 훽 while retaining conjugacy, respectively. We use bp
u and
bl
i to parameterize the initial states of the user and item chains. 2.1 Conjugate Gamma Markov Chain When
ratio < 1 ( ratio > 1 ), the probability mass is shifted towards
the interval 𝜃t < 𝜃t−1 (𝜃t > 𝜃t−1) and the chain exhibit a sys-
tematic negative (positive drift). 2.1 Conjugate Gamma Markov Chain Therefore, the basic idea is to introduce
latent auxiliary variables Pt
n,i between 휃t
n,i and 휃t−1
n,i , which
restore positive correlation between 휃t−1
n,i , 휃t+1
n,i while preserv-
ing conjugacy. This allows straightforward implementation
of a Gibbs sampler or a variational algorithm. Our Conjugate
Gamma Markov Chain is defined by: 1. We propose a conjugate Gamma–Markov chain con-
struction with dampened, positively correlated states 1. We propose a conjugate Gamma–Markov chain con-
struction with dampened, positively correlated states 1 3 International Journal of Computational Intelligence Systems (2021) 14:185 Page 3 of 10
185 185 Fig. 1 The dynamic of uses’ preferences and items’ attributes with
Gamma Markov Chain (or neutral) influence on the user. But we should keep in mind
that it is true in real-world scenarios as the impact degree
of users’ actions on their friends usually decays over time. We model the effect of previous user-item interactions on the
tendency of user u to adopt item i at time t as: (or neutral) influence on the user. But we should keep in mind
that it is true in real-world scenarios as the impact degree
of users’ actions on their friends usually decays over time. f (or neutral) influence on the user. But we should keep in mind
that it is true in real-world scenarios as the impact degree
of users’ actions on their friends usually decays over time. f We model the effect of previous user-item interactions on the
tendency of user u to adopt item i at time t as: (3)
휒(t, u, i) =
∑
v∈N(u,i)
T
∑
t=1
휆u,vrt
v,if(t, t)
f(t, t) =
{ 0
otherwise
exp
(
−(t−t)
휈∕5
)
t ≤t ≤t + 휈 (3) where N(u) is the set of indices of other users connected to
u, 휆u,v represents the per-neighbor non-negative latent influ-
ence of user v on user u and rt′
v,i is the number of times user
v clicked on item i at time t′ . The trust factor 휆u,휐 is equal
to zero for all users that are not connected in the social net-
work. In order to ensure the effect of interaction decreas-
ing over time, we uses exponential decay function f(t, t ) to
realize this purpose. 2.2 Time‑Evolving Impact of Social Network i
Instead of introducing a new variable solely to rescale
the nonnegative multiplicative gamma chains, independently
from the generating distribution, we exploits compound
Poisson to draw the variables from exponential dispersion
models (EDM) that we can leverage for that purpose, which
also provides more flexibility with ratings data types [17]. A
random variable that is the sum of 휔 independent and iden-
tically distributed Gamma random variables is compound
Poisson distributed with an element random distribution
p휓(휀,휅) with fixed scale parameter 휀 and natural parameter 휅
[18]. Here, the independent and identically distributed vari-
ables are a sequence of additive EDM distributions and 휔 is
a Poisson-distributed random variable of mean Λ . By virtue
of the additivity property of EDM distributions, a compound Many applications of recommendation contain an additional
source of information: a social network. In order to jointly
model the dynamic interests of users and popularity of items
over time and peer influence among users in social network,
we further consider to model latent Gamma factor for social
trust in the user social network, combining all factors in the
rate of a Poisson likelihood of the user-item interaction, which
assumes the actions of user are either triggered by intrinsic
intensity or her friends in social network to engage with some
item. The intuition behind the effect of social network is that
users tend to interact with items presented by their peers,
where items offered through the social network have a positive 1 3 3 International Journal of Computational Intelligence Systems (2021) 14:185 Page 4 of 10 185 Fig. 2 The left represents the
actions are triggered by user
intrinsic interest on different
items. The social relations are
also depicted in the right top. Latent triggers are shown by
arrows between users in the
right bottom Fig. 2 The left represents the
actions are triggered by user
intrinsic interest on different
items. The social relations are
also depicted in the right top. Latent triggers are shown by
arrows between users in the
right bottom Poisson random variable has a distribution p휓(휀,휔휅) , which
is also an EDM distribution with scale parameter 휔휅 and
natural parameter 휀 [2]. Our model rescales of the contribution of the latent fac-
tors towards the generating distributions to ensure the chains
don’t grow uncontrollably in time and thus lead to numeri-
cal stability during the inference routine. 2.2 Time‑Evolving Impact of Social Network Figure 3 presents
the graphical model of our Evolutionary Social Poisson
Factorization ( EPF_Social ) model, and the full generative
process of the Evolutionary Social Poisson Factorization
( EPF_Social ) is detailed in Algorithm 1. This generative
process describes the statistical assumptions behind the
model. 4 Stochastic Variational Inference The key computation for Poisson factorization is to infer the
posterior distribution of the user preferences 휃t
uk , item attrib-
utes 훽t
uk and user trust, given the social network and a set of
observed matrix of user ratings for items across time. As for
many Bayesian models of interest, however, the posterior is
not tractable to compute exactly. We therefore introduce how
to efficiently approximate the posterior based on mean-field
variational inference [23]. Variational inference is an optimization-based strategy
for approximating posterior distributions in complex proba-
bilistic models. Variational inference algorithm introduces a
family of distributions over the target latent variable that is
indexed by a set of variational parameters, and then optimiz-
ing those parameters to find the member of this family that
is close to the posterior in terms of Kullback–Liebler (KL)
divergence. This inference problem thus becomes an optimi-
zation problem that can be scaled to massive data sets using
stochastic optimization, leading to stochastic variational
inference. By combining stochastic gradient algorithms and
variational inference, stochastic variational inference (SVI) 1 3 Page 5 of 10
185 International Journal of Computational Intelligence Systems (2021) 14:185 185 Fig. 3 Graphical model of
social evolutionary poisson
factorization Fig. 3 Graphical model of
social evolutionary poisson
factorization Fig. 3 Graphical model of
social evolutionary poisson
factorization Fig. 3 Graphical model of
social evolutionary poisson
factorization (4)
q
(
휃t
u,k|휇휃
u,k,t
)
q
(
pt
u,k|휇p
u,k,t
)
q(bp
u|휇bp
u
)
q
(
훽t
i,k|휇훽
i,k,t
)
q
(
lt
i,k|휇l
i,k,t
)
q(bl
i|휇bl
u
)
q
(
휆t
u,v|휇휆
u,v,t
)
q
(
st
u,i|휑u,i,t
)
q
(
휔t
u,i
) (4)
q
(
휃t
u,k|휇휃
u,k,t
)
q
(
pt
u,k|휇p
u,k,t
)
q(bp
u|휇bp
u
)
q
(
훽t
i,k|휇훽
i,k,t
)
q
(
lt
i,k|휇l
i,k,t
)
q(bl
i|휇bl
u
)
q
(
휆t
u,v|휇휆
u,v,t
)
q
(
st
u,i|휑u,i,t
)
q
(
휔t
u,i
) provides a new framework for approximating model poste-
riors with only a small number of passes through the data,
enabling such models to be fit at scale. (4) i
So the latent variables in the model are user preferences
휃 , item attributes 훽 , their corresponding auxiliary chain
variables p and l, the rate parameters of the initial states of
each chain bp and bl , user influences 휆 . 5.1 Datasets We evaluate the performance of EPF_Social on two real
data sets: Netflix-time Dataset consists of users’ ratings for movies
along with the timestamp of the rating and spans 86 months. We follow a similar procedure as described in Li et al. [19]
by filtering users and movies who are active between the
first and last time periods(10/01/98 to 12/31/05) and obtain a
subset of the Netflix data set. The resulting data set contains
7400 users, 3500 movies, and 2 million non-zero ratings. Last.fm Dataset is used to make personalized radio and
music recommendations for their online radio station. This
data set also contains time stamped records of users’ music
listening activity with music streaming logs between 1200
users and 3000 artists, which makes it appropriate bench-
mark for comparing the proposed method to the other state-
of-the-art methods. The SVI algorithm for the general EPF_social framework 4 Stochastic Variational Inference For convenience,
we introduce the auxiliary latent Poisson variables s. Recall
that each su,i is a K-vector of Poisson counts that sum to the
observation 휔u,i . In mean-field variational inference, each
latent variable is assumed to be independent from each other
and is governed by its own free variational distribution,
which simplifies the optimization procedure. The mean-
field variational family for EPF_social is defined as follows: where the variational factors for 휃 , 훽 , p, l, bp , bl , and 휆 follow
all Gamma distribution, with freely set shape and rate vari-
ational parameters. We denote shape with the superscript s
and rate with the superscript r . For example, the variational
distribution for user preferences 휃 is Gamma(휇휃;휇휃,s, 휇휃,r) . The variational distribution of s is a multinomial distribution. where the variational factors for 휃 , 훽 , p, l, bp , bl , and 휆 follow
all Gamma distribution, with freely set shape and rate vari-
ational parameters. We denote shape with the superscript s
and rate with the superscript r . For example, the variational
distribution for user preferences 휃 is Gamma(휇휃;휇휃,s, 휇휃,r) . The variational distribution of s is a multinomial distribution. 1 3 1 3 185
Page 6 of 10 International Journal of Computational Intelligence Systems (2021) 14:185 Page 6 of 10 185 is summarized in Algorithm 2. This algorithm is very effi-
cient on sparse matrices. The variational parameter 휑 , sums
over users and items only need to update over the non-zero
observations 휔 . Otherwise, the update for each user or item
parameter is independent of all the other users or items,
respectively. Accordingly, this inference routine is eas-
ily implemented in parallel. Note the learning rate power
휉∈(0.5, 1] controls how quickly old information is forgot-
ten, and the leaning rate delay 휏≥0 down-weights early
iterations. We terminate the algorithm when the variational
distribution converges. Convergence is measured by comput-
ing the prediction accuracy on a validation set. In this paper,
we declare convergence when the change in predictive likeli-
hood is less than 0.0001%. 5.2 Baselines We compare the performance of our proposed model to three
state-of-the-art models in recommendation listed as follows: DPF Dynamic Poisson factorization is a new dynamic
matrix factorization probabilistic model that based on the
Poisson factorization model. The aim is to capture the evolv-
ing preferences of users as well as the evolution of item
popularity over time [8]. 5.3 Compare EPF_Social to DPF For each user the NDCG metric is discounted cumula-
tive gain For each user the NDCG metric is discounted cumula-
tive gain (6)
DCG@k =
k∑
i=1
2reli−1
log2(i+1), (6) divided by the ideal DCG value, or (7)
NDCG@k = DCG@k
IDCG@k, (7) where IDCG@k is a normalization factor that ensures
NDCG lies between zero and perfect ranking and reli is the
ground-truth relevance of item i for user u. The Precision@k and NDCG@k results are summa-
rized in Table 1 for each method and data sets. As seen in
Table 1, EPF_Social performs marginally better than DPF
in both data sets based on all metrics. We believe this is
mostly due to the fact that DPF adopts a simple Gaussian
state-space model to replace the gamma priors, which lacks
the expressive transition structure of the linear dynamical
system. Explicitly, Gaussian priors at each time step fail to
capture the empirical response distribution and the long-
tailed Gamma distributions. However our conjugate gamma
markov chains keep the gamma-Poisson structure to enable
us to effect the long-tailed gamma prior’s better fit to user
and item factors in sparse matrices. SPF Social Poisson factorization incorporates social net-
work information into a traditional factorization method, by
adding a degree of trust variable and making all user-item
interaction conditionally dependent on the user friends [4]. TSR Time-sensitive recommendation is a convex formu-
lation and an efficient learning algorithm to recommend rel-
evant services, which connects self-exciting point processes TSR Time-sensitive recommendation is a convex formu-
lation and an efficient learning algorithm to recommend rel-
evant services, which connects self-exciting point processes The SVI algorithm for the general EPF_social framework The SVI algorithm for the general EPF_social framework 1 3 3 International Journal of Computational Intelligence Systems (2021) 14:185 Page 7 of 10
185 185 and low-rank models to predict the next returning-time of
users to existing services in the collection of user-item con-
sumption pairs [20]. 5.4 Compare EPF_Social to SPF and TSR In this subsection, our quantitative goal is to predict which
test items each user will interact with. We compare the
Recall@k and NDCG@k versus our EPF_Social and SPF. Recall@k is a standard information retrieval measure, which
consider user recall given k top-items to recommend. For
user u RecallL@ is equivalent to computing 5.3 Compare EPF_Social to DPF Recall@k is a standard information retrieval measure, which
consider user recall given k top-items to recommend. For
user u RecallL@ is equivalent to computing To show the performance of our proposed model in captur-
ing the dynamic preferences, we compare the MAP@k and
NDCG@k versus our proposed model and DPF. Mean Aver-
age Precision at top k (MAP@k) is a metric often used in
information retrieval and recommendation system. MAP@k
calculates the mean of users’ average precision. MAP@k for
user u is equivalent to computing (8)
Recall@k = ∑
i∈ytest
u
I(rank(u,i)≤k)
min(k, ytest
u ) (8) Figure 4a, b show the comparison results of Recall@20
and NDCG@20 for EPF_social , SPF and TSR models
over Netflix-time , and Fig. 4e, f over Last.fm, respectively. As it can be seen, TSR model performs margin better than
SPF because it continue improves the performance by tak-
ing information evaporation into consideration, which is
a more proper way to treat time. Overall, our EPF_social
exhibits particularly obvious improvements over SPF and
TSR model. We believe this is mostly due to the fact that
TSR pays more attention to the impact of previous events
of the user, and SPF is not able to consider decay degree of
users’ actions on their friends over time. These two models
pay less or no attention to capture the evolving preferences
of users as well as the evolution of item popularity over
time. Figure 4c, d show the performance of the NDCG@k
and Recall@k with varying k over Netflix −time , Fig. 4g, h
over Last.fm, respectively. All models show an early increase
in NDCG@k and Recall@k with increase of k. Moreover,
EPF_social model consistently being the best one. The
results demonstrate further evidence for the importance of
modeling jointly the dynamic interests of users and popu-
larity of items over time and peer influence among users in
social network. (5)
MAP@k =
k∑
i=1
precision@i
min(i, ytest
u ), (5) where ytest
u is the set of items in the heldout test set for user u. where ytest
u is the set of items in the heldout test set for user u. For each user the NDCG metric is discounted cumula-
tive gain where ytest
u is the set of items in the heldout test set for user u. 5.5 Returning Time Prediction we evaluated the performance of different methods in pre-
dicting the time when a user will return to the system by
computing the mean absolute error (MAE) and root mean
squared error (RMSE) between the estimated time and the
ground-truth time. Otherwise, Suppose the testing event 1 3
Table 1 Performance
comparison of EPF_Social and
DPF on MAP and NDCG
Dataset
Model
MAP@10
MAP@100
NDCG@10
NDCG@100
EPF_social
0.051
0.029
0.063
0.097
Netflix-time
DPF
0.034
0.020
0.047
0.080
EPF_social
0.191
0.176
0.0296
0.251
Last.fm
DPF
0.073
0.068
0.092
0.085 Table 1 Performance
comparison of EPF_Social and
DPF on MAP and NDCG 1 International Journal of Computational Intelligence Systems (2021) 14:185 185
Page 8 of 10 Page 8 of 10 185
Page 185 International Journal of Computational Intelligence Systems (2021) 14:185
3
Page 8 of 10
0
0.1
0.2
0.3
0.4
0.5
0.6
NDCG@20
(a)
(c)
0
0.1
0.2
0.3
0.4
0.5
0.6
NDCG@20
(e)
(g)
0
0.1
0.2
0.3
0.4
0.5
0.6
Recall@20
(b)
(d)
0
0.1
0.2
0.3
0.4
0.5
0.6
Recall@20
(f)
(h)
Performance comparison of EPF_Social , SPF and TSR on Recall and NDCG 0
0.1
0.2
0.3
0.4
0.5
0.6
Recall@20
(b) 0
0.1
0.2
0.3
0.4
0.5
0.6
NDCG@20
(a) (b) (a) (d) (c) (c) (d) (d)
0
0.1
0.2
0.3
0.4
0.5
0.6
Recall@20
(f) 0
0.1
0.2
0.3
0.4
0.5
0.6
NDCG@20
(e) (f) (e) (e)
(g) (f)
(h) (h) (g) Fig. 4 Performance comparison of EPF_Social , SPF and TSR on Recall and NDCG 1 3 Page 9 of 10 International Journal of Computational Intelligence Systems (2021) 14:185 185 belongs to the user-item pair (u, i). Ideally item i should
rank top at the testing moment. We record its predicted rank
among all items. The expressions for MAE and RMSE are
defined as follows: We report the prediction accuracy results over all users in
Fig. 5. Figure 5a, b show that all the Social-based Poisson
Factorization methods (EPF_Social to SPF) perform bet-
ter than the other models (TSRS and DPF). This is due to
the fact that the proposed social-based poisson factorization
methods model the dynamics of user interests and consider
the peer influence of the neighboring users consuming a
product. Hence, they are more expressive than other models
which do not consider the peer influence among users in
social network. Similar results were obtained in Fig. 5c, d. Declarations 17. Basbug, M.E., Engelhardt, B.E.: Hierarchical Compound Poisson
Factorization. ICML, New York (2016) Conflict of interest The authors declared that they have no conflicts of
interest to this work. We declare that we do not have any commercial or
associative interest that represents a conflict of interest in connection
with the work submitted. 18. Cemgil, A.T., Yilmaz, K.: Learning the Beta-Divergence in
Tweedie Compound Poisson Matrix Factorization Models. ICML,
Atlanta (2013) 19. Li, B., Zhu, X., Li, R., Zhang, C., Xue, X., Wu, X.: Cross-domain
Collaborative Filtering Over Time. IJCAI, Barcelona (2011) Open Access This article is licensed under a Creative Commons Attri-
bution 4.0 International License, which permits use, sharing, adapta-
tion, 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/. 20. Du, N., Wang, Y., He, N., Song, L.: Time-sensitive Recommenda-
tion from Recurrent User Activities. NIPS, Montreal (2015) 21. Zhang, Q., Wu, D., Zhang, G., Lu, J.: Fuzzy User-interest Drift
Detection-based Recommender Systems. IEEE, Vancouver (2016) 22. Zhang, Q., Lu, J., Wu, D., Zhang, G.: A cross-domain recom-
mender system with kernel-induced knowledge transfer for over-
lapping entities. IEEE Trans. Neural Netw. Learn. Syst. 30(7),
1998–2012 (2018) 23. Parvin, H., Moradi, P., Esmaeili, S., Qader, N.N.: A scalable and
robust trust-based nonnegative matrix factorization recommender
using the alternating direction method. Knowl. Based Syst. 166,
92–107 (2019) 24. Zhao, J., Geng, X., Zhou, J., Sun, Q., Xiao, Y., Zhang, Z., Fu, Z.:
Attribute mapping and autoencoder neural network based matrix
factorization initialization for recommendation systems. Knowl. Based Syst. 166, 132–139 (2019) 5.5 Returning Time Prediction Figure 5 clearly shows that our EPF_Social model can bet-
ter explain the observed data compared to the other survival
analysis models. (9)
MAE = 1
n
N
i=1
(fi −yi)
RMSD =
∑N
i=1 (fi −yi)2
N
, (9) where fi represents the predicted value, and yi represents
the true value. where fi represents the predicted value, and yi represents
the true value. 1 3
Fig. 5 MAE and RMSE of returning-time and predicted rankings Fig. 5 MAE and RMSE of returning-time and predicted rankings 185
Page 10 of 10 185
Page 10 of 10 International Journal of Computational Intelligence Systems (2021) 14:185 (2021) 14:185 6 Conclusion 6. Yi, X., Hong, L., Zhong, E., Liu, N.N., Rajan, S.: Beyond Clicks:
Dwell Time for Personalization. ACM, California (2014) 7. Charlin, L., Ranganath, R., Mcinerney, J., Blei, D.M.: Dynamic
Poisson Factorization. ACM, Vienna (2015) In this paper we address the problem of modeling the
evolution of user and item factors within the context of
Poisson factorization. We have developed EPF_Social , a
new Bayesian factorization model, which can effectively
model the smoothly drifting latent factors using Conjugate
Gamma–Markov chains. Otherwise, EPF_Social further
considers the time-evolving impact of social network for
how users consume items. Precision, Recall and NDCG
experiments were performed on Netflix-time and Last.fm
datasets and showed that our model is superior to other state-
of-the-art topic models. 8. Chua, F., Oentaryo, R.J., Lim, E.P.: Modeling Temporal Adop-
tions Using Dynamic Matrix Factorization. IEEE, Dallas (2013) 9. Ju, B., Qian, Y., Ye, M. , Ni, R., Zhu, C.: Using dynamic multi-
task non-negative matrix factorization to detect the evolution of
user preferences in collaborative filtering. PLoS ONE 10(8), 1–20
(2015) 10. Koren, Y.: Collaborative filtering with temporal dynamics. ACM,
New York (2010) 11. Ren, L., Dunson, D.B., Carin, L.: The Dynamic Hierarchical Dir-
ichlet Process. ICML, Helsinki (2008) 12. Chatzis, S.: Dynamic Bayesian Probabilistic Matrix Factorization. AAAI, Québec (2014) 13. Luo, C., Cai, X.: Bayesian Wishart matrix factorization. Data Min. Knowl. Discov. 30(5), 1–26 (2016) Acknowledgements This work is supported by Key scientific
research platform of Guangdong Provincial Nos. 2020ZDZX3033,
2021ZDZX1030; Scientific and Technological Project of Zhanjiang
Nos. 2020B01252, 2020B01272; Lingnan Normal University Scientific
and Technological Project of YB2105; The project of human social
science of Guangdong Provincial No. GD20XXW05. 14. Xiong, L., Chen, X., Huang, T., Schneider, J., Carbonell, J.: Tem-
poral Collaborative Filtering with Bayesian Probabilistic Tensor
Factorization. SDM, Columbus (2010) 15. Pan, J., Ma, Z., Pang, Y., Yuan, Y.: Robust probabilistic tensor
analysis for time-variant collaborative filtering. Neurocomputing
119(7), 139–143 (2013) 16. Gopalan, P., Hofman, J.M., Blei, D.M.: Scalable Recommendation
with Hierarchical Poisson Factorization. AUAI, Virginia (2015) References 25. Zhao, J., Wang, W., et al.: TrustTF: A tensor factorization model
using user trust and implicit feedback for context-aware recom-
mender systems. Knowl. Based Syst. 209, 106418 (2020) 1. Gopalan, P., Hofman, J.M., Blei, D.M.: Scalable recommendation
with poisson factorization. Comput. Sci. (2014) 2. Koren, Y., Bell, R., Volinsky, C.: Matrix factorization techniques
for recommender systems. Computer 8, 30–37 (2009) 26. Bobadilla, J., Ortega, F., Hernando, A., Gutierrez, A.: Recom-
mender systems survey. Knowl. Based Syst. 46, 109–132 (2013) 3. Ma, H., Yang, H., et al.: SoRec: social recommendation using
probabilistic matrix factorization. Comput. Intell. 28(3), 289–328
(2008) 27. Yera, R., Martínez, L.: Fuzzy tools in recommender systems: a
survey. Int. J. Comput. Intell. Syst. 10(1), 776–803 (2017) 4. Chaney, A., Blei, D., Eliassi-Rad, T.: A Probabilistic Model for
Using Social Networks in Personalized Item Recommendation. ACM, Vienna (2015) Publisher's Note Springer Nature remains neutral with regard
tojurisdictional claims in published maps and institutional affiliations. Publisher's Note Springer Nature remains neutral with regard
tojurisdictional claims in published maps and institutional affiliations. 5. Koren, Y., Sill, J.: Ordrec: An Ordinal Model for Predicting Per-
sonalized Item Rating Distributions. ACM, New York (2011) 1 3
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ОТКРЫТИЕ ЕДИНОГО ИНФОРМАЦИОННОГО ЦЕНТРА ПОЛИКЛИНИКИ В РАМКАХ ПРОГРАММЫ УПРАВЛЕНИЯ КАЧЕСТВОМ МЕДИЦИНСКОЙ ПОМОЩИ
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THEORY AND PRACTICE OF SCIENCE: KEY ASPECTS
DOI 10.51582/interconf.19-20.06.2022.009
Шевкоплясова Анастасия
MBA, заместитель директора по медицинской части,
ТОО «Поликлиника №8» г. Караганда,
Республика Казахстан
ОТКРЫТИЕ ЕДИНОГО ИНФОРМАЦИОННОГО ЦЕНТРА
ПОЛИКЛИНИКИ В РАМКАХ ПРОГРАММЫ
УПРАВЛЕНИЯ КАЧЕСТВОМ МЕДИЦИНСКОЙ ПОМОЩИ Аннотация: В данной статье описываются произведённые шаги со стороны руководства
медицинской организации в части нововведений для разрешения основных имеющихся
проблем по взаимодействию между пациентом, посетителем поликлиники с медицинским
персоналом,
а
также
внутреннему
взаимодействию
между
структурными
подразделениями поликлиники в рамках исполнения программы управления качеством
медицинской помощи Ключевые слова: качество, частный, реформа, взаимодействие Ключевые слова: качество, частный, реформа, взаимодействие Одной из главных целей привлечения частного сектора в систему
здравоохранения на основе государственно-частного партнёрства являлось
повышение качества оказываемой медицинской помощи за счёт увеличения
финансирования сектора частным партнёром, покрытия части рисков
государственным партнёром, а также улучшения сервиса в целом. На
сегодняшний день в сферу здравоохранения уже активно вовлечён частный
бизнес, только преимущественным направлением здесь является не столько
фактическое государственно-частное партнёрство, сколько исполнение
государственного заказа частными медицинскими центрами в обеспечении
необходимыми
медицинскими
услугами
население
и
достоверное
направление государственного финансирования частнику. При этом в массе
своей качество предоставляемой медицинской помощи не изменилось в
лучшую сторону. При оценке данного показателя необходимо рассматривать
не только оказанную медицинскую услугу частным центром, а и 96 SCIENTIFIC COLLECTION «INTERCONF» | № 113 SCIENTIFIC COLLECTION «INTERCONF» | № 113 непосредственно весь процесс её реализации с момента возникновения
потребности в ней и до принятых действий по её результатам. И вот здесь
выявляются множество скрытых проблем, на решении которых особо никто
не концентрируется. Следует отметить, что проводимое реформирование
задевает
очень
актуальные
точки
функционирования
системы
здравоохранения. Таковыми, например, являются совершенствование
службы сестринского ухода [1], развитие превентивного направления
медицины, повышение доступности медицинской помощи, снижение
реализации возможных рисков при оказании медицинской помощи. В свете
рассмотрения
вышеперечисленных
пунктов
выявляются
исходные
проблемы, как малая доля участия среднего медицинского персонала в
профилактике, лечении, восстановлении и уходе за пациентами, высокая
стоимость медицинской помощи в разрезе совершенствования научно-
технического оснащения и дефицита квалифицированных человеческих
ресурсов
при
растущем
числе
населения,
высокая
физическая
и
психологическая нагрузка у медицинских работников при реализации
экстренных и неотложных ситуаций, увеличение объёма заболеваемости в
виду роста количества населения, низкой осведомлённости о мерах
профилактики заболеваний среди населения, а также роста вредных
факторов внешней среды в том числе и по результатам деятельности
человека. Безусловно, решение таких проблем на государственном уровне
весомо, аргументировано и даёт свой результат. Однако, и на внутреннем
уровне медицинской организации возможно создавать условия для
разрешения таких глобальных, казалось бы, задач. В данной статье
рассматривается такой пример внутреннего подхода администрации
поликлиники, осуществляющей свою деятельность в рамках государственно-
частного партнёрства, для реализации утверждённой программы управления
качеством медицинской помощи в части повышения удовлетворённости
населения предоставляемыми медицинскими услугами. В условиях существующего дефицита квалифицированных врачебных
кадров в частной медицинской организации, выполняющей государственный 97 THEORY AND PRACTICE OF SCIENCE: KEY ASPECTS заказ в виде оказания медицинской помощи прикреплённому населению,
остро встал вопрос о делегировании части врачебных полномочий среднему
медицинскому персоналу. И согласно вводимым инновациям со стороны
Министерства
здравоохранения
в
разрезе
Программы
управления
заболеванием (ПУЗ), Универсальной прогрессивной модели патронажной
медсестры (УПМПМ), Триаж-системы, Интегрированного ведения болезней
детского возраста (ИВБДВ), Службы поддержки пациента и внутреннего
контроля (СППВК) явно вырисовывалась потребность в расширении участия
среднего медицинского персонала в жизни пациента. Ранее медсестра
выполняла роль помощника врача, осуществляя только письменную
фиксацию проведённого осмотра, выдачу справок, медикаментов, причём её
работа была строго координирована и связана непосредственно с работой
врача. На сегодняшний день роль и статус медсестры существенно выросли и,
безусловно, сестринская служба занимает теперь по праву серьёзную и
решающую позицию в оказании качественной медицинской помощи. Что же
изменилось в рамках реформирования системы здравоохранения? В первую
очередь, участковая служба была усилена штатом средних медицинских
работников, а именно: на одном участке врача общей практики с
прикреплённым населением до 1700 человек теперь работают 3 средних
медработника, за каждым из которых закреплён определённый функционал. Следующим изменением стало разделение врача и медсестры территориально,
в первую очередь это было сделано с целью соблюдения приватности
пациентов, однако, в последующем сыграло важную роль в открытии нового
вида предоставляемых услуг - приём медсестры [2]. Всё это стало возможным,
благодаря
внедрению
в
работу
медицинских
организаций
единой
медицинской информационной системы с определёнными уровнями доступа
медицинских специалистов в зависимости от того функционала, который
закреплён за ними (рис. 1). Таким образом, теперь доктор концентрируется непосредственно на
осмотре пациента, беседе с ним и достижении комплаенса, причём для этого
не приходится увеличивать продолжительность приёма. В виду того, что 98 98 SCIENTIFIC COLLECTION «INTERCONF» | № 113 медсестра теперь территориально находится отдельно от доктора, она может
параллельно доктору вести свой собственный приём, что является очень
актуальным в рамках мониторинга состояния пациента при диспансеризации:
согласно утверждённым стандартам при определённых нозологиях требуется
до 6 плановых осмотров пациента в год средним медперсоналом и лишь 2
врачебных осмотра. медсестра теперь территориально находится отдельно от доктора, она может
параллельно доктору вести свой собственный приём, что является очень
актуальным в рамках мониторинга состояния пациента при диспансеризации:
согласно утверждённым стандартам при определённых нозологиях требуется
до 6 плановых осмотров пациента в год средним медперсоналом и лишь 2
врачебных осмотра. Рис. 1. Распределение функционала медперсонала и приёма пациентов
на участке врача общей практики в течение рабочей смены Рис. 1. Распределение функционала медперсонала и приёма пациентов
на участке врача общей практики в течение рабочей смены Рис. 1. Распределение функционала медперсонала и приёма пациентов
на участке врача общей практики в течение рабочей смены Для обеспечения высокой частоты осмотров медсестрой теперь нет
необходимости в сокращении продолжительности врачебного приёма и в
тоже
время
увеличилась
доступность
медицинской
помощи
для
прикреплённого населения. Тем не менее, при проведении анализа работы
поликлиники были выявлены множественные точки пересечения потоков
разных групп посетителей медорганизации [3], а также регулярная высокая
загруженность кабинета медсестры, регистратуры и аптеки поликлиники с
формированием очерёдности ожидания. А взаимодействие центра семейного
здоровья (ЦСЗ: врачи общей практики, педиатры) и специализированного
диагностического отделения (СДО: узкие специалисты, диагностические
исследования) по сути осуществлялось через пациента, что обусловливало
затягивание процессов обследования и выработки тактики ведения на
амбулаторном этапе, что негативно сказывалось на удовлетворённости
посетителей и пациентов качеством предоставляемой поликлиникой
медицинской помощи (рис. 2). 99 99 THEORY AND PRACTICE OF SCIENCE: KEY ASPECTS Рис. 2. Схема взаимодействия между подразделениями поликлиники
с вектором заинтересованности в результате на пациенте Рис. 2. Схема взаимодействия между подразделениями поликлиники
с вектором заинтересованности в результате на пациенте Из предоставленной выше схемы очевидно, что для получения
направления на какое-либо обследование или консультацию профильного
специалиста пациенту необходимо обратиться в ЦСЗ, после чего через МИС
врач или медсестра формирует направление в СДО, в дальнейшем пациент
обращается в СДО. Результаты оказанной услуги в СДО также через МИС
отправляются в ЦСЗ и для получения последующих рекомендаций пациенту
вновь необходимо обратиться на приём в ЦСЗ. В результате пациент
совершает для одной процедуры 3 действия, из которых 2 являются
неоправданными, занимают время самого пациента, врача и медсестры, тем
самым необоснованно увеличивая очередность на приём. SCIENTIFIC COLLECTION «INTERCONF» | № 113 Исходя из полученной в результате аналитики информации, руководство
медорганизации
столкнулось
с
необходимостью
срочно
разрешить
выявленные проблемы, а с учётом работы в рамках государственно-частного
партнёрства и потребностью соблюсти интересы также и частной стороны, не
выйти за пределы запланированного и имеющегося финансирования. Первым
значимым изменением стал перенос вектора заинтересованности в результате
с пациента на участковую службу. Таким действием было решено сразу
несколько критических направлений: своевременность обследований и
консультаций, сокращение продолжительности обследований, своевременное
лекарственное обеспечение пациентов, сокращение потоков пациентов и
посетителей и их упорядочение непосредственно в медорганизации, 100 SCIENTIFIC COLLECTION «INTERCONF» | № 113 SCIENTIFIC COLLECTION «INTERCONF» | № 113 повышение удовлетворённости пациентов и посетителей поликлиники. На
практике это выглядело работой медперсонала в МИС, интегрированной для
реализации данных потребностей с CRM-системой (customer relationship
management), позволяющей при имеющихся разработанных чек-листах для
мониторинга состояния пациентов с хроническими неинфекционными
заболеваниями, состояния беременных и родильниц, а также других групп
пациентов получать уведомления на рабочий стол о предстоящих и
исполненных обследованиях и консультациях, потребности в лекарственном
обеспечении в ближайшее время. По итогам данного нововведения пациент
теперь приходил в поликлинику только 1 раз для прохождения обследования,
а не в качестве связующего звена (рис. 3). Рис. 3. Внедрённая схема взаимодействия пациента и структурных
подразделений поликлиники с перенаправлением вектора
заинтересованности в результате от пациента к медицинскому
персоналу Рис. 3. Внедрённая схема взаимодействия пациента и структурных
подразделений поликлиники с перенаправлением вектора
заинтересованности в результате от пациента к медицинскому
персоналу В
целях
совершенствования
оказания
медицинской
помощи
в
поликлинике внедрение CRM-системы позволило также сократить поток
посетителей и пациентов за получением рецептов на лекарственное
обеспечение с последующим направлением в аптеку (рис. 4). 101 THEORY AND PRACTICE OF SCIENCE: KEY ASPECTS Рис. 4. Путь пациента для получения медикаментов в сравнении
до и после проведённых изменений Рис. 4. Путь пациента для получения медикаментов в сравнении
до и после проведённых изменений Важным аспектом, не смотря на внедрение CRM-системы, всё-таки
являлось изменение подхода в самой работе среднего медперсонала. Дело в
том, что работа участковой службы ЦСЗ сводится к разделению рабочей
смены на две составляющие: работа на приёме и работа непосредственно на
участке. За счёт чего все три медсестры участка после 4-х часов работы в
поликлинике должны были выходить на участок при том, что потребность в
них ещё на приёме как для пациентов, так и для взаимодействия с СДО,
аптекой, регистратурой, СППВК в поликлинике сохранялась. По факту же,
при наличии мотивации и потребности у медсестёр в продолжении своей
работы непосредственно в поликлинике, отсутствовала возможность
территориального расположения в виду формирования работы участковой
службы с локацией в одном кабинете 2-х участков. Для урегулирования
данных процессов было решено открыть информационный центр, в котором
формировалась единая связующая между пациентами и медперсоналом
разных структурных подразделений поликлиники (рис. 5). Таким образом проведена передислокация и концентрация в одном месте
персонала поликлиники: службы поддержки пациента и внутреннего
контроля, регистратуры с Call-центром, школ здоровья, а также рабочие места
среднего медперсонала центра семейного здоровья и специализированного
диагностического отделения с определёнными уровнями доступа к МИС и 102 102 THEORY AND PRACTICE OF SCIENCE: KEY ASPECTS повысилось
качество
лекарственного
амбулаторного
обеспечения
и
диспансерного наблюдения. Всё это позволило обеспечить слаженную работу
всей медицинской организации в целом. SCIENTIFIC COLLECTION «INTERCONF» | № 113 CRM. Пациент при самостоятельном очном обращении в поликлинику в
первую очередь контактирует с встречающим администратором, который
имеет прямую связь с информационным центром, для обеспечения
правильного направления потоков пациентов. Рис. 5. Взаимодействие пациента и структурных подразделений
поликлиники при работе единого информационного центра Рис. 5. Взаимодействие пациента и структурных подразделений
поликлиники при работе единого информационного центра При обращении в поликлинику по телефону, посредством мессенджеров
или социальных интернет площадок обеспечивается одномоментное
взаимодействие всех заинтересованных для предоставления качественной
услуги. По результатам проведённых изменений уровень удовлетворённости
предоставляемой населению медицинской и консультативной помощи
повысился, снизилась очередность непосредственно в самой поликлинике, 103 THEORY AND PRACTICE OF SCIENCE: KEY ASPECTS Список источников: 1. Зурнаджьянц Ю.А., Кашкарова И.А. Современные аспекты совершенствования
деятельности сестринского персонала в условиях медицинской организации //
Материалы Всероссийской научно-практической конференции: Актуальные вопросы
управления сестринской деятельностью в системе управления здравоохранения,
Астрахань, 2016. С. 88-92 2. Каусова Г.К., Толегенова С.У. и др. Опыт делегирования полномочий среднему
медицинскому персоналу на примере смешанной поликлиники при ГКБ №5 г.Алматы
[Электронный ресурс]. URL: https://articlekz.com/article/25382? (Дата обращения:
17.05.2022) 3. Лепина М. Идеальная 64-я: нам показали, какой должна быть поликлиника будущего
[Электронный ресурс]. URL: https://www.miloserdie.ru/article/? (Дата обращения:
17.05.2022) 3. Лепина М. Идеальная 64-я: нам показали, какой должна быть поликлиника будущего
[Электронный ресурс]. URL: https://www.miloserdie.ru/article/? (Дата обращения:
17.05.2022) 104
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https://openalex.org/W2620489131
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https://rgu-repository.worktribe.com/file/1085415/1/SUN%202017%20Gravitation-based%20edge%20%28VOR%29
|
English
| null |
Gravitation-Based Edge Detection in Hyperspectral Images
|
Remote sensing
| 2,017
|
cc-by
| 20,098
|
SUN, G., ZHANG, A., REN, J., MA, J., WANG, P., ZHANG, Y. and JIA, X. 2017. Gravitation-based edge detection in
hyperspectral images. Remote sensing [online], 9(6), article 592. Available from: https://doi.org/10.3390/rs9060592
Gravitation-based edge detection in
hyperspectral images.
SUN, G., ZHANG, A., REN, J., MA, J., WANG, P., ZHANG, Y. and JIA, X.
2017 SUN, G., ZHANG, A., REN, J., MA, J., WANG, P., ZHANG, Y. and JIA, X. 2017. Gravitation-based edge detection in
hyperspectral images. Remote sensing [online], 9(6), article 592. Available from: https://doi.org/10.3390/rs9060592 Genyun Sun 1,2, Aizhu Zhang 1,2,*, Jinchang Ren 3, Jingsheng Ma 4, Peng Wang 1,2,
Yuanzhi Zhang 5 and Xiuping Jia 6 1
School of Geosciences, China University of Petroleum (East China), Qingdao 266580, China;
genyunsun@163.com (G.S.); wangpeng187@126.com (P.W.) 1
School of Geosciences, China University of Petroleum (East China), Qingdao 266580, China;
genyunsun@163.com (G.S.); wangpeng187@126.com (P.W.) 1
School of Geosciences, China University of Petroleum (East China), Qingdao 266580, China;
genyunsun@163.com (G.S.); wangpeng187@126.com (P.W.) 2
Laboratory for Marine Mineral Resources Qingdao National Laboratory for Marine Science and Technology
Qingdao 266071, China 2
Laboratory for Marine Mineral Resources Qingdao National Laboratory for Marine Science and Technology,
Qingdao 266071, China 3
Department of Electronic and Electrical Engineering, University of Strathclyde, Glasgow G1 1XQ, UK;
jinchang.ren@strath.ac.uk 3
Department of Electronic and Electrical Engineering, University of Strathclyde, Glasgow G1 1XQ, UK;
jinchang.ren@strath.ac.uk 4
Institute of Petroleum, Heriot-Watt University, Edinburgh EH14 4AS, UK; jingsheng.ma@hw.ac.uk
5
Key Lab of Lunar Science and Deep-Exploration, Chinese Academy of Sciences, Beijing 100012, China;
yuanzhizhang@hotmail.com Institute of Petroleum, Heriot Watt University, Edinburgh EH14 4AS, UK; jingsheng.ma@hw.ac.uk
5
Key Lab of Lunar Science and Deep-Exploration, Chinese Academy of Sciences, Beijing 100012, China;
yuanzhizhang@hotmail.com 6
School of Engineering and Information Technology, The University of New South Wales at Canberra, Canberra ACT 2600, Australia; x.jia@adfa.edu.au Canberra ACT 2600, Australia; x.jia@adfa.edu.au Canberra ACT 2600, Australia; x.jia@adfa.edu.au *
Correspondence: zhangaizhu789@163.com; Tel./Fax: +86-532-8698-1952 Academic Editors: Eyal Ben-Dor and Prasad S. Thenkabail
Received: 28 March 2017; Accepted: 8 June 2017; Published: 11 June 2017 Academic Editors: Eyal Ben-Dor and Prasad S. Thenkabail eived: 28 March 2017; Accepted: 8 June 2017; Publishe Received: 28 March 2017; Accepted: 8 June 2017; Published: 11 June 2017 Abstract: Edge detection is one of the key issues in the field of computer vision and remote sensing
image analysis. Although many different edge-detection methods have been proposed for gray-scale,
color, and multispectral images, they still face difficulties when extracting edge features from
hyperspectral images (HSIs) that contain a large number of bands with very narrow gap in the spectral
domain. Inspired by the clustering characteristic of the gravitational theory, a novel edge-detection
algorithm for HSIs is presented in this paper. In the proposed method, we first construct a joint feature
space by combining the spatial and spectral features. Each pixel of HSI is assumed to be a celestial
object in the joint feature space, which exerts gravitational force to each of its neighboring pixel. Accordingly, each object travels in the joint feature space until it reaches a stable equilibrium. Genyun Sun 1,2, Aizhu Zhang 1,2,*, Jinchang Ren 3, Jingsheng Ma 4, Peng Wang 1,2,
Yuanzhi Zhang 5 and Xiuping Jia 6 At the
equilibrium, the image is smoothed and the edges are enhanced, where the edge pixels can be easily
distinguished by calculating the gravitational potential energy. The proposed edge-detection method
is tested on several benchmark HSIs and the obtained results were compared with those of four
state-of-the-art approaches. The experimental results confirm the efficacy of the proposed method. Keywords: edge detection; hyperspectral image; gravitation; remote sensing; feature space Gravitation-Based Edge Detection in
Hyperspectral Images Genyun Sun 1,2, Aizhu Zhang 1,2,*, Jinchang Ren 3, Jingsheng Ma 4, Peng Wang 1,2,
Yuanzhi Zhang 5 and Xiuping Jia 6 Gravitation-based edge detection in
hyperspectral images. 2017 © 2017 by the authors. Licensee MDPI, Basel, Switzerland. © 2017 by the authors. Licensee MDPI, Basel, Switzerland. © 2017 by the authors. Licensee MDPI, Basel, Switzerland. This document was downloaded from
https://openair.rgu.ac.uk remote sensing 1. Introduction That is to say, different spectral bands may even contain inconsistent edges, which can easily leads
to false edges by simple combination. To overcome this drawback caused by the combination rules,
Lei and Fan combined the first principal component and hue component of color images to obtain
complete object edges [16]. Different from monochromatic approaches, vector-based approaches consider each pixel as a
spectral vector and then use vector operations to detect edges [17–24]. In these approaches, the gradient
magnitude and direction are defined in a vector field by extending the gray-scale edge definition [11]. Di Zenzo’s gradient operator [17], which uses the tensor gradient to define the edge magnitude and
direction, is a widely used vector-based method. However, Di Zeno’s method is sensitive to small
changes in intensity because it is based on the measure of the squared local contrast variation of
multispectral images. To overcome this problem, in Drewniok [19], Di Zeno’s method is combined
with the Canny edge detector with a Gaussian pre-filter model applied to each band for smoothing
the image. Drewniok’s method has better effectiveness, but it results in a localization error when the
Gaussian scale parameter is large [25]. To remove both the effect of noise and the localization error
caused by pre-filtering, a novel method based on robust color morphological gradient (RCMG) is
proposed, where the edge strength of a pixel is defined as the maximum distance between any two
pixels in its surrounding window [24]. The RCMG is robust to noise and helps top reserve accurate
spatial structures due to a non-linear scheme is implied in the process [10]. Generally speaking, vector based approaches are superior to monochromatic approaches in
multispectral edge detection since they take the spectral correlation of the bands into consideration. Different from color images or multispectral images, however, HSIs usually consist of hundreds
of contiguous spectral bands. Although the high spectral resolution of HSIs provides potential for
more accurate identification of targets, it still suffers from three problems in edge detection, various
interferences that lead to low homogeneity inside regions [26], spectral mixtures or edges that appear in
a small subset of bands often cause weak edges [26] and inconsistent edges from narrow and abundant
channels [27], which have made edge detection more challenging in HSIs. 1. Introduction With the development of optic-electronics and data transmission technologies, hyperspectral
sensors are able to capture hundreds of spectral bands simultaneously with good spatial resolution. A few well-known hyperspectral imaging systems, such as AVIRIS (the Airborne Visible Infrared
Imaging Spectrometer), HYDICE (the Hyperspectral Digital Imagery Collection Experiment), and
EnMap (the Environmental Mapping and Analysis Program), can provide 126–512 spectral bands with
a spatial resolution from 3 m to 30 m. The growing availability of hyperspectral images (HSIs) with
both fine spectral and spatial resolution has opened the door to numerous new applications in remote
sensing [1]. In many application areas for HSIs, segmentation is required as an important image processing
step because it divides an image into meaningful homogeneous regions [2]. However, because Remote Sens. 2017, 9, 592; doi:10.3390/rs9060592 www.mdpi.com/journal/remotesensing 2 of 23 Remote Sens. 2017, 9, 592 the imaged scenes are complex scenes and may not have clear boundaries, image segmentation is
always a big challenging task. Effective edge detection algorithms have been found useful for image
segmentation since they delineate boundaries of regions and determine segmentation results [2,3]. Especially for the advanced HSIs with high spatial resolution, the clearly depicted edges could be of
great benefit in characterizing the spatial structures of landscapes [4]. Over the past few decades, a large number of edge-detection techniques have been proposed, such
as Canny [5], Marr [6], SUSAN (Univalue Segment Assimilating Nucleus) [7] and active contour based
methods [8]. However, these approaches are designed primarily for gray, color or multispectral images
and few studies have paid attention to edge detection on HSIs [9,10]. In previous studies [10,11],
edge-detection tasks are completed by adapting color or multispectral approaches to hyperspectral
images (HSIs). Accordingly, existing approaches for color or multispectral based edge detection are
discussed as follows. In general, most approaches for color or multispectral based edge detection can be grouped into
three categories: (1) monochromatic approaches; (2) vector based approaches; and (3) feature-space
based approaches. Monochromatic approaches combine all edge maps after applying well-established
gray-scale edge detectors on each band, and typical rules for combining edges include the maximum
rule [12], the summation rule [13] and the logic OR operation [14]. Although the fine spectral resolution
of HSI provides invaluable and abundant information regarding the physical nature of different
materials [10], edge features may be only observable over a small subset of bands in the HSI [15]. 1. Introduction Dimension reduction has
been investigated using signal subspace approach and proposed the local rank based edge detection
method [28], however the performance is limited. It is necessary to consider the spatial relationships
between spectral vectors [29]. In Reference [30], a modified Simultaneous Spectral/Spatial Detection
of Edges (SSSDE) [31] for HSIs was proposed to exploit spectral information and identify boundaries
(edges) between different materials. This is a kind of constructive attempt to joint utilization of 3 of 23 Remote Sens. 2017, 9, 592 spatial and spectral information. Recently, many researchers have tried to develop feature-based
new edge-detection methods because edge pixels have saliency in the feature space [32]. In Lin [32],
color image edge detection is proposed based on a subspace classification in which multivariate
features are utilized to determine edge pixels. In Dinh [27], MSI edge detection is achieved via
clustering the gradient feature. The feature space is suitable to analyze HSIs because it is capable of
showing data from various land cover types in groups [33]. In particular, a spatial-spectral joined
feature space, which takes into consideration both the spatial and spectral correlation between pixels,
has attracted increasing attention [1,29]. In addition to edge detection, feature space has also been
widely used in other fields of image processing, such as hyperspectral feature selection [34,35], target
detection [36], and image segmentation [37,38]. However, few studies focus on edge detection for HSIs
using feature space. g
p
Physical models that mimic real-world systems can be used to analyze complex scientific
and engineering problems [37,39,40]. The ongoing research of physical models has motivated the
exploration of new ways to handle image-processing tasks. As a typical physical model, Newton’s
law of universal gravitation [41] has received considerable attention, based on which, a series of
image-processing algorithms has been proposed, such as the stochastic gravitational approach to
feature-based color image segmentation (SGISA) [37], the improved gravitational search algorithm
for image multi-level image thresholding [42] and the gravitational collapse (CTCGC) approach for
color texture classification [43]. Other gravitation model based approaches for classification include
data gravitation classification (DGC) [44–46] and gravitational self-organizing maps (GSOM) [47]. The advantages of the gravity field model have inspired the development of new edge-detection
method for gray-scale images [48,49]. Although the image-processing procedure using gravitational
theory is relatively easy for understanding, this theory is rarely used in edge detection for HSIs. 1. Introduction As the
gravitational theory describes the movements of objects in the three-dimensional feature space of the
real world, we can easily extend it to n-dimensional feature space and apply it for edge detection
in HSIs. In this paper, a novel edge-detection method for HSIs based on gravitation (GEDHSI) is proposed. The main ideas of the proposed GEDHSI are as follows: (1) there exists a type of “force”, called
gravitation, between any two pixels in the feature space; (2) computational gravitation obeys the law
of gravitation in the physical world; (3) all pixels move in the feature space according to the law of
motion until the stopping criteria are satisfied, and then the image system reaches a stable equilibrium;
and (4) the edge pixels and non-edge pixels are classified into two different clusters. Therefore, in
the equilibrium system, edge responses can be obtained by computing the gravitational potential
energy due to the relative positions. Different from our previous approach for gravitation based edge
detection in gray-scale images [49], the proposed method features a dynamic scheme. By imitating
the gravitational theory, all pixels move in the joint spatial-spectral feature space, which makes the
GEDHSI more attractive and effective for HSIs. The experiment results illustrate both the efficiency
and efficacy of the proposed method for HSI edge-detection problems. The rest of the paper is organized as follows: The law of universal gravity is briefly reviewed
in Section 2. The proposed gravitation-based edge detection method is presented in Section 3. The experimental validations of the proposed algorithm using both artificial and real HSIs are given in
Section 4. Finally, the paper is concluded in Section 5. 2. Background of the Universal Law of Gravitation According to gravitational theory [35], any two objects exert gravitational force onto each other. The forces between them are equal to each other but reverse in direction. The force is proportional to
the product of the two masses and inversely proportional to the square of the distance between them,
as shown in Equation (1): Remote Sens. 2017, 9, 592 4 of 23 →
F i,j = G
MiMj
→
Zi −
→
Zj
3 (
→
Zi −
→
Zj),
(1) (1) where
→
F i,j is the vector-form magnitude of the gravitational force between objects i and j, and their
masses are denoted as Mi and Mj (Mi < Mj), respectively. G is the universal gravitational constant,
→
Zi
and
→
Zj represent the position vector of objects i and j. Newton’s second law says that when a force,
→
F i,j, is applied to object i, its acceleration,
→ai, depends only on the force and its mass, Mi [50]: where
→
F i,j is the vector-form magnitude of the gravitational force between objects i and j, and their
masses are denoted as Mi and Mj (Mi < Mj), respectively. G is the universal gravitational constant,
→
Zi
and
→
Zj represent the position vector of objects i and j. Newton’s second law says that when a force,
→
F i,j, is applied to object i, its acceleration,
→ai, depends only on the force and its mass, Mi [50]: →ai =
→
Fi,j
Mi
. (2) (2) For Mi, two types of mass are defined in theoretical physics: For Mi, two types of mass are defined in theoretical physics:
G Gravitational mass, MG
i , is a measure of the strength of the gravitational field due to a particular
object. The gravitational field of an object with small gravitational mass is weaker than an object with
larger gravitational mass. Inertial mass, MI
i , is a measure of an object’s resistance to changing its state of motion when a
force is applied. An object with large inertial mass changes its motion more slowly, and an object with
small inertial mass changes it rapidly. In physics, gravitational mass and inertial mass are usually treated as the same. In this paper,
they are treated separately to enhance the adaptability of gravitational theory for HSI edge detection. 2. Background of the Universal Law of Gravitation It can be concluded that the force is the nega
differential of the SPE [52], which is expressed as follows: Similar to the gravitational force, the SPE also obeys the superposition principle. The SPE is closely linked with forces. It can be concluded that the force is the
differential of the SPE [52], which is expressed as follows: Similar to the gravitational force, the SPE also obeys the superposition principle. The SPE is closely linked with forces. It can be concluded that the force is the negative differential
of the SPE [52], which is expressed as follows:
y
g
differential of the SPE [52], which is expressed as follows:
,
,
i j
i j
F
φ
→
= −∇
(7) The SPE is closely linked with forces. It can be concluded that the force is the negative differentia
he SPE [52], which is expressed as follows:
[
],
p
,
i j
i j
F
φ
→
= −∇
(7) →
F i,j = −∇φi,j,
(7)
function is extendable to high dimensional feature space (7)
pace where ∇is the delta operator. The SPE function is extendable to high dimensional feature space and
capable of reflecting the inhomogeneity of data, which is a key point for edge detection. and capable of reflecting the inhomogeneity of data, which is a key point for edge detection. In gravitational systems, all objects move under the gravitational force until an equilibrium
h
i
h d I
th
ilib i
h
ll i
il
bj
t
t t
f
l
t
Diff
t In gravitational systems, all objects move under the gravitational force until an equilibrium
phase is reached. In the equilibrium phase, all similar objects aggregate to form a cluster. Different
clusters have different properties, such as SPE. Some gravitation-based algorithms are inspired from
the characteristics of gravitational theory [37,50]. In this paper, we assume that edge and non-edge
pixels are by nature two different clusters. Therefore, they will have different SPEs. These properties
constitute the background of the proposed method. phase is reached. In the equilibrium phase, all similar objects aggregate to form a cluster. Different
clusters have different properties, such as SPE. Some gravitation-based algorithms are inspired from
the characteristics of gravitational theory [37,50]. In this paper, we assume that edge and non-edge
pixels are by nature two different clusters. Therefore, they will have different SPEs. 2. Background of the Universal Law of Gravitation These properties
constitute the background of the proposed method. 3 Gravitation-Based Edge Detection in Hyperspectral Image (GEDHSI) 3.1. Overview
GEDHSI GEDHSI is proposed in this study based on the Newton’s law of universal gravitation. It contains
three main phases as illustrated in Figure 1. The first phase is image mapping which maps a HSI
into a joint spatial-spectral feature space, as detailed in Section 3.2. In the joint space, every pixel is
considered as an object which attracts each other via the gravitational force. The second phase is the
pixel traveling phase as detailed in Section 3.3 where an iterative procedure is used to allow all pixels
move within the joint spatial-spectral feature space under the influence of the gravitational force until
the image system reaches a stable equilibrium. In this state, the edge pixels and non-edge pixels will
have different scalar potential energy (SPEs) based on their relative positions. Consequently, in the
third phase, the edge response is obtained based on the determined SPE of each pixel. contains three main phases as illustrated in Figure 1. The first phase is image mapping which maps
a HSI into a joint spatial-spectral feature space, as detailed in Section 3.2. In the joint space, every
pixel is considered as an object which attracts each other via the gravitational force. The second
phase is the pixel traveling phase as detailed in Section 3.3 where an iterative procedure is used to
allow all pixels move within the joint spatial-spectral feature space under the influence of the
gravitational force until the image system reaches a stable equilibrium. In this state, the edge pixels
and non-edge pixels will have different scalar potential energy (SPEs) based on their relative
positions. Consequently, in the third phase, the edge response is obtained based on the determined
SPE of each pixel. Figure 1. Overview of the proposed GEDHSI (Gravitation-Based Edge Detection in Hyperspectral Image)
Figure 1. Overview of the proposed GEDHSI (Gravitation-Based Edge Detection in Hyperspectral Image). gure 1. Overview of the proposed GEDHSI (Gravitation-Based Edge Detection in Hyperspectral Image
Figure 1. Overview of the proposed GEDHSI (Gravitation-Based Edge Detection in Hyperspectral Image). 2. Background of the Universal Law of Gravitation → That is to say, the gravitational force,
→
F i,j, that acts on object i by object j is proportional to the product
of the gravitational mass of objects j and i and inversely proportional to the square distance between
them. →ai is proportional to
→
F i,j and inversely proportional to inertia mass of object i. More precisely,
we rewrite Equations (1) and (2) as follows: →
F i,j = G
MG
i MG
j
→
Zi −
→
Zj
3 (
→
Zi −
→
Zj),
(3)
→ai =
→
F i,j
MI
i
,
(4) (3) →ai = F i,j
MI
i
,
(4) (4) where MG
i and MG
j represent the gravitational mass of objects i and j, respectively. MI
i represents the
inertial mass of object i. The gravitational force obeys the superposition principle. Thus, for any object k in the space,
the gravitational force exerted by objects i and j is calculated by Equation (5). →
Fk =
→
F i,k +
→
F j,k = G MG
i MG
k
→
Zi −
→
Zk
3
→
(Zi −
→
Zk) + G MG
i MG
k
→
Zj −
→
Zk
3
→
(Zj −
→
Zk),
(5) (5) where
→
Zk is the position of object k in space Ω, and MG
k is the gravitational mass of object k. The resultant
force forms the gravitational field. where
→
Zk is the position of object k in space Ω, and MG
k is the gravitational mass of object k. The resultant
force forms the gravitational field. Because the gravitational field is conservative, there is a scalar potential energy (SPE) per unit
mass at each point in the space associated within the gravitational fields. The SPE of object i exerted by
object j is determined by Equation (6) [51] as follows: Remote Sens. 2017, 9, 592 5 of 23 φi,j = −G
MG
i MG
j
→
Zi −
→
Zj
. (6)
,
. i j
j
i
G
Z
Z
φ
→
→
−
(6)
the SPE also obeys the superposition principle. fo
e
It
a
be
o
luded that the fo
e i
the
e ati e (6) Similar to the gravitational force, the SPE also obeys the superposition principle. The SPE is closely linked with forces. 3.2. Image Mapping
Th
i
i
3.2. Image Mapping The gravitational theory is derived from three-dimensional real-world space. To simulate the
theory, we first need to map the HSI into a feature space. For a HSI denoted by
B
W
H
I
×
×
, the
parameters H, W and B represent height, the width of the image and the number of bands,
respectively. A simple feature space is composed only of its bands. This type of feature space
The gravitational theory is derived from three-dimensional real-world space. To simulate the
theory, we first need to map the HSI into a feature space. For a HSI denoted by IH×W×B, the parameters
H, W and B represent height, the width of the image and the number of bands, respectively. A simple
feature space is composed only of its bands. This type of feature space primarily uses spectral 6 of 23 Remote Sens. 2017, 9, 592 information. To make full use of spatial and spectral information, inspired by Reference [37], we define
a spatial-spectral jointed feature space containing (B + 2) features (dimensions). In the feature space,
each pixel addresses a location in the space: information. To make full use of spatial and spectral information, inspired by Reference [37], we define
a spatial-spectral jointed feature space containing (B + 2) features (dimensions). In the feature space,
each pixel addresses a location in the space: →
Zi = (
→
Vi,
→
Si) = (v1
i , v2
i ..., vb
i , · · · , vB
i , xi, yi), for i = 1, 2, . . . , N,
(8) (8) where N = H × W is the total number of pixels in the image,
→
Vi = (v1
i , v2
i , ..., vB
i ) is the spectral
components of the pixel i, and
→
Si = (xi, yi) is its spatial position. where N = H × W is the total number of pixels in the image,
→
Vi = (v1
i , v2
i , ..., vB
i ) is the spectral
components of the pixel i, and
→
Si = (xi, yi) is its spatial position. 3.3. Pixel Traveling Once the joint spatial-spectral feature space is constructed, we consider each pixel as a mass
object in the space. Thereby, as introduced in Section 2, each pixel can attract others through the
gravitational force and then moves in the joint feature space according to the gravitational theory. In other words, in the traveling stage, all objects move step by step within the joint feature space under
the gravitational force until the stopping criteria are met. Specifically, the pixel traveling phase contains
the following three steps in an iterative way until the whole image based system becomes stable: •
Calculation of the acceleration; •
Calculation of the traveling step size; •
Location updating. (1)
Calculation of the Acceleration (1)
Calculation of the Acceleration In GEDHSI, for a pixel i at time t, its acceleration
→ai(t) is computed according to Equation (9) as
follow:
→ →ai(t) =
→
Fi(t)
MI
i (t),
(9) (9) where
→
Fi(t) is the gravitational force of the pixel i exerted by the other pixels at time t, and MI
i (t) is the
inertial mass of the pixel i at time t. According to Equation (7), the gravitational force is proportional to the delta of SPE and along the
decreased direction of SPE. Therefore, we first calculate the SPE of the pixels i produced by a pixel j
as follows: φi,j(t) = −G
MG
j (t) · MG
i (t)
(dij(t))
,
(10) (10) where dij(t) is the distance between the pixels i and j, and MG
i (t) and MG
j (t) are the gravitational mass
of the pixels i and j. In this paper, to simplify the calculation, gravitational mass of each object is set as
a unit value. where dij(t) is the distance between the pixels i and j, and MG
i (t) and MG
j (t) are the gravitational mass
of the pixels i and j. In this paper, to simplify the calculation, gravitational mass of each object is set as
a unit value. →
→ Obviously, the distance dij is a dominant factor in Equation (10). The distance measure 1/
→
Zi −
→
Zj
used in Equation (1) will result in singularity. To avoid this problem and make
1
di,j smooth and finite,
the distance in GEDHSI is redefined by Equation (11), as follows: di,j(t) = 1 + (∥
→
Zi(t) −
→
Zj(t)∥
σ
)2,
(11) (11) where ∥
→
Zi(t) −
→
Zj(t)∥is the Euclidean distance between pixels i and j, and σ is an influence factor. Remote Sens. 2017, 9, 592 7 of 23 Then, by substituting Equations (10) and (11) into Equation (7), we can obtain the gravitational
force of pixel i exerted by the pixel j as follows: Then, by substituting Equations (10) and (11) into Equation (7), we can obtain the gravitational
force of pixel i exerted by the pixel j as follows: →
Fi,j(t) = −∇φi,j = 2G
MG
i (t) · MG
j (t)
σ
·
(
→
Zj(t) −
→
Zi(t))
(1 + (∥
→
Zi(t) −
→
Zj(t)∥/σ)2)2
. (1)
Calculation of the Acceleration (12) (12) Because the gravitational force obeys the superposition principle, for a dataset D that consists of
m pixels, the gravitational force of pixel i exerted by the dataset D is determined as the accumulated
force for each pixel within D as follows: →
Fi = ∑
j∈D
2G
MG
i · MG
j
σ
·
(
→
Zj −
→
Zi)
(1 + (∥
→
Zi −
→
Zj∥/σ)2)2
. (13) (13) According to Equation (13), the dataset D is a key factor for computing gravitational force. In general cases, all pixels are involved to compute the gravitational force. However, this will lead to
high computational time. According to Equation (13), the gravitational force attenuates toward zero
when the distance increases. Thus, for those pixels that exceed a pre-specified range, we can safely
assume that their gravitational forces are zero to simplify the computation. To this end, the dataset D can be constructed by D =
j
∥
→
Si −
→
Sj∥< r and ∥
→
Vi −
→
Vj∥< δ
,
(14) (14) where r is the radius of a local circular window in the spatial domain, δ is the radius in the spectral
domain, and j is the neighbor pixel around the pixel i. r and δ construct a local suprasphere in the
feature space. Here, to balance the effect of the spatial and spectral distance, the position of each pixel
is stretched in spectral domain by
→
V′
i =
→
Vi · r
δ. Thus, the influence factor σ is equal to r, and
→
Zi is
replaced by
→
Zi = (
→
V′
i ,
→
Si). All pixels inside the suprasphere compose the dataset D. i
According to Equation (9), in addition to resultant force, inertial mass is critical in the traveling
procedure. In the proposed method, the inertial mass MI
i (t) of pixel i, is defined as follows: MI
i (t) = ∑
j∈D
exp(−(∥
→
Zj(t) −
→
Zi(t)∥
σ
)2). (15) (15) Obviously, the inertial mass is capable of capturing the local information with self-adaptation
when traveling. Obviously, the inertial mass is capable of capturing the local information with self-adaptation
when traveling. Calculation of the Traveling Step (2)
Calculation of the Traveling Step (2)
Calculation of the Traveling Step (2)
Calculation of the Traveling Step After obtaining the acceleration, we can calculate the traveling step size using the law of motion
(Equation (16)). ∆
→
Zi(t) = 1
2
→ai(t)∆t2 +
→vi(t)∆t,
(16) (16) where
→v i(t) is the velocity of pixel i at time t. To make the pixel move directly toward the potential
center,
→v i(t) is considered zero in ∆t time. ∆t is the time interval in two adjacent iterations and is set
to 1. Thus, Equation (16) is replaced by Equation (17): where
→v i(t) is the velocity of pixel i at time t. To make the pixel move directly toward the potential
center,
→v i(t) is considered zero in ∆t time. ∆t is the time interval in two adjacent iterations and is set
to 1. Thus, Equation (16) is replaced by Equation (17): ∆
→
Zi(t) = 1
2
→ai(t). (17) (17) (3)
Location Updating (3)
Location Updating (3)
Location Updating Remote Sens. 2017, 9, 592 8 of 23 Based on the traveling step size, the location of the pixel i can be updated as follows: Based on the traveling step size, the location of the pixel i can be updated as follows: →
Zi(t + 1) =
→
Zi(t) + ∆
→
Zi(t) =
→
Zi(t) +
→
F(t)
2MI
i (t). (18) (18) The traveling procedure is an iterative procedure and will be repeated until the following stopping
criteria are met: (1) the maximum step size Smax(t) < ε where Smax(t) =
max
i∈[1,2,..,H×W](abs(
→
Zi(t)));
or (2) the iterations reach a maximum T. In this stage, each pixel moves along the direction of the
associated gravitational force. By traveling, they can find other, similar pixels, which remove the
influence of noise and preserves the structural detail. 4. Experimental Results and Discussion
T d
t
t th
ffi
d ffi
4. Experimental Results and Discussion To demonstrate the efficacy and efficiency of the proposed method, we carried out three groups
of experimental validation, including quantitative and qualitative evaluation and CPU runtime. Group 1, was designed to quantitatively compare the performances of different edge detectors by
using two artificial images, AI 1 and AI 2 (Section 4.3). Group 2 was designed to validate the
performance of the proposed approach on two real remote sensing HSIs and two ground scene HSIs
(Section 4.4). Group 3 was designed to test the computational efficiency of different edge detectors
using the two remote sensing HSIs (Section 4.5). Detailed introduction of the tested data sets used
in experiments and evaluation methods are given in Sections 4.1 and 4.2, respectively. In all experiments, all edge results were compared between the proposed method and four
To demonstrate the efficacy and efficiency of the proposed method, we carried out three groups of
experimental validation, including quantitative and qualitative evaluation and CPU runtime. Group 1,
was designed to quantitatively compare the performances of different edge detectors by using two
artificial images, AI 1 and AI 2 (Section 4.3). Group 2 was designed to validate the performance of
the proposed approach on two real remote sensing HSIs and two ground scene HSIs (Section 4.4). Group 3 was designed to test the computational efficiency of different edge detectors using the two
remote sensing HSIs (Section 4.5). Detailed introduction of the tested data sets used in experiments
and evaluation methods are given in Sections 4.1 and 4.2, respectively. other state-of-the-art multispectral edge detection methods, which include those from Di Zenzo [17],
Drewniok [19], RCMG [24] and SSSDE [30]. Di Zenzo initially used the Sobel operator as
channel-wise gradient technique [17], which is sensitive to noise. To improve the anti-noise anility,
we prefer to use the ANDD-based gradient [46,54] considering its superiority in noise robustness. It
is worth noting that the implementation codes of the comparative methods are directly taken from
the authors for fair and consistent comparison. We selected their optimized parameters by trial-error
methods. The parameters of GEDHSI were set as
0.0001
ε =
,
20
T =
,
4
r =
and
50
=
δ
in this
paper. The parameter σ was set to the same value as r as discussed in Section 3.3. 4. Experimental Results and Discussion
T d
t
t th
ffi
d ffi
4. Experimental Results and Discussion All the
experiments were conducted under the following settings: Matlab2013a, 64 bit Windows 7 system,
2.9 GHz Intel Pentium CPU with 6 GB RAM. 4.1. Test Datasets
In all experiments, all edge results were compared between the proposed method and four
other state-of-the-art multispectral edge detection methods, which include those from Di Zenzo [17],
Drewniok [19], RCMG [24] and SSSDE [30]. Di Zenzo initially used the Sobel operator as channel-wise
gradient technique [17], which is sensitive to noise. To improve the anti-noise anility, we prefer to use
the ANDD-based gradient [46,54] considering its superiority in noise robustness. It is worth noting that
the implementation codes of the comparative methods are directly taken from the authors for fair and
consistent comparison. We selected their optimized parameters by trial-error methods. The parameters
of GEDHSI were set as ε = 0.0001, T = 20, r = 4 and δ = 50 in this paper. The parameter σ was
set to the same value as r as discussed in Section 3.3. All the experiments were conducted under
the following settings: Matlab2013a, 64 bit Windows 7 system, 2.9 GHz Intel Pentium CPU with
6 GB RAM. 3.4. Edge Detection When the traveling stops, the image system reaches a stable equilibrium where similar pixels will
cluster together. Thereby, due to the relative positions, different pixels have various SPEs. The SPE of
each pixel can be calculated. For edge pixels, the SPE is relatively high, whereas for non-edge pixels,
the SPE is relatively low. Thus, we take the SPE of each pixel as the edge response. The calculation of
the SPE of each object is also restricted to the dataset D and using Equation (19). Φi = ∑
j∈D
ϕi,j = ∑
j∈D
−G
MG
j MG
i
(1 + (∥
→
Zi −
→
Zj∥/σ)2)
. (19) (19) Apparently, finding an appropriate threshold will classify the edge and non-edge pixels into two
categories and the edge will be finally detected. Apparently, finding an appropriate threshold will classify the edge and non-edge pixels into two
categories and the edge will be finally detected. According to Reference [35], a good edge detection method should keep a thin and accurate
localization of edges. To satisfy such requirements, information about edges’ directions is necessary
for non-maximum suppression. In accordance with Reference [49], we first project the SPE to the
horizontal and vertical directions in the spatial domain, as shown in Equation (20). ΦX
i = ∑
j∈D
ϕi,j ·
xj
q
(xj + yj)2 and ΦY
i = ∑
j∈D
φi,j ·
yj
q
(xj + yj)2 ,
(20) (20) (20) where ΦX
i
and ΦY
i are the SPE of pixel i projected onto the horizontal and vertical directions,
respectively. Then, the edge direction is estimated by Equation (21) as follows: where ΦX
i
and ΦY
i are the SPE of pixel i projected onto the horizontal and vertical directions,
respectively. Then, the edge direction is estimated by Equation (21) as follows: θi = arctan(ΦX
i /ΦY
i ). (21) (21) With Φ and θ, non-maximum suppression and automatic hysteresis thresholding [53] can be
applied to obtain the exact edge. Figure 2 depicts the flowchart of the proposed GEDHSI. With Φ and θ, non-maximum suppression and automatic hysteresis thresholding [53] can be
applied to obtain the exact edge. Figure 2 depicts the flowchart of the proposed GEDHSI. 9 of 23 Remote Sens. 2017, 9, 592 Figure 2. Flowchart of the proposed GEDHSI. Figure 2. Flowchart of the proposed GEDHSI. Figure 2. Flowchart of the proposed GEDHSI. Figure 2. Flowchart of the proposed GEDHSI. Six hypers
d
4.1. Test Datasets scenes are used in our experiments for performance assessment as listed in Table 1. The first two
artificial images are synthesized using the HYDRA [55] which were used to evaluate the
performance in dealing with background clutter and weak edges. The real HSIs include two remote
Six hyperspectral data sets in three groups that include two artificial ones and four real natural
scenes are used in our experiments for performance assessment as listed in Table 1. The first two
artificial images are synthesized using the HYDRA [55] which were used to evaluate the performance 10 of 23 Remote Sens. 2017, 9, 592 in dealing with background clutter and weak edges. The real HSIs include two remote sensing HSIs
and two ground scene HSIs. The first remote sensing HSI is Farmland image is an AVIRIS flight line
over a farmland of east-central Indiana [56]. Another remote sensing HSI is a HYDICE flight line over
the Washington DC Mall [57]. The two ground scene HSIs are chosen from the Foster’s HSI dataset [58]
to further investigate the performance of the proposed method on real ground scenes. All these images
are suitable to evaluate the performance of connectivity, edge resolution and the ability to suppress
false edges. Remote Sens. 2017, 9, 592
10 of 23
sensing HSIs and two ground scene HSIs. The first remote sensing HSI is Farmland image is an
AVIRIS flight line over a farmland of east-central Indiana [56]. Another remote sensing HSI is a
HYDICE flight line over the Washington DC Mall [57]. The two ground scene HSIs are chosen from
the Foster’s HSI dataset [58] to further investigate the performance of the proposed method on real
ground scenes. All these images are suitable to evaluate the performance of connectivity, edge
l ti
d th
bilit
t
f l
d Table 1. Properties of the data sets used in experiments. Datasets
Data
Source
Characteristic
Spatial
Resolution
No. of
Bands
Size (Pixels)
1
AI 1
Artificial
background clutter
N/A
224
180 × 180
2
AI 2
weak edge, spectral mixture
224
180 × 180
3
Farmland
Remote
sensing
straight lined boundary, textured
High
224
400 × 200
4
Washington DC Mall
many tiny objects, cluttered
191
466 × 307
5
Foster’s Scene 5
Ground
scene
man-made objects, textured wall,
desk, ball, blocks, etc. Very
high
31
820 × 820
6
Foster’s Scene 7
textured wall, grass, shadow, etc. Six hypers
d
4.1. Test Datasets 31
690 × 425
4.2. Evaluation Methods
resolution and the ability to suppress false edges. Table 1. Properties of the data sets used in experiments. Datasets
Data
Source
Characteristic
Spatial
Resolution
No. of
Bands
Size
(Pixels)
1
AI 1
Artificial
background clutter
N/A
224
180 × 180
2
AI 2
weak edge, spectral mixture
224
180 × 180
3
Farmland
Remote
sensing
straight lined boundary, textured
High
224
400 × 200
4
Washington DC Mall
many tiny objects, cluttered
191
466 × 307
5
Foster’s Scene 5
Ground
scene
man-made objects, textured wall,
desk, ball, blocks, etc. Very high
31
820 × 820
6
Foster’s Scene 7
textured wall, grass, shadow, etc. 31
690 × 425
4.2. Evaluation Methods
M
t d
d t
ti
t
h i
tili
thi
i
d bi
i
ti
t
bt i
th fi
l d
lt Table 1. Properties of the data sets used in experiments. y
pp
g
Table 1 Properties of the data sets used in experiments Table 1. Properties of the data sets used in experiments. y
pp
g 4.2. Evaluation Methods
4.2. Evaluation Methods
M
t d
d t
ti Most edge detection techniques utilize thinning and binarization to obtain the final edge
results [59]. In this study, we put all these edge strength maps into the same thinning process. In this process, a pixel is only considered as an edge if it is a local maximum in the horizontal or
vertical direction. Then, we applied the automatic threshold selection and hysteresis thresholding
technique [53] on the edge strength maps to generate the binary edges. Most edge detection techniques utilize thinning and binarization to obtain the final edge results
[59]. In this study, we put all these edge strength maps into the same thinning process. In this
process, a pixel is only considered as an edge if it is a local maximum in the horizontal or vertical
direction. Then, we applied the automatic threshold selection and hysteresis thresholding technique
[53] on the edge strength maps to generate the binary edges. We investigated edge detection results with both subjective and quantitative evaluations. For We investigated edge detection results with both subjective and quantitative evaluations. A. Experiment on AI 1
A. Experiment on AI 1 A channel in the AI 1 dataset: (a) The content of the synthetic image without noise (object is
located in the middle); and (b) a corrupted image with SNR = 16 dB (where SNR stands Signal Noise
Ratio) in the object region and SNR = 0.2 dB in the background region. The dark color indicates low
intensity. Figure 4. A channel in the AI 1 dataset: (a) The content of the synthetic image without noise (object
is located in the middle); and (b) a corrupted image with SNR = 16 dB (where SNR stands Signal
Noise Ratio) in the object region and SNR = 0.2 dB in the background region. The dark color indicates
low intensity. Figure 5 shows an edge-detection example for AI 1, where the background noise is SNR = 0.2
dB. The edge-strength maps and corresponding binary edge maps were shown in the first row and
the second row, respectively, in Figure 5. For Di Zenzo’s method, the strength of the edge pixels is
slightly larger than the background (Figure 5b). Although we can set a pair of thresholds to
separate edge and non-edge pixels, severe false positive detections cannot be avoided, as shown in
Figure 5g. For the RCMG, the strength of noisy pixels is larger than that of edge pixels, as shown in
Figure 5c; thus, the boundaries of the object were concealed from the noisy background (Figure 5h). According to Figure 5d,i, the results of Drewniok’s method are better than those of Di Zenzo’s and
the RCMG method, but they blurred the edges to some extent (Figure 5d). Therefore, the object’s
edges were blurred with burrs, as shown in Figure 5i. For the SSSDE, the strength of almost all the
edge pixels is larger than that of the noise pixels as depicted in Figure 5e. However, some false
edges are detected in the object, as shown in Figure 5j. Compared to the other four methods, the
better performance of the presented detector is clearly perceivable, and the strength of the edge
pixels is larger than that of the noise pixels (Figure 5a). It is therefore easy to set appropriate
thresholds to separate the edge and non-edge pixels, as shown in Figure 5e. Figure 5 shows an edge-detection example for AI 1, where the background noise is SNR = 0.2 dB. A. Experiment on AI 1
A. Experiment on AI 1 In this experiment, we investigated the behaviors of the five edge-detection methods when the
objects in an image are embedded by background clutter. The tested image AI 1 is composed of one
object (in the middle) and a background, where the object is located between column 60 and column
120. Thus, edge pixels are located at columns 60 and 120. To simulate different levels of background
clutter, we added Gaussian noises with various Signal Noise Ratio (SNR) to AI 1. The object noise is
fixed to SNR = 16 dB, whereas the background noise varies from 0.1 to 3.0 dB. Figure 4a shows the
content of the synthetic image without noise. Figure 4b shows a channel for the background with
SNR = 0.2 dB. In this experiment, we investigated the behaviors of the five edge-detection methods when the
objects in an image are embedded by background clutter. The tested image AI 1 is composed of one
object (in the middle) and a background, where the object is located between column 60 and column
120. Thus, edge pixels are located at columns 60 and 120. To simulate different levels of background
clutter, we added Gaussian noises with various Signal Noise Ratio (SNR) to AI 1. The object noise is
fixed to SNR = 16 dB, whereas the background noise varies from 0.1 to 3.0 dB. Figure 4a shows the
content of the synthetic image without noise. Figure 4b shows a channel for the background with
SNR = 0.2 dB. (a)
(b)
Figure 4. A channel in the AI 1 dataset: (a) The content of the synthetic image without noise (object is
located in the middle); and (b) a corrupted image with SNR = 16 dB (where SNR stands Signal Noise
Ratio) in the object region and SNR = 0.2 dB in the background region. The dark color indicates low
intensity. Figure 4. A channel in the AI 1 dataset: (a) The content of the synthetic image without noise (object
is located in the middle); and (b) a corrupted image with SNR = 16 dB (where SNR stands Signal
Noise Ratio) in the object region and SNR = 0.2 dB in the background region. The dark color indicates
low intensity. (b) (a) (a) (b) Figure 4. Six hypers
d
4.1. Test Datasets 11 of 23 11 of 23 Remote Sens. 2017, 9, 592 4.3. Evaluation on Artificial Datasets In practice, two cases make edge detection a challenge. The first one is objects that are embedded
by background clutter, and the second is weak edges caused by spectral mixture or just appearing in a
small subset of bands. As a result, we constructed two artificial images, AI 1 and AI 2, to evaluate the
performance of our proposed edge detection approach under these two cases. Remote Sens. 2017, 9, 592
11 of 23 Six hypers
d
4.1. Test Datasets For quantitative evaluation, as shown in Figure 3, pixels in the candidate edge image were classified as
TP (True Positive), TN (True Negative), FP (False Positive) and FN (False Negative), from which the
precision and recall evolutions could be obtained as Prec and Rec. The Prec shows the correct rate of
the detected edges while the Rec implies the sensitivity of an edge detector to true edges. Because the
two evaluation methods may be unfaithful when the number of objects and backgrounds has a great
different, according to Reference [25], the F-measure derived from Prec and Rec is used to evaluate the
overall performance. We investigated edge detection results with both subjective and quantitative evaluations. For
quantitative evaluation, as shown in Figure 3, pixels in the candidate edge image were classified as
TP (True Positive), TN (True Negative), FP (False Positive) and FN (False Negative), from which the
precision and recall evolutions could be obtained as Prec and Rec. The Prec shows the correct rate of
the detected edges while the Rec implies the sensitivity of an edge detector to true edges. Because
the two evaluation methods may be unfaithful when the number of objects and backgrounds has a
great different, according to Reference [25], the F-measure derived from Prec and Rec is used to
evaluate the overall performance. Fα =
Prec × Rec
α · Prec + (1 −α) · Rec,
(22)
,
(1- )
Prec Rec
F
Prec
Rec
α
α
α
×
=
⋅
+
⋅
(22) (22)
(22) where α ∈[0,1] is a parameter weighing the contributions of Prec and Rec. In this paper, α was set to
0.5 to balance the Prec and Rec. As a statistical error measure, F-measure works well for FP and FN,
where a good detection has higher F-measure value. where α ∈[0,1] is a parameter weighing the contributions of Prec and Rec. In this paper, α was set
to 0.5 to balance the Prec and Rec. As a statistical error measure, F-measure works well for FP and
FN, where a good detection has higher F-measure value. Figure 3. Confusion matrix for the edge detection problem. Figure 3. Confusion matrix for the edge detection problem. Figure 3. Confusion matrix for the edge detection problem. Figure 3. Confusion matrix for the edge detection problem. Figure 3. Confusion matrix for the edge detection problem. Figure 3. Confusion matrix for the edge detection problem. A. Experiment on AI 1
A. Experiment on AI 1 The edge-strength maps and corresponding binary edge maps were shown in the first row and the
second row, respectively, in Figure 5. For Di Zenzo’s method, the strength of the edge pixels is slightly
larger than the background (Figure 5b). Although we can set a pair of thresholds to separate edge and
non-edge pixels, severe false positive detections cannot be avoided, as shown in Figure 5g. For the
RCMG, the strength of noisy pixels is larger than that of edge pixels, as shown in Figure 5c; thus,
the boundaries of the object were concealed from the noisy background (Figure 5h). According to
Figure 5d,i, the results of Drewniok’s method are better than those of Di Zenzo’s and the RCMG
method, but they blurred the edges to some extent (Figure 5d). Therefore, the object’s edges were
blurred with burrs, as shown in Figure 5i. For the SSSDE, the strength of almost all the edge pixels is
larger than that of the noise pixels as depicted in Figure 5e. However, some false edges are detected
in the object, as shown in Figure 5j. Compared to the other four methods, the better performance of
the presented detector is clearly perceivable, and the strength of the edge pixels is larger than that of
the noise pixels (Figure 5a). It is therefore easy to set appropriate thresholds to separate the edge and
non-edge pixels, as shown in Figure 5e. 12 of 23
priate Remote Sens. 2017, 9, 592
pixels is larger tha e
o
o epa a e
e e ge a
o
e ge pi e , a
o
i
igu e e
(a)
(b)
(c)
(d)
(e)
Remote Sens. 2017, 9, 592
12 of 23
(f)
(g)
(h)
(i)
(j)
Figure 5. Edge-strength maps and corresponding binary edges of AI 1 with SNR = 0.2 dB. Edge-strength maps of: (a) GEDHSI; (b) Di Zenzo’s method; (c) the Robust Color Morphological
Gradient (RCMG) method; (d) Drewniok’s method; and (e) the modified Simultaneous Spectral/Spatial
Detection of Edges (SSSDE) method. (f–j) Binary edge maps of corresponding results of (a–e). Figure 5. Edge-strength maps and corresponding binary edges of AI 1 with SNR = 0.2 dB. Edge-strength
maps of: (a) GEDHSI; (b) Di Zenzo’s method; (c) the Robust Color Morphological Gradient (RCMG)
method; (d) Drewniok’s method; and (e) the modified Simultaneous Spectral/Spatial Detection of
Edges (SSSDE) method. (f–j) Binary edge maps of corresponding results of (a–e). A. Experiment on AI 1
A. Experiment on AI 1 mote Sens. 2017, 9, 592
12 of 2
(f)
(g)
(h)
(i)
(j)
Figure 5. Edge-strength maps and corresponding binary edges of AI 1 with SNR = 0.2 dB. Edge-strength maps of: (a) GEDHSI; (b) Di Zenzo’s method; (c) the Robust Color Morphological
Gradient (RCMG) method; (d) Drewniok’s method; and (e) the modified Simultaneous Spectral/Spatial
Detection of Edges (SSSDE) method. (f–j) Binary edge maps of corresponding results of (a–e). (b) (c) (b) (d) (e) (c) (a)
ens. 2
s. 201 (f)
(f) (i)
(i) (h)
(h) (g)
(g) (f)
f) (i)
(i (g)
(g) (j) (h)
(h) Figure 5. Edge-strength maps and corresponding binary edges of AI 1 with SNR = 0.2 dB. Edge-strength maps of: (a) GEDHSI; (b) Di Zenzo’s method; (c) the Robust Color Morphological
Gradient (RCMG) method; (d) Drewniok’s method; and (e) the modified Simultaneous Spectral/Spatial
Detection of Edges (SSSDE) method. (f–j) Binary edge maps of corresponding results of (a–e). Figure 5. Edge-strength maps and corresponding binary edges of AI 1 with SNR = 0.2 dB. Edge-strength
maps of: (a) GEDHSI; (b) Di Zenzo’s method; (c) the Robust Color Morphological Gradient (RCMG)
method; (d) Drewniok’s method; and (e) the modified Simultaneous Spectral/Spatial Detection of
Edges (SSSDE) method. (f–j) Binary edge maps of corresponding results of (a–e). Figure 5. Edge-strength maps and corresponding binary edges of AI 1 with SNR = 0.2 dB. Edge-strength maps of: (a) GEDHSI; (b) Di Zenzo’s method; (c) the Robust Color Morphological
Gradient (RCMG) method; (d) Drewniok’s method; and (e) the modified Simultaneous Spectral/Spatial
Detection of Edges (SSSDE) method. (f–j) Binary edge maps of corresponding results of (a–e). under different background-noise levels from 0.1 dB to 3 dB. From Figure 6b we can find that the
GEDHSI produces better Rec values in almost all the noise levels. This indicates the successful
detection of almost all the edges, which is also confirmed by Figure 5f. Although the Prec values
obtained by the GEDHSI are occasionally slightly worse than that of Di Zenzo’s method, it increases
quickly as SNR raised as shown in Figure 6a. For the F-measure in Figure 6c, we can see that under
high background-noise levels (SNR approximately 0.1 dB), the GEDHSI produced an F-measure
value larger than 0.9, whereas the F-measure values produced by the other four methods are all less
than 0.7. A. Experiment on AI 1
A. Experiment on AI 1 When the SNR surpasses 0.9, the F-measure value for the GEDHSI reaches approximately
1.0, whereas for the other four methods the F-measure values increased slowly. It is obvious that the
other four methods are weaker than the GEDHSI in addressing such severe background clutter. With severe noise interference, the large difference between a noisy pixel and its neighbors usually
leads to a high gradient magnitude for that noisy pixel, a magnitude even larger than that of the
true edge pixels. As a result, this will inevitably cause incorrect edges because of failing to make the
full use of the spatial information of the image, especially inadequate consideration of the
relationships among the pixels within a local window. The robustness to the background clutter of the GEDHSI was further demonstrated in Figure 6,
under different background-noise levels from 0.1 dB to 3 dB. From Figure 6b we can find that the
GEDHSI produces better Rec values in almost all the noise levels. This indicates the successful detection
of almost all the edges, which is also confirmed by Figure 5f. Although the Prec values obtained by
the GEDHSI are occasionally slightly worse than that of Di Zenzo’s method, it increases quickly
as SNR raised as shown in Figure 6a. For the F-measure in Figure 6c, we can see that under high
background-noise levels (SNR approximately 0.1 dB), the GEDHSI produced an F-measure value
larger than 0.9, whereas the F-measure values produced by the other four methods are all less than
0.7. When the SNR surpasses 0.9, the F-measure value for the GEDHSI reaches approximately 1.0,
whereas for the other four methods the F-measure values increased slowly. It is obvious that the other
four methods are weaker than the GEDHSI in addressing such severe background clutter. With severe
noise interference, the large difference between a noisy pixel and its neighbors usually leads to a high
gradient magnitude for that noisy pixel, a magnitude even larger than that of the true edge pixels. As a
result, this will inevitably cause incorrect edges because of failing to make the full use of the spatial
information of the image, especially inadequate consideration of the relationships among the pixels
within a local window. The robustness to the background clutter of the GEDHSI was further demonstrated in Figure 6,
under different background-noise levels from 0.1 dB to 3 dB. A. Experiment on AI 1
A. Experiment on AI 1 From Figure 6b we can find that the
GEDHSI produces better Rec values in almost all the noise levels. This indicates the successful
detection of almost all the edges, which is also confirmed by Figure 5f. Although the Prec values
obtained by the GEDHSI are occasionally slightly worse than that of Di Zenzo’s method, it increases
quickly as SNR raised as shown in Figure 6a. For the F-measure in Figure 6c, we can see that under
high background-noise levels (SNR approximately 0.1 dB), the GEDHSI produced an F-measure
value larger than 0.9, whereas the F-measure values produced by the other four methods are all less
than 0.7. When the SNR surpasses 0.9, the F-measure value for the GEDHSI reaches approximately
1.0, whereas for the other four methods the F-measure values increased slowly. It is obvious that the
other four methods are weaker than the GEDHSI in addressing such severe background clutter. With severe noise interference, the large difference between a noisy pixel and its neighbors usually
leads to a high gradient magnitude for that noisy pixel, a magnitude even larger than that of the
true edge pixels. As a result, this will inevitably cause incorrect edges because of failing to make the
full use of the spatial information of the image, especially inadequate consideration of the
relationships among the pixels within a local window. (a)
(b)
(c)
Figure 6. Performance evaluation on AI 1 under various noise levels. Performance of: (a) Precision;
(b) Recall; and (c) F-measure. B. Experiment on AI 2
In this experiment we analyze the behaviors of the five edge detection methods in dealing with
(a)
(b)
(c)
Figure 6. Performance evaluation on AI 1 under various noise levels. Performance of: (a) Precision;
(b) Recall; and (c) F-measure. Figure 6. Performance evaluation on AI 1 under various noise levels. Performance of: (a) Precision;
(b) Recall; and (c) F-measure. (b)
on on AI 1 under various noise levels
(b) (c)
erformance of: (a) Precision;
(c) (a)
Figure 6. Performance evalua
(b) Recall; and (c) F-measure. (a) (b) )
(c) ll; and
(a) . Experiment on AI 2
Figure 6. Performance evaluation on AI 1 under various noise levels. Performance of: (a) Precision;
(b) Recall; and (c) F-measure. Figure 6. Performance evaluation on AI 1 under various noise levels. Performance of: (a) Precision;
(b) Recall; and (c) F-measure. weak edges between o
objects or edges app
B. Experiment on AI 2
B. Experiment on AI 2 objects or edges appearing in only a small subset of bands [17]. To tackle this problem, we
generated a well-designed image AI 2 with a spatial dimension of 180 × 180 in nine regions. The size
of each region is 60 × 60 pixels. We first selected three types of endmembers from the USGS Spectral
Lib
[60]
h
i
Fi
7
In this experiment, we analyze the behaviors of the five edge-detection methods in dealing with
weak edges between objects. For HSIs, weak edges are usually caused by spectral mixture between
objects or edges appearing in only a small subset of bands [17] To tackle this problem we
In this experiment, we analyze the behaviors of the five edge-detection methods in dealing with
weak edges between objects. For HSIs, weak edges are usually caused by spectral mixture between 13 of 23 Remote Sens. 2017, 9, 592 objects or edges appearing in only a small subset of bands [17]. To tackle this problem, we generated a
well-designed image AI 2 with a spatial dimension of 180 × 180 in nine regions. The size of each region
is 60 × 60 pixels. We first selected three types of endmembers from the USGS Spectral Library [60],
as shown in Figure 7a. Remote Sens. 2017, 9, 592
13 of 23 (a)
(b)
(c)
Figure 7. Materials for AI 2: (a) curve of endmembers; (b) diagram of AI 2; and (c) a band of AI 2
with SNR = 10 dB. Figure 7. Materials for AI 2: (a) curve of endmembers; (b) diagram of AI 2; and (c) a band of AI 2 with
SNR = 10 dB. (b) (a) (c) (b) (a) (c) Figure 7. Materials for AI 2: (a) curve of endmembers; (b) diagram of AI 2; and (c) a band of AI 2
with SNR = 10 dB. Figure 7. Materials for AI 2: (a) curve of endmembers; (b) diagram of AI 2; and (c) a band of AI 2 with
SNR = 10 dB. According to Figure 7a, the two endmembers A and C are found to be very similar in the range
of 0.6 µm to 1.4 µm, whereas the two endmembers B and C are very similar in the range of 1.5 µm
to 2.1 µm. A diagram of AI 2 is shown in Figure 7b, where the symbol of each region denotes which
endmembers the region is formed. weak edges between o
objects or edges app
B. Experiment on AI 2
B. Experiment on AI 2 For example, the “A” of Region A means the region was
generated only using endmember A, Region AB was generated by using endmembers A and B,
whereas Region BA was composed of endmembers B and A, etc. Except for the three regions
located in the principal diagonal, all regions were mixed by different endmembers. The mixed ratio
was fixed as 0.4:0.6 for these regions. We then added Gaussian noise to AI 2 and result in a varying
SNR between 1 and 30 dB. Figure 7c shows an example band with SNR = 10 dB. Due to the spectral
mixture and spectral similarity in many bands, there are several weak edges. Under such
conditions, weak edges may be easily vanished in noise when white Gaussian noise was added. For the noise-degraded AI 2 with a SNR of 10 dB Figure 8 shows the edge-strength maps (the
According to Figure 7a, the two endmembers A and C are found to be very similar in the range
of 0.6 µm to 1.4 µm, whereas the two endmembers B and C are very similar in the range of 1.5 µm
to 2.1 µm. A diagram of AI 2 is shown in Figure 7b, where the symbol of each region denotes which
endmembers the region is formed. For example, the “A” of Region A means the region was generated
only using endmember A, Region AB was generated by using endmembers A and B, whereas Region
BA was composed of endmembers B and A, etc. Except for the three regions located in the principal
diagonal, all regions were mixed by different endmembers. The mixed ratio was fixed as 0.4:0.6 for
these regions. We then added Gaussian noise to AI 2 and result in a varying SNR between 1 and
30 dB. Figure 7c shows an example band with SNR = 10 dB. Due to the spectral mixture and spectral
similarity in many bands, there are several weak edges. Under such conditions, weak edges may be
easily vanished in noise when white Gaussian noise was added. For the noise-degraded AI 2 with a SNR of 10 dB, Figure 8 shows the edge-strength maps (the
first row) and their corresponding binary edge maps (the second row) generated by the five
methods. weak edges between o
objects or edges app
B. Experiment on AI 2
B. Experiment on AI 2 As seen from the edge-strength maps generated by Di Zenzo’s method (Figure 8b), the
RCMG (Figure 8c), Drewniok’s method (Figure 8d) and SSSDE method (Figure 8e), the strength of
upper horizontal and left vertical edge pixels is close to that of the noisy pixels. Therefore, it is
difficult to set appropriate thresholds for these edge-strength maps. According to Figure 8g, Di Zenzo’s
method seems to produce discontinuous edges and severe false edges. Although RCMG is slightly better
than Di Zenzo’s as shown in Figure 8h, the results still contains some spurious edges. The similar
problems exist in the results of SSSDE as illustrated in Figure 8j through the SSSDE generate much better
results than RCMG. Although Drewniok’s method has removed almost all noise, the edges are hackly
due to the displacement caused by Gaussian smoothing in each band. For comparison, the intensity of
the edge pixels is dramatically larger than that of the noisy pixels, as shown in Figure 8a. Thus,
thresholding can easily classify edge and non-edge pixels, as shown in Figure 8f. For the noise-degraded AI 2 with a SNR of 10 dB, Figure 8 shows the edge-strength maps
(the first row) and their corresponding binary edge maps (the second row) generated by the five
methods. As seen from the edge-strength maps generated by Di Zenzo’s method (Figure 8b), the
RCMG (Figure 8c), Drewniok’s method (Figure 8d) and SSSDE method (Figure 8e), the strength of
upper horizontal and left vertical edge pixels is close to that of the noisy pixels. Therefore, it is difficult
to set appropriate thresholds for these edge-strength maps. According to Figure 8g, Di Zenzo’s method
seems to produce discontinuous edges and severe false edges. Although RCMG is slightly better
than Di Zenzo’s as shown in Figure 8h, the results still contains some spurious edges. The similar
problems exist in the results of SSSDE as illustrated in Figure 8j through the SSSDE generate much
better results than RCMG. Although Drewniok’s method has removed almost all noise, the edges are
hackly due to the displacement caused by Gaussian smoothing in each band. For comparison, the
intensity of the edge pixels is dramatically larger than that of the noisy pixels, as shown in Figure 8a. Thus, thresholding can easily classify edge and non-edge pixels, as shown in Figure 8f. Figure 9 shows the performance comparison of the five methods under noise from 1.0 dB to 30 dB. weak edges between o
objects or edges app
B. Experiment on AI 2
B. Experiment on AI 2 As seen in Figure 9c, due to the simplicity of the tested image, all five methods produced good results
when the level of noise is low (SNR > 10 db). With the increase of noise level, especially when SNR < 7 db,
the F-measure values of the four compared algorithms decreased rapidly while the proposed GEDHSI
still produce much better results. Furthermore, GEDHSI obtained better results on both Prec and Rec
values than Di Zenzo’s, RCMG and SSSDE methods as illustrated in Figure 9a,b. This demonstrates
the GEDHSI can detect the edges in the HSIs with higher accuracy in the complicated environments. For Drewniok’s method, although its Rec values are better than that of the GEDHSI to some extent, 14 of 23
etter
milar Remote Sens. 2017, 9, 592
p
than Di Zenzo’s as its Prec values are remarkably worse. This implies that Drewniok’s method is easily affected by the
noise, which is also confirmed by the F-measure shown in Figure 9c. Moreover, the jagged boundaries
between different edges in Figure 8i also confirm that Drewniok’s method produces many false edges. To this end, we can reasonably conclude that GEDHSI is more robust to severe noise and weak edges
than the other four methods. results than RCMG. Although Drewniok s method has removed almost all noise, the edges are hackly
due to the displacement caused by Gaussian smoothing in each band. For comparison, the intensity of
the edge pixels is dramatically larger than that of the noisy pixels, as shown in Figure 8a. Thus,
thresholding can easily classify edge and non-edge pixels, as shown in Figure 8f. (a)
(b)
(c)
(d)
(e)
Remote Sens. 2017, 9, 592
14 of 23
(f)
(g)
(h)
(i)
(j)
Figure 8. Edge-strength maps and corresponding binary edges of AI 2 with SNR = 10 dB. Edge-strength maps of: (a) GEDHSI; (b) Di Zenzo’s method; (c) the RCMG method; (d) Drewniok’s
method; and (e) SSSDE method. (f–j) Binary edge maps of corresponding results of (a–e) by
applying automatic hysteresis thresholding and thinning processes. Figure 8. Edge-strength maps and corresponding binary edges of AI 2 with SNR = 10 dB. Edge-strength
maps of: (a) GEDHSI; (b) Di Zenzo’s method; (c) the RCMG method; (d) Drewniok’s method; and
(e) SSSDE method. (f–j) Binary edge maps of corresponding results of (a–e) by applying automatic
hysteresis thresholding and thinning processes. (f)
(g)
(h)
(i)
(j)
Figure 8. weak edges between o
objects or edges app
B. Experiment on AI 2
B. Experiment on AI 2 20
igure (d)
(e)
(i)
(j)
methods under noise from 1.0 dB to 30
image, all five methods produced good
h the increase of noise level, especially
red algorithms decreased rapidly while
Furthermore, GEDHSI obtained better
G and SSSDE methods as illustrated in
edges in the HSIs with higher accuracy
although its Rec values are better than
k bl
Thi
i
li
th t (e)
(j)
er noise from 1.0 dB to 30
methods produced good
of noise level, especially
decreased rapidly while
GEDHSI obtained better
methods as illustrated in
SIs with higher accuracy
Rec values are better than
Thi
i
li
th t (a)
(f)
Figure 9 shows
dB. As seen in Figu
results when the le
when SNR < 7 db,
the proposed GED
results on both Pre
Figure 9a,b. This de
in the complicated
that of the GEDH (g)
s the performance co
re 9c, due to the sim
vel of noise is low
the F-measure value
HSI still produce m
c and Rec values tha
emonstrates the GED
environments. For D
SI t
t
t (h)
omparison of the five
mplicity of the tested
(SNR > 10 db). With
es of the four compar
much better results. an Di Zenzo’s, RCMG
DHSI can detect the
Drewniok’s method,
it
P
l (h)
ok’s (f)
com (j)
bet (i)
gh it (g)
nme Figure 8. Edge-strength maps and corresponding binary edges of AI 2 with SNR = 10 dB. Edge-strength maps of: (a) GEDHSI; (b) Di Zenzo’s method; (c) the RCMG method; (d) Drewniok’s
method; and (e) SSSDE method. (f–j) Binary edge maps of corresponding results of (a–e) by
applying automatic hysteresis thresholding and thinning processes. Figure 8. Edge-strength maps and corresponding binary edges of AI 2 with SNR = 10 dB. Edge-strength
maps of: (a) GEDHSI; (b) Di Zenzo’s method; (c) the RCMG method; (d) Drewniok’s method; and
(e) SSSDE method. (f–j) Binary edge maps of corresponding results of (a–e) by applying automatic
hysteresis thresholding and thinning processes. y
p
Drewniok’s method is easily affected by the noise, which is also confirmed by the F-measure shown
n Figure 9c. Moreover, the jagged boundaries between different edges in Figure 8i also confirm
hat Drewniok’s method produces many false edges. To this end, we can reasonably conclude tha
GEDHSI is more robust to severe noise and weak edges than the other four methods. weak edges between o
objects or edges app
B. Experiment on AI 2
B. Experiment on AI 2 s under noise from 1.0 dB to 30
ll five methods produced good
rease of noise level, especially
rithms decreased rapidly while
more, GEDHSI obtained better
SSDE methods as illustrated in
the HSIs with higher accuracy
h its Rec values are better than
ably worse. This implies that
irmed by the F-measure shown
(c) Figure 9 shows the performance comparison of the five methods under noise from 1.0 dB to 30
dB. As seen in Figure 9c, due to the simplicity of the tested image, all five methods produced good
results when the level of noise is low (SNR > 10 db). With the increase of noise level, especially
when SNR < 7 db, the F-measure values of the four compared algorithms decreased rapidly while
the proposed GEDHSI still produce much better results. Furthermore, GEDHSI obtained better
results on both Prec and Rec values than Di Zenzo’s, RCMG and SSSDE methods as illustrated in
Figure 9a,b. This demonstrates the GEDHSI can detect the edges in the HSIs with higher accuracy
in the complicated environments. For Drewniok’s method, although its Rec values are better than
that of the GEDHSI to some extent, its Prec values are remarkably worse. This implies that
Drewniok’s method is easily affected by the noise, which is also confirmed by the F-measure shown
in Figure 9c. Moreover, the jagged boundaries between different edges in Figure 8i also confirm
that Drewniok’s method produces many false edges. To this end, we can reasonably conclude that
GEDHSI is more robust to severe noise and weak edges than the other four methods. (a)
(b)
(c)
Figure 9. Performance evaluation on AI 2 under various noise levels: (a) precision; (b) recall; and (c)
F-measure. Figure 9. Performance evaluation on AI 2 under various noise levels: (a) precision; (b) recall;
and (c) F-measure. nce comparison of the five method
he simplicity of the tested image, a
s low (SNR > 10 db). With the in
values of the four compared algo
uce much better results. Furtherm
ues than Di Zenzo’s, RCMG and S
e GEDHSI can detect the edges in
. For Drewniok’s method, althoug
xtent, its Prec values are remark
ted by the noise, which is also con
(b) Figure 9 shows the performa
dB. weak edges between o
objects or edges app
B. Experiment on AI 2
B. Experiment on AI 2 As seen in Figure 9c, due to t
results when the level of noise i
when SNR < 7 db, the F-measure
the proposed GEDHSI still prod
results on both Prec and Rec valu
Figure 9a,b. This demonstrates th
in the complicated environments
that of the GEDHSI to some e
Drewniok’s method is easily affec
(a) This
F-me
(c) valu
e, wh
(b) GEDH
ethod
(a) n Figure 9c. Moreover, the jagged boundaries between different edges in Figure 8i also confirm
hat Drewniok’s method produces many false edges. To this end, we can reasonably conclude that
EDHSI is more robust to severe noise and weak edges than the other four methods
Figure 9. Performance evaluation on AI 2 under various noise levels: (a) precision; (b) recall; and (c)
F-measure. Figure 9. Performance evaluation on AI 2 under various noise levels: (a) precision; (b) recall;
and (c) F-measure. 4.4.1. Experiments on Remote Sensing Datasets
F
t
i
i
d
t
th
4.4.1. Experiments on Remote Sensing Datasets (a)
(b)
(c)
For remote sensing images, due to the complexity of land cover based surface feature,
illumination and atmospheric effects as well as spectral mixing of pixels, edges are always
deteriorated for proper detection. More seriously, there usually exist some noisy bands in remote
sensing HSIs, which make edge detection very difficult. To validate the performance of edge
detection on remote sensing HSIs, we selected two remote sensing HSIs, the Farmland and
Washington DC Mall image as shown in Figures 10 and 12, to conduct experiments in this section. A E
i
t
th F
l
d HSI
For remote sensing images, due to the complexity of land cover based surface feature, illumination
and atmospheric effects as well as spectral mixing of pixels, edges are always deteriorated for proper
detection. More seriously, there usually exist some noisy bands in remote sensing HSIs, which make
edge detection very difficult. To validate the performance of edge detection on remote sensing HSIs,
we selected two remote sensing HSIs, the Farmland and Washington DC Mall image as shown in
Figures 10 and 12, to conduct experiments in this section. weak edges between o
objects or edges app
B. Experiment on AI 2
B. Experiment on AI 2 Edge-strength maps and corresponding binary edges of AI 2 with SNR = 10 dB. Edge-strength maps of: (a) GEDHSI; (b) Di Zenzo’s method; (c) the RCMG method; (d) Drewniok’s
method; and (e) SSSDE method. (f–j) Binary edge maps of corresponding results of (a–e) by
applying automatic hysteresis thresholding and thinning processes. Figure 9 shows the performance comparison of the five methods under noise from 1.0 dB to 30
dB. As seen in Figure 9c, due to the simplicity of the tested image, all five methods produced good
results when the level of noise is low (SNR > 10 db). With the increase of noise level, especially
when SNR < 7 db, the F-measure values of the four compared algorithms decreased rapidly while
the proposed GEDHSI still produce much better results. Furthermore, GEDHSI obtained better
results on both Prec and Rec values than Di Zenzo’s, RCMG and SSSDE methods as illustrated in
Figure 9a,b. This demonstrates the GEDHSI can detect the edges in the HSIs with higher accuracy
in the complicated environments. For Drewniok’s method, although its Rec values are better than
that of the GEDHSI to some extent, its Prec values are remarkably worse. This implies that
Drewniok’s method is easily affected by the noise, which is also confirmed by the F-measure shown
in Figure 9c. Moreover, the jagged boundaries between different edges in Figure 8i also confirm
that Drewniok’s method produces many false edges. To this end, we can reasonably conclude that
GEDHSI is more robust to severe noise and weak edges than the other four methods. (e)
14 of 23
(j)
SNR = 10 dB. (d) Drewniok’s
ults of (a–e) by
se from 1 0 dB to 30 (d)
(i)
edges of AI 2 with
(c) the RCMG method;
of corresponding resu
esses. methods under nois (a)
Remote Sens. 2017, 9, 592
(f)
Figure 8. Edge
Edge-strength m
method; and (e
applying autom
Fi
9 h (c)
(h)
corresponding binary
b) Di Zenzo’s method;
j) Binary edge maps
ding and thinning proc
mparison of the five (b)
(g)
-strength maps and
maps of: (a) GEDHSI; (b
) SSSDE method. (f–
atic hysteresis threshol
s the pe fo
a ce co (b)
rform (c)
on o (d)
ds un (e)
1 0 d (a)
ens. 2
igur (a)
ens. Figure 9. Performance evaluation on A
F-measure
A. Experiment on the Farmland HSIs
A. Experiment on the Farmland HSIs Farmland image: (a) noisy image in gray-scale (band 1); (b) noiseless image in gray-scale
(band 87); and (c) three-band false color composite (bands 25, 60, 128). Figure 10. Farmland image: (a) noisy image in gray-scale (band 1); (b) noiseless image in gray-scale
(band 87); and (c) three-band false color composite (bands 25, 60, 128). Figure 11 illustrates the results of edge detection from the four methods. Overall, the better
performance of the GEDHSI on the image is clearly perceivable. The proposed GEDHSI has
produced continuous, compact and clear edges, where the most edge structures are well preserved
as shown in Figure 11a. For Di Zenzo's method, almost all of edges produced are broken as shown
in Figure 11b. Drewniok’s method generates many spurious edges within regions and some line
edges are distorted at the place where it should be straight according to Figure 11d. RCMG and
SSSDE produce similar results as GEDHSI. However, there are many grainy edges with some fine
edge structures missing or distorted to some degree, as shown in Figure 11c,e. Specifically, as
labeled by the red rectangles, Di Zenzo’s method has detected serious broken edges. Drewniok’s
method generates many small and severe spurious edges and fails to capture the main structure. Moreover, Drewniok’s method results in distortion and flexuous edges. The RCMG produces some
grainy and slightly distorted edges. In contrast, the edges produced by the proposed GEDHSI are
straight and continuous. Figure 11 illustrates the results of edge detection from the four methods. Overall, the better
performance of the GEDHSI on the image is clearly perceivable. The proposed GEDHSI has produced
continuous, compact and clear edges, where the most edge structures are well preserved as shown in
Figure 11a. For Di Zenzo's method, almost all of edges produced are broken as shown in Figure 11b. Drewniok’s method generates many spurious edges within regions and some line edges are distorted
at the place where it should be straight according to Figure 11d. RCMG and SSSDE produce similar
results as GEDHSI. However, there are many grainy edges with some fine edge structures missing
or distorted to some degree, as shown in Figure 11c,e. Specifically, as labeled by the red rectangles,
Di Zenzo’s method has detected serious broken edges. Drewniok’s method generates many small
and severe spurious edges and fails to capture the main structure. Moreover, Drewniok’s method
results in distortion and flexuous edges. Figure 9. Performance evaluation on A
F-measure
A. Experiment on the Farmland HSIs
A. Experiment on the Farmland HSIs 4.4. Evaluation on Real World Datasets
Figure 10 is the Farmland image obtained by AVIRIS. The AVIRIS instrument contains 224
different detectors, where the spectral band width is approximately 0.01 µm, allowing it to cover
Figure 10 is the Farmland image obtained by AVIRIS. The AVIRIS instrument contains 224 different
detectors, where the spectral band width is approximately 0.01 µm, allowing it to cover the entire 15 of 23
f 15 of 23
1
f 23 Remote Sens. 2017, 9, 592 range between 0.4 µm and 2.5 µm. There are several low SNR bands in the AVIRIS image, and one
example is shown in Figure 10a where the edges are nearly invisible. Even in some noiseless bands,
as shown in Figure 10b, the edges are still very vague. Composited with three low correlation bands,
the edge structure can be much clearer as shown in Figure 10c. This is mainly due to the effective
utilization of spectral information. In addition, as shown in Figure 10c, the boundaries between regions
are clearer, which make it appropriate for the evaluation of edge detection algorithms. However, in the
composited image, the pixels inside the region are heavily textured, which makes edge detection a
difficult problem. the entire range between 0.4 µm and 2.5 µm. There are several low SNR bands in the AVIRIS image,
and one example is shown in Figure 10a where the edges are nearly invisible. Even in some
noiseless bands, as shown in Figure 10b, the edges are still very vague. Composited with three low
correlation bands, the edge structure can be much clearer as shown in Figure 10c. This is mainly
due to the effective utilization of spectral information. In addition, as shown in Figure 10c, the
boundaries between regions are clearer, which make it appropriate for the evaluation of edge
detection algorithms. However, in the composited image, the pixels inside the region are heavily
textured, which makes edge detection a difficult problem. (a)
(b)
(c)
Figure 10. Farmland image: (a) noisy image in gray-scale (band 1); (b) noiseless image in gray-scale
(band 87); and (c) three-band false color composite (bands 25, 60, 128). Figure 10. Farmland image: (a) noisy image in gray-scale (band 1); (b) noiseless image in gray-scale
(band 87); and (c) three-band false color composite (bands 25, 60, 128). (b) (a) (c) (c) (b) (a) Figure 10. B. Experiment on the Washington DC Mall HSIs
Fi
12 i
i
bt i
d b
HYDICE
B. Experiment on the Washington DC Mall HSIs
B Experiment on the Washington DC Mall HSIs Figure 12 is an image obtained by HYDICE, which has 210 bands ranging from 0.4 µm to 2.4
µm and covers the visible and (near) infrared spectrum. Bands in the 0.9 µm and 1.4 µm regions
where the atmosphere is opaque have been omitted from the dataset, leaving 191 bands for
experiments. As shown in Figure 12a, the dataset contains complex and cluttered landcovers. Especially in the middle of Figure 12a, as highlighted within a dotted rectangle, various roofs,
roads, trails, grass, trees and shadow are cluttered. For better visual effect, the enlarged version of
this part of the image is shown in Figure 12b. This is used for testing as it contains abundant tinny
edges which is difficult to be detected completely. Examples of these tiny structures can be found in
the two ellipses in Figure 12b, where local details such as the boxes on the roof and the footpath are
clearly visible yet hard for detection. Figure 12 is an image obtained by HYDICE, which has 210 bands ranging from 0.4 µm to 2.4 µm
and covers the visible and (near) infrared spectrum. Bands in the 0.9 µm and 1.4 µm regions where
the atmosphere is opaque have been omitted from the dataset, leaving 191 bands for experiments. As shown in Figure 12a, the dataset contains complex and cluttered landcovers. Especially in the
middle of Figure 12a, as highlighted within a dotted rectangle, various roofs, roads, trails, grass, trees
and shadow are cluttered. For better visual effect, the enlarged version of this part of the image is
shown in Figure 12b. This is used for testing as it contains abundant tinny edges which is difficult
to be detected completely. Examples of these tiny structures can be found in the two ellipses in
Figure 12b, where local details such as the boxes on the roof and the footpath are clearly visible yet
hard for detection. . E pe i
e t o
t e
as i gto
C Ma
SIs
Figure 12 is an image obtained by HYDICE, which has 210 bands ranging from 0.4 µm to 2.4
µm and covers the visible and (near) infrared spectrum. Bands in the 0.9 µm and 1.4 µm regions
where the atmosphere is opaque have been omitted from the dataset, leaving 191 bands for
experiments. As shown in Figure 12a, the dataset contains complex and cluttered landcovers. B. Experiment on the Washington DC Mall HSIs
Fi
12 i
i
bt i
d b
HYDICE
B. Experiment on the Washington DC Mall HSIs
B Experiment on the Washington DC Mall HSIs Especially in the middle of Figure 12a, as highlighted within a dotted rectangle, various roofs,
roads, trails, grass, trees and shadow are cluttered. For better visual effect, the enlarged version of
this part of the image is shown in Figure 12b. This is used for testing as it contains abundant tinny
edges which is difficult to be detected completely. Examples of these tiny structures can be found in
the two ellipses in Figure 12b, where local details such as the boxes on the roof and the footpath are
clearly visible yet hard for detection. (a)
(b)
Figure 12. False color representation of the Washington DC Mall hyperspectral data: (a) the whole
data; and (b) the detail display of the doted rectangle region of (a) with two regions emphasized in
ellipses to show local tiny structures. Figure 13 illustrates the edge results for Figure 13b produced by the five methods. According
to the criteria of Reference [54]
a good detection should keep good edge resolution and
(a)
(b)
Figure 12. False color representation of the Washington DC Mall hyperspectral data: (a) the whole
data; and (b) the detail display of the doted rectangle region of (a) with two regions emphasized in
ellipses to show local tiny structures. Figure 12. False color representation of the Washington DC Mall hyperspectral data: (a) the whole
data; and (b) the detail display of the doted rectangle region of (a) with two regions emphasized in
ellipses to show local tiny structures. (b)
hington DC Mall hyperspectral data: (a) the
angle region of (a) with two regions emphasi
(b) (a)
re 12. False color represen
a d (b) the detail di
lay
(a) ypersp
(b) e colo
(a) p y
g
g
g
p
ellipses to show local tiny structures. Figure 13 illustrates the edge results for Figure 13b produced by the five methods. According
th
it
i
f R f
[54]
d d t
ti
h
ld k
d
d
l ti
d
Figure 12. False color representation of the Washington DC Mall hyperspectral data: (a) the whole
data; and (b) the detail display of the doted rectangle region of (a) with two regions emphasized in
ellipses to show local tiny structures. Figure 12. Figure 9. Performance evaluation on A
F-measure
A. Experiment on the Farmland HSIs
A. Experiment on the Farmland HSIs The RCMG produces some grainy and slightly distorted
edges. In contrast, the edges produced by the proposed GEDHSI are straight and continuous. Remote Sens. 2017, 9, 592 16 of 23 16 of 23 (a)
(b)
(c)
(d)
(e)
Figure 11. The binary edge maps of the Farmland image by: (a) GEDHSI; (b) Di Zenzo’s method; (c)
the RCMG method; (d) Drewniok’s method (
0. 1
=
σ
); and (e) SSSDE method. Figure 11. The binary edge maps of the Farmland image by: (a) GEDHSI; (b) Di Zenzo’s method;
(c) the RCMG method; (d) Drewniok’s method (σ = 1.0); and (e) SSSDE method. mote Sens. 2017, 9, 592
16 of 23
(a)
(b)
(c)
(d)
(e)
Figure 11. The binary edge maps of the Farmland image by: (a) GEDHSI; (b) Di Zenzo’s method; (c)
the RCMG method; (d) Drewniok’s method (
0
1
=
σ
); and (e) SSSDE method (a)
te Sens. 2017, 9, 592 (b) (c) (e)
16 of 23 (d) (a) (d) (e) (c) (b) Figure 11. The binary edge maps of the Farmland image by: (a) GEDHSI; (b) Di Zenzo’s method; (c)
the RCMG method; (d) Drewniok’s method (
0. 1
=
σ
); and (e) SSSDE method. Figure 11. The binary edge maps of the Farmland image by: (a) GEDHSI; (b) Di Zenzo’s method;
(c) the RCMG method; (d) Drewniok’s method (σ = 1.0); and (e) SSSDE method. (a)
(b)
(c)
(d)
(e)
Figure 11. The binary edge maps of the Farmland image by: (a) GEDHSI; (b) Di Zenzo’s method; (c)
th RCMG
th d (d) D
i k’
th d (
)
d ( ) SSSDE
th d B. Experiment on the Washington DC Mall HSIs
Fi
12 i
i
bt i
d b
HYDICE
B. Experiment on the Washington DC Mall HSIs
B Experiment on the Washington DC Mall HSIs False color representation of the Washington DC Mall hyperspectral data: (a) the whole
data; and (b) the detail display of the doted rectangle region of (a) with two regions emphasized in
ellipses to show local tiny structures. connectivity. The superiority of the proposed method on these criteria is clearly visible as shown in
g
g
g
p
y
g
to the criteria of Reference [54], a good detection should keep good edge resolution and
connectivity. The superiority of the proposed method on these criteria is clearly visible as shown in
Figure 13 illustrates the edge results for Figure 13b produced by the five methods. According
to the criteria of Reference [54], a good detection should keep good edge resolution and connectivity. 17 of 23 17 of 23 Remote Sens. 2017, 9, 592 The superiority of the proposed method on these criteria is clearly visible as shown in Figure 13. The edge image of Di Zenzo’s method is poor and contains many broken edges as shown in Figure 13b. The RCMG and SSSDE are better than Di Zenzo’s method, but they still produce many grainy and
spurious edges (Figure 13c,e). Compared with RCMG and SSSDE, the edge map produced by the
Drewniok’s method in Figure 13d contains more grainy edges within regions and more spurious edges. In contrast, the edges in Figure 13a produced by our approach are clearer and of better connectivity,
where most of fine edge structures from different land covers are successfully detected. Remote Sens. 2017, 9, 592
17 of 23
Figure 13. The edge image of Di Zenzo’s method is poor and contains many broken edges as shown
in Figure 13b. The RCMG and SSSDE are better than Di Zenzo’s method, but they still produce many
grainy and spurious edges (Figure 13c,e). Compared with RCMG and SSSDE, the edge map produced
by the Drewniok’s method in Figure 13d contains more grainy edges within regions and more spurious
edges. In contrast, the edges in Figure 13a produced by our approach are clearer and of better
connectivity, where most of fine edge structures from different land covers are successfully detected. (a)
(b)
(c)
(d)
(e)
Figure 13. The binary edge maps of the Washington DC Mall by: (a) GEDHSI; (b) Di Zenzo’s method;
(c) the RCMG method; (d) Drewniok’s method (σ = 1.0); and (e) SSSDE method. Figure 13. B. Experiment on the Washington DC Mall HSIs
Fi
12 i
i
bt i
d b
HYDICE
B. Experiment on the Washington DC Mall HSIs
B Experiment on the Washington DC Mall HSIs The binary edge maps of the Washington DC Mall by: (a) GEDHSI; (b) Di Zenzo’s method;
(c) the RCMG method; (d) Drewniok’s method (σ = 1.0); and (e) SSSDE method. (e) (d) (a) (c) (b) (d) (b) (a) (e) (c) Figure 13. The binary edge maps of the Washington DC Mall by: (a) GEDHSI; (b) Di Zenzo’s method;
(c) the RCMG method; (d) Drewniok’s method (σ = 1.0); and (e) SSSDE method. Figure 13. The binary edge maps of the Washington DC Mall by: (a) GEDHSI; (b) Di Zenzo’s method;
(c) the RCMG method; (d) Drewniok’s method (σ = 1.0); and (e) SSSDE method. g
,
p
p
p
obviously perceivable. In the upper left ellipse of Figure 13b,d, the Di Zenzo’s and RCMG methods
have missed the edge structures, while the proposed GEDHSI preserves such tiny structures very
well. In the lower right corner ellipse of Figure 13c–e, the RCMG, Drewniok’s, and SSSDE methods
not only fail to obtain continuous edge but also miss one side of the edge. On the contrary, the
proposed GEDHSI can detect complete edges at both side of the path. Overall, the proposed
approach is more effective than the other four methods for detecting edges from these HSIs. 4 4 2 Experiments on Ground Scene Datasets
For the edge resolution criterion, the better performance of the proposed method is also obviously
perceivable. In the upper left ellipse of Figure 13b,d, the Di Zenzo’s and RCMG methods have missed
the edge structures, while the proposed GEDHSI preserves such tiny structures very well. In the lower
right corner ellipse of Figure 13c–e, the RCMG, Drewniok’s, and SSSDE methods not only fail to obtain
continuous edge but also miss one side of the edge. On the contrary, the proposed GEDHSI can detect
complete edges at both side of the path. Overall, the proposed approach is more effective than the
other four methods for detecting edges from these HSIs. A. Experiment on the Scene 5 dataset
A. Experiment on the Scene 5 dataset
A. Experiment on the Scene 5 dataset For the Scene 5 dataset, Figure 15 illustrates the binary edge maps generated by the five
methods. Obviously, Di Zenzo’s edge image is poor and contains many small and spurious edges,
and the details of the wall are poorly detected as shown in Figure 15b. Compared to Figure 15b, the
RCMG method can detect more edges on the wall, while there are still some spurious edges
especially on the chair, under the ball and at the bottom right of the wall due to the variance in
intensity of the surface as shown in Figure 15c. Drewniok’s method performs better than Di Zenzo’s
method on the wall, but it has produced more spurious and grainy edges than either the RCMG or
GEDHSI. Some fine edges on the wall grid and racket bag are missing (Figure 15d). Performance of
SSSDE is better than that of Di Zenzo’s and Drewniok’s on the left wall, while it misses many edges
on the blue and yellow boxes as well as the basketball, as shown in Figure 15e. In contrast, almost
complete edges of most objects are attained by the proposed method as shown in Figure 15a. The
details in the circle from Figure 15 also illustrate the efficacy of the proposed method, where the
proposed GEDHSI method has generated the best edge results in keeping the real structure. For the Scene 5 dataset, Figure 15 illustrates the binary edge maps generated by the five methods. Obviously, Di Zenzo’s edge image is poor and contains many small and spurious edges, and the details
of the wall are poorly detected as shown in Figure 15b. Compared to Figure 15b, the RCMG method
can detect more edges on the wall, while there are still some spurious edges especially on the chair,
under the ball and at the bottom right of the wall due to the variance in intensity of the surface as
shown in Figure 15c. Drewniok’s method performs better than Di Zenzo’s method on the wall, but it
has produced more spurious and grainy edges than either the RCMG or GEDHSI. Some fine edges
on the wall grid and racket bag are missing (Figure 15d). Performance of SSSDE is better than that
of Di Zenzo’s and Drewniok’s on the left wall, while it misses many edges on the blue and yellow
boxes as well as the basketball, as shown in Figure 15e. p
Both of the two tested real ground scene
4.4.2. Experiments on Ground Scene Datasets µm, and are sampled at 0.01 µm intervals and result in 31 bands. For simplicity, we selected two
images, Scenes 5 and Scenes 7 as illustrated, because of the following two reasons: First, as shown in
Figure 14, the spatial resolution is higher than the four other datasets and they contain many
man-made objects, thus we know where the edges occur exactly. Therefore, they are rather
appropriate for performance evaluation. Second, there are heavily textured objects in the datasets,
such as the objects surrounded by a heavily textured wall in Scene 5 (Figure 14a), and finely
textured walls and grass in Scene 7 (Figure 14b). Edge detection under these circumstances is still a
challenge. Both of the two tested real ground scenes images cover the wavelength from 0.4 µm to 0.72 µm,
and are sampled at 0.01 µm intervals and result in 31 bands. For simplicity, we selected two images,
Scenes 5 and Scenes 7 as illustrated, because of the following two reasons: First, as shown in Figure 14,
the spatial resolution is higher than the four other datasets and they contain many man-made objects,
thus we know where the edges occur exactly. Therefore, they are rather appropriate for performance
evaluation. Second, there are heavily textured objects in the datasets, such as the objects surrounded
by a heavily textured wall in Scene 5 (Figure 14a), and finely textured walls and grass in Scene 7
(Figure 14b). Edge detection under these circumstances is still a challenge. Remote Sens. 2017, 9, 592 18 of 23 18 of 23 (b)
(b) (a)
(b)
Figure 14. RGB representations of two hyperspectral scene dataset: (a) Scene 5; and (b) Scene 7. Figure 14. RGB representations of two hyperspectral scene dataset: (a) Scene 5; and (b) Scene 7. (a)
(b)
Figure 14. RGB representations of two hyperspectral scene dataset: (a) Scene 5; and (b) Scene 7. (a)
( ) (a)
(a) (b)
(b) Figure 14. RGB representations of two hyperspectral scene dataset: (a) Scene 5; and (b) Scene 7. Figure 14. RGB representations of two hyperspectral scene dataset: (a) Scene 5; and (b) Scene 7. Figure 14. RGB representations of two hyperspectral scene dataset: (a) Scene 5; and (b) Scene 7. B. Experiment on Scene 7
Figure 16 shows the b Figure 16 shows the binary edge maps of Scene 7 produced by the five methods. According to
Figure 16b, Di Zenzo’s method produces many spurious edges and performs poorly on connectivity. The RCMG preserves most significant spatial structures, but it also brings many grainy edges especially
in the grass region as shown in Figure 16c. The Drewniok’s method can detect many edges on the wall
and grass as depicted in Figure 16d, but the interval between edges on the windows is enlarged due
to edges displacement caused by Gaussian smoothing. Connectivity of edges generated by SSSDE is
better than that of Di Zenzo’s while some edges between the upper and lower windows are missed,
as can be seen in Figure 16e. Comparatively, the better performance of the proposed method is
perceivable. Most of the fine edge structures such as the edges located at the windows are attained
and the edges are clear and connective as shown in Figure 16a. More specifically, as indicated by the
circle at the bottom of the image, Di Zenzo’s and Drewniok’s methods produce undesirable edges
for the grass in the circle. The RCMG almost misses and the SSSDE totally misses the edge structure. Comparatively, the fine structure is well preserved by the proposed GEDHSI method. At the right
bottom ellipse, Drewniok’s method has missed both the edge of the left sidewalls and the edge between
the middle white door and the dark shadow. On the contrary, the proposed GEDHSI can again detect
correct edges without missing main structure of that region. g
y
g
p
p
y
g
Figure 16b, Di Zenzo’s method produces many spurious edges and performs poorly on connectivity. The RCMG preserves most significant spatial structures, but it also brings many grainy edges
especially in the grass region as shown in Figure 16c. The Drewniok’s method can detect many
edges on the wall and grass as depicted in Figure 16d, but the interval between edges on the
windows is enlarged due to edges displacement caused by Gaussian smoothing. Connectivity of
edges generated by SSSDE is better than that of Di Zenzo’s while some edges between the upper
and lower windows are missed, as can be seen in Figure 16e. Comparatively, the better
performance of the proposed method is perceivable. A. Experiment on the Scene 5 dataset
A. Experiment on the Scene 5 dataset
A. Experiment on the Scene 5 dataset In contrast, almost complete edges of most
objects are attained by the proposed method as shown in Figure 15a. The details in the circle from
Figure 15 also illustrate the efficacy of the proposed method, where the proposed GEDHSI method has
generated the best edge results in keeping the real structure. For the Scene 5 dataset, Figure 15 illustrates the binary edge maps generated by the five
methods. Obviously, Di Zenzo’s edge image is poor and contains many small and spurious edges,
and the details of the wall are poorly detected as shown in Figure 15b. Compared to Figure 15b, the
RCMG method can detect more edges on the wall, while there are still some spurious edges
especially on the chair, under the ball and at the bottom right of the wall due to the variance in
intensity of the surface as shown in Figure 15c. Drewniok’s method performs better than Di Zenzo’s
method on the wall, but it has produced more spurious and grainy edges than either the RCMG or
GEDHSI. Some fine edges on the wall grid and racket bag are missing (Figure 15d). Performance of
SSSDE is better than that of Di Zenzo’s and Drewniok’s on the left wall, while it misses many edges
on the blue and yellow boxes as well as the basketball, as shown in Figure 15e. In contrast, almost
complete edges of most objects are attained by the proposed method as shown in Figure 15a. The
details in the circle from Figure 15 also illustrate the efficacy of the proposed method, where the
proposed GEDHSI method has generated the best edge results in keeping the real structure. (b)
(b) (a)
(b)
(a)
(b)
Figure 15. Cont. (a)
(a) (b)
(b) (a)
(a) Figure 15. Cont. Remote Sens. 2017, 9, 592
Remote Sens. 2017, 9, 592 19 of 23
19 of 23 19 of 23
19 of 23 (a)
(b)
(c)
(d)
(e)
Figure 15. The binary edge maps generated for the Scene 5 dataset by: (a) GEDHSI; (b) Di Zenzo’s
method; (c) the RCMG method; (d) Drewniok’s method (σ = 1.0); and (e) SSSDE. Figure 15. The binary edge maps generated for the Scene 5 dataset by: (a) GEDHSI; (b) Di Zenzo’s
method; (c) the RCMG method; (d) Drewniok’s method (σ = 1.0); and (e) SSSDE. (c)
(d)
(e)
Figure 15. A. Experiment on the Scene 5 dataset
A. Experiment on the Scene 5 dataset
A. Experiment on the Scene 5 dataset The binary edge maps generated for the Scene 5 dataset by: (a) GEDHSI; (b) Di Zenzo’s
method; (c) the RCMG method; (d) Drewniok’s method (σ = 1.0); and (e) SSSDE. . Experiment on Scene 7 (d)
(d)
s generated for the Scene 5 datase ( )
(e)
(e)
y (a) GEDHSI (b) Di Ze
o’ (c)
(c)
Figure 15 The binary edge m (d)
(d)
h (e)
(e
I (b) (c)
(c)
Th Figure 15. The binary edge maps generated for the Scene 5 dataset by: (a) GEDHSI; (b) Di Zenzo’s
method; (c) the RCMG method; (d) Drewniok’s method (σ = 1.0); and (e) SSSDE. Figure 15. The binary edge maps generated for the Scene 5 dataset by: (a) GEDHSI; (b) Di Zenzo’s
method; (c) the RCMG method; (d) Drewniok’s method (σ = 1.0); and (e) SSSDE. g
y
g
p g
y ( )
; ( )
method; (c) the RCMG method; (d) Drewniok’s method (σ = 1.0); and (e) SSSDE. Experiment on Scene 7 B. Experiment on Scene 7
Figure 16 shows the b Most of the fine edge structures such as the
edges located at the windows are attained and the edges are clear and connective as shown in
Figure 16a. More specifically, as indicated by the circle at the bottom of the image, Di Zenzo’s and
Drewniok’s methods produce undesirable edges for the grass in the circle. The RCMG almost
misses and the SSSDE totally misses the edge structure. Comparatively, the fine structure is well
preserved by the proposed GEDHSI method. At the right bottom ellipse, Drewniok’s method has
missed both the edge of the left sidewalls and the edge between the middle white door and the dark
shadow. On the contrary, the proposed GEDHSI can again detect correct edges without missing
main structure of that region. 1
(a)
(b)
(c)
(d)
(e)
Figure 16. The binary edge maps generated for the Scene 7 by: (a) GEDHSI; (b) Di Zenzo’s method;
(c) the RCMG method; (d) Drewniok’s method (σ = 1.0); and (e) SSSDE. Figure 16. The binary edge maps generated for the Scene 7 by: (a) GEDHSI; (b) Di Zenzo’s method;
(c) the RCMG method; (d) Drewniok’s method (σ = 1.0); and (e) SSSDE. (e) (d) (a) (b) 1
(c) (b) (d) (a) (c) (e) Figure 16. The binary edge maps generated for the Scene 7 by: (a) GEDHSI; (b) Di Zenzo’s method;
(c) the RCMG method; (d) Drewniok’s method (σ = 1.0); and (e) SSSDE. Figure 16. The binary edge maps generated for the Scene 7 by: (a) GEDHSI; (b) Di Zenzo’s method;
(c) the RCMG method; (d) Drewniok’s method (σ = 1.0); and (e) SSSDE. Overall, the comparative experiments above have validated the superiority of the proposed
GEDHSI for edge detection in HSIs. The proposed method is more robust to background clusters
and weak edges, as illustrated by the experiments on artificial images. For real-world images, it is
robust to noisy bands in keeping complete and reasonable spatial structures and performs well on
Overall, the comparative experiments above have validated the superiority of the proposed
GEDHSI for edge detection in HSIs. The proposed method is more robust to background clusters
and weak edges, as illustrated by the experiments on artificial images. For real-world images, it is 20 of 23 Remote Sens. 2017, 9, 592 robust to noisy bands in keeping complete and reasonable spatial structures and performs well on
edge localization and resolution. 5. Conclusions In recent years, various heuristic and physically inspired image-understanding methods have been
developed. To extract the edges from HSIs, a novel edge-detection method, namely Gravitation-based
Edge Detection on HSIs (GEDHSI), is presented in this paper. In GEDHSI, each pixel is modeled
as a movable celestial object in the joined spatial-spectral feature space where all objects travel in
the feature space under the gravitational field force. Due to the relative positions, the edge pixels
and non-edge pixels can be separated by computing their potential energy fields. In addition to
its simplicity and generality, GEDHSI has promising theoretical properties that make it a solid tool
for edge detection. The performance of the proposed algorithm has been benchmarked with four
state-of-the-art methods, including Di Zenzo’s, Drewniok’s, RCMG, and SSSD detectors. Experiments
on a variety of datasets have validated the efficacy of the proposed algorithm in edge-detection for
HSIs under various levels of noise. The future work will focus on further improving the efficiency of
the approach and investigating the relationship between the edge scale and the parameter settings for
edge results in the multiscale space. Acknowledgments: This work was supported by Chinese Natural Science Foundation Projects (41471353) and
National Key Research and Development Program of China (2016YFB0501501). Author Contributions: Genyun Sun, Aizhu Zhang, and Peng Wang conceived, designed and performed the
experiments, analyzed the data, and wrote the paper. Jinchang Ren, Jingsheng Ma, and Yuanzhi Zhang help
organize and revise the paper. Xiuping Jia helps analyze the experimental data and improve the presentation of
the second version. Conflicts of Interest: The authors declare no conflict of interest. B. Experiment on Scene 7
Figure 16 shows the b As a result, GEDHSI is more suitable for HSI edge detection than
other state-of-the-art multi-band approaches. 4.5. Comparison of Runtime In this section, we compare the computational efficiency of the above five algorithms. The mean
computational time (in seconds) required by each algorithm for five independent runs in solving the
two tested images, the Farmland and Washington DC Mall, are presented for comparison. As seen in Table 2, RCMG takes the least time, and one main reason is its C++ based implementation
in comparison to the MATLAB-based implementation for the other four methods. The proposed
GEDHSI, unfortunately, consumes the longest runtime as a whole. The long time consumption is
largely caused by the iterative computation in traveling procedure. For each particle, the repeating
calculation of gravitational force for all particles in dataset D leads to heavy burden of runtime. How
to speed up the process and improve the efficiency of GEDHSI will be investigated in the future. Table 2. The time efficiency evaluation. Image
GEDHSI 1
Di Zenzo
RCMG 2
Drewniok
SSSDE 3
Farmland
32.17s
15.65s
4.04s
13.55s
21.53s
Washington DC Mall
36.56s
22.45s
4.81s
14.28s
26.75s
1 GEDHSI (Gravitation-Based Edge Detection in Hyperspectral Image); 2 RCMG (Robust Color Morphological
Gradient); 3 SSSDE (modified Simultaneous Spectral/Spatial Detection of Edges). Table 2. The time efficiency evaluation. 1 GEDHSI (Gravitation-Based Edge Detection in Hyperspectral Image); 2 RCMG (Robust Color Morphological
Gradient); 3 SSSDE (modified Simultaneous Spectral/Spatial Detection of Edges). 1.
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Library Splib06a; USGS: Reston, VA, USA, 2007. © 2017 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/). © 2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).
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Development of a core outcome set and identification of patient-reportable outcomes for primary brain tumour trials: protocol for the COBra study
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Keywords: eco-anxiety; eating behaviors; climate change; questionnaire development; mental health Copyright:
© 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article
distributed
under
the
terms
and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/). Department of Nutrition, University of North Carolina at Chapel Hill, Chapel Hill, NC 27516, USA
*
Correspondence: melissa_chernoff@med.unc.edu; Tel.: +1-984-974-3788 *
Correspondence: melissa_chernoff@med.unc.edu; Tel.: +1-984-974-3788 Citation: Qi, B.; Presseller, E.K.;
Cooper, G.E.; Kapadia, A.; Dumain,
A.S.; Jayawickreme, S.M.;
Bulik-Sullivan, E.C.; van Furth, E.F.;
Thornton, L.M.; Bulik, C.M.; et al. Development and Validation of an
Eating-Related Eco-Concern
Questionnaire. Nutrients 2022, 14,
4517. https://doi.org/10.3390/
nu14214517
Academic Editor: Ata Ghaderi
Received: 31 August 2022
Accepted: 25 October 2022
Published: 27 October 2022 Citation: Qi, B.; Presseller, E.K.;
Cooper, G.E.; Kapadia, A.; Dumain,
A.S.; Jayawickreme, S.M.;
Bulik-Sullivan, E.C.; van Furth, E.F.;
Thornton, L.M.; Bulik, C.M.; et al. Development and Validation of an
Eating-Related Eco-Concern
Questionnaire. Nutrients 2022, 14,
4517. https://doi.org/10.3390/
nu14214517
Academic Editor: Ata Ghaderi
Received: 31 August 2022
Accepted: 25 October 2022
Published: 27 October 2022 Citation: Qi, B.; Presseller, E.K.;
Cooper, G.E.; Kapadia, A.; Dumain,
A.S.; Jayawickreme, S.M.;
Bulik-Sullivan, E.C.; van Furth, E.F.;
Thornton, L.M.; Bulik, C.M.; et al. Development and Validation of an
Eating-Related Eco-Concern
Questionnaire. Nutrients 2022, 14,
4517. https://doi.org/10.3390/
nu14214517 Abstract: Eco-concern, the distress experienced relating to climate change, is associated with mental
health, yet no study has examined disordered eating related to eco-concern. This study developed
and validated a 10-item scale assessing Eating-Related Eco-Concern (EREC). Participants (n = 224)
completed the EREC, Climate Change Worry Scale (CCWS), and Eating Disorder Examination-
Questionnaire (EDE-Q). Construct validity, convergent validity, and internal consistency were evalu-
ated. Sex differences in EREC were evaluated using t-tests. Associations among the EREC, CCWS,
and EDE-Q were evaluated using linear regression models. Sensitivity analyses were conducted
in individuals below EDE-Q global score clinical cut-offs. Factor analysis suggested that all items
loaded adequately onto one factor. Pearson’s correlation and Bland–Altman analyses suggested
strong correlation and acceptable agreement between the EREC and CCWS (r = 0.57), but weak
correlation and low agreement with the EDE-Q global score (r = 0.14). The EREC had acceptable
internal consistency (α = 0.88). No sex difference was observed in the EREC in the full sample;
females had a significantly higher mean score than males in sensitivity analysis. The EREC was
significantly positively associated with the CCWS and EDE-Q global and shape concern scores,
but not in sensitivity analysis. The EREC is a brief, validated scale that can be useful to screen for
eating-related eco-concern. Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations. Article
Development and Validation of an Eating-Related
Eco-Concern Questionnaire Munn-Chernoff 4,* 1
Department of Epidemiology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27516, USA 1
Department of Epidemiology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27516, USA
2
Department of Psychological and Brain Sciences, Drexel University, Philadelphia, PA 19104, USA
3
Center for Weight, Eating, and Lifestyle Science, Drexel University, Philadelphia, PA 19104, USA
4
Department of Psychiatry, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
5
Department of Clinical Psychology, National Institute of Mental Health and Neurosciences,
Bangalore 560030, India
6 Department of Epidemiology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27516, USA
2
Department of Psychological and Brain Sciences, Drexel University, Philadelphia, PA 19104, USA
3
Center for Weight, Eating, and Lifestyle Science, Drexel University, Philadelphia, PA 19104, USA
4
Department of Psychiatry, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
5
Department of Clinical Psychology, National Institute of Mental Health and Neurosciences, p
p
gy
y
p
p
2
Department of Psychological and Brain Sciences, Drexel University, Philadelphia, PA 19104, USA
3
Center for Weight, Eating, and Lifestyle Science, Drexel University, Philadelphia, PA 19104, USA 6
Department of Psychology and Neuroscience, University of North Carolina at Chapel Hill,
Chapel Hill, NC 27599, USA 7
Marsico Lung Institute and Cystic Fibrosis Research Center, University of North Carolina at Chapel Hill,
Chapel Hill, NC 27599, USA 8
Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill,
Chapel Hill, NC 27599, USA 9
GGZ Rivierduinen Eating Disorders Ursula, 2333 ZZ Leiden, The Netherlands 10
Department of Psychiatry, Leiden University Medical Center, 2333 ZB Leiden, The Netherlands p
y
y
y
11
Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, 171 77 Stockholm, Sweden
12
Department of Nutrition, University of North Carolina at Chapel Hill, Chapel Hill, NC 27516, USA
*
Correspondence: melissa chernoff@med.unc.edu; Tel.: +1-984-974-3788 11
Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, 171 77 Stockholm, Sweden 11
Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, 171 77 Stockholm, Sweden
12
Department of Nutrition University of North Carolina at Chapel Hill Chapel Hill NC 27516 USA nutrients nutrients Article
Development and Validation of an Eating-Related
Eco-Concern Questionnaire Baiyu Qi 1
, Emily K. Presseller 2,3
, Gabrielle E. Cooper 4, Avantika Kapadia 5, Alexis S. Dumain 6,
Shantal M. Jayawickreme 4, Emily C. Bulik-Sullivan 7,8, Eric F. van Furth 9,10, Laura M. Thornton 4
,
Cynthia M. Bulik 4,11,12
and Melissa A. Citation Citation
Retzer, A., Sivell, S., Scott, H., Nelson, A., Bulbeck, H., Seddon, K., … Byrne, A. (2022). Development of a core outcome set and identification of patient-reportable outcomes for
primary brain tumour trials: protocol for the COBra study. Bmj Open, 12(9). doi:10.1136/bmjopen-2021-057712 Version:
Publisher's Version
License:
Creative Commons CC BY 4.0 license
Downloaded from:
https://hdl.handle.net/1887/3502259 License: Note: To cite this publication please use the final published version (if applicable). Note: To cite this publication please use the final published version (if applica nutrients nutrients 1. Introduction Climate change is defined as alterations in weather patterns and temperatures caused
by both natural disasters and human activity [1]. The world has encountered progressively
worsening climate change, which is known to have a significant impact on food insecurity,
housing, and physical health [2]. More recently, the adverse impact of climate change on
mental health has been documented, with 25−50% percent of individuals who are exposed https://www.mdpi.com/journal/nutrients Nutrients 2022, 14, 4517. https://doi.org/10.3390/nu14214517 Nutrients 2022, 14, 4517 2 of 14 to extreme climate-related events subsequently developing psychiatric conditions [3]. As
a result, the more general emotional distress induced by climate change, known as eco-
concern, is rapidly gaining attention. Prior studies have indicated an association between
eco-concern and psychopathology; however, the connection between eco-concern and
disordered eating (e.g., dietary restraint, eating concern, weight concern, and shape concern)
remains understudied [3,4]. Thus, the current study explored this potential relationship,
proposing that an association may exist between eco-concern and disordered eating. p
p
g
y
g
Eco-concern is a general term for the distress one experiences specifically related to
the climate crisis; it may include emotional disturbance due to environmental changes,
symptoms of anxiety and distress (e.g., rumination), helplessness, dread, guilt, sadness,
doom, and frustration [5,6]. Eco-concern may also be associated with specific coping and
affect regulation strategies, such as cognitive reframing of threats and altering consumer
habits, as well as other mental health conditions, such as anxiety, depression, substance use
disorders, and post-traumatic stress disorder [3,7–10]. Numerous factors may influence the association between eco-concern and mental
health outcomes. Age may be an important factor: eco-concern is known to impact chil-
dren, adolescents, and young adults the most, particularly those with preexisting mental
health conditions and lacking social support [4,11,12]. In addition, constant media expo-
sure has exacerbated eco-concern [13]. Relatedly, the recently examined phenomenon of
“doomscrolling”—the act of compulsively scrolling through distressing information—may
worsen anxiety about climate change [14]. Moreover, individuals who are more invested in
and aware of climate change and sustainability and those who live in areas more prone
to extreme weather events and natural disasters are also at higher risk of experiencing
worsening mental health outcomes [15,16]. Lastly, feelings of diminished control, along
with uncertainty and powerlessness regarding one’s future related to climate change fore-
casts, may contribute to the association between eco-concern and adverse mental health
outcomes [11,17,18]. 2.2.1. Demographic Information 2.2.1. Demographic Information Participants self-reported demographic information, including age, sex assigned at
birth, gender, race, and ethnicity. Participants self-reported their current height and weight
from which body mass index was calculated. 2.1. Participants Participants were recruited from across the United States via flyers and online re-
cruitment strategies, including posting on social media, ResearchMatch, and University
of North Carolina at Chapel Hill listservs. The only inclusion criterion for the present
study was that individuals were at least 18 years old; there were no exclusion criteria. Participants provided online consent and then completed all questionnaires via an online
survey. Participation was anonymous. A total of 257 adults took the survey. We excluded
participants who had incorrect answers to quality assessment questions (n = 6), those who
had missing data on eating-related eco-concern (n = 24), and those with inflammatory
responses (n = 3), resulting in a final sample size of 224. Compared with participants who
were excluded (n = 33), the analytical sample had a higher proportion of White/Caucasian
participants (82% vs. 50%), but did not differ in age or sex assigned at birth. A total
of 31 participants had missing data on any EDE-Q subscales, and thus the global score. Compared with these participants, those without missing data on EDE-Q scores were
significantly older (Mean = 39.01, SD = 16.97 vs. Mean = 27.30 years, SD = 11.59), more
likely to be male (22% vs. 13%), and had higher current BMI (Mean = 25.79, SD = 5.74 vs. Mean = 20.87, SD = 4.56). This study was reviewed and approved by The University of North Carolina at
Chapel Hill Biomedical Institutional Review Board. Due to the anonymous nature of the
questionnaires, the committee determined this study to be exempt from further review. 2.2.3. Eating-Related Eco-Concern Scale Item Development 2.2.3. Eating-Related Eco-Concern Scale Item Development We developed the Eating-Related Eco-Concern (EREC) scale, which is a 10-item assess-
ment regarding the degree to which individuals consider ecological impact when making
food choices due to concerns about the changing climate. The items were based on our
clinical observations, the previously published CCWS [21], and a literature review on
eco-friendly eating and sustainable eating. Items were scored on a 5-point Likert scale
with the anchors 1 = “Never”, 2 = “Rarely”, 3 = “Sometimes”, 4 = “Often”, and 5 = “Always”. The score was calculated by summing all items, which ranged from 10 to 50. The full
questionnaire is included in the Supplementary Material (S1). 2.2.4. Eating Disorder Examination-Questionnaire 2.2.2. Climate Change Worry Scale The Climate Change Worry Scale (CCWS) is a 10-item scale assessing worry about
climate change [21]. Items were scored on a 1 = Never to 5 = Always Likert scale. The
score was computed as the sum of all 10 items. The scale has demonstrated good internal
consistency (Cronbach’s α = 0.95), factor structure invariance, and test–retest reliability
(r = 0.91) [21]. The CCWS also demonstrates convergent and divergent validity with widely
used clinical measures of worry, anxiety, and weather-related fear [21]. In this study, it had
an internal consistency of 0.93. 1. Introduction The manner in which eco-concern impacts disordered eating and the magnitude of
this effect are unknown. However, studies have examined associations between specific
eating behaviors related to climate change. Undergraduate students, particularly female
students, may adopt behaviors such as eliminating meat, seeking organic and/or local
foods, and avoiding food waste in response to climate change concerns [19]. Although
such behaviors are not harmful themselves, and are, in fact, beneficial for the environment,
when taken to the extreme, they could represent more concerning behavioral patterns
(e.g., elimination of entire food groups, rigidity of eating choices) similar to established
patterns of disordered eating (e.g., dietary restraint). A survey of 2000 children and teens
in the UK found that 17% reported that climate concerns impact their sleeping and eating
behaviors [20]. However, no study to our knowledge has comprehensively explored the
specific association of concerns about climate change with eating behaviors in the general
population and whether those eating behaviors reflect disordered eating. Emerging from
clinical observations of individuals with eating disorders (e.g., anorexia nervosa, bulimia
nervosa, and binge-eating disorder) who endorse disordered eating related to concerns
about climate change, the goal of this study was to provide insight into specific changes to
eating behavior prompted by eco-concern and its relationship with disordered eating in a
general population sample. The current study addressed research gaps by: (1) developing and validating the
first questionnaire assessing eating behaviors related to eco-concern, the eating-related
eco-concern (EREC) scale; (2) examining sex differences in climate change worry and eating-
related eco-concern; and (3) examining associations among climate change worry, eating-
related eco-concern, and disordered eating. In this initial study, there were three hypotheses:
(1) the EREC scale would have good psychometric properties (construct validity, convergent
validity, and internal consistency) in this sample; (2) female participants would score higher
on both climate change worry and eating-related eco-concern; and (3) eating-related eco-
concern would be associated with both climate change worry and disordered eating. This Nutrients 2022, 14, 4517 3 of 14 3 of 14 study provides the first questionnaire to screen for eating behaviors related to eco-concern
and brings awareness to eco-concern as a potentially important contributing factor in the
development of disordered eating. 2.3.1. Psychometric Properties of the Eating-Related Eco-Concern Scale 2.3.1. Psychometric Properties of the Eating-Related Eco-Concern Scale To examine the factor structure of the EREC scale, polychoric correlations were cal-
culated and tested using Bartlett’s test of sphericity to ensure they did not constitute an
identity matrix. Results of Bartlett’s test of sphericity were not significant. Factor anal-
ysis was conducted with an unweighted least squares factor extraction procedure [33]. The number of factors extracted was determined using multiple approaches, including
conducting a parallel analysis and evaluating factor analytic results using Kaiser’s Rule
(i.e., eigenvalues > 1), the scree plot, and factor loading interpretations [34,35]. Factor
loadings ≥0.40 indicated that the item loaded adequately onto the factor [36,37]. Items
with factor loadings < 0.40 were removed from subsequent analyses. Pearson correlation coefficients were calculated between the EREC score with the
CCWS score, and between the EREC score with the EDE-Q global score to examine the
strength of the relationship between two scales. Correlations of 0.10 were considered small,
0.30 were medium, and 0.50 or higher were large [38]. Bland–Altman analysis was conducted to examine the level of agreement between
the EREC with the CCWS scores, and between the percentages of the EREC score and
the EDE-Q global score. Due to different ranges of the EREC score (range 10–50) and
EDE-Q global score (range 0–6), these two scores were converted to percentages using the
following equations: •
1 percentage value on the EREC score: (1/50) × 100 = 2
•
1 percentage value on the EDE-Q global score: (1/6) × 100 = 16.67 •
1 percentage value on the EREC score: (1/50) × 100 = 2 •
1 percentage value on the EDE-Q global score: (1/6) × 100 = 16.67 •
1 percentage value on the EDE-Q global score: (1/6) × 100 = 16.67 Thus, for each individual, the EREC percentage was calculated as 2 × the EREC score,
and the EDE-Q percentage was calculated as 16.67 × the EDE-Q global score. Thus, for each individual, the EREC percentage was calculated as 2 × the EREC score,
and the EDE-Q percentage was calculated as 16.67 × the EDE-Q global score. Thus, for each individual, the EREC percentage was calculated as 2 × the EREC score,
and the EDE-Q percentage was calculated as 16.67 × the EDE-Q global score. 2.2.4. Eating Disorder Examination-Questionnaire The Eating Disorder Examination-Questionnaire (EDE-Q) 6.0 is a 28-item self-report ques-
tionnaire designed to measure disordered eating over the last 28days [22]. Twenty-two items Nutrients 2022, 14, 4517 4 of 14 were included, measuring four subscales: dietary restraint (5 items; measures restraint
over eating, avoidance of eating, and dietary avoidance), eating concern (5 items; mea-
sures the preoccupation with food, eating in secret, and guilt about eating), shape concern
(8 items; measures the desire for a flat stomach, the importance of body shape, and fear
of weight gain), and weight concern (5 items; measures the importance of weight, dissat-
isfaction with weight, and the desire to lose weight). Responses were on a 7-point rating
scale (0–6). Subscale scores were calculated by taking the mean of all items in each subscale,
with higher scores indicating a greater degree of disordered eating. The global score was
calculated as the mean of the four subscales scores. The EDE-Q has been validated in
non-clinical [23–26] and clinical eating disorder samples [27,28]. The EDE-Q demonstrates
high internal consistency [24,25,29], test–retest reliability [25,28], and good discriminant
validity [30,31]. In this study, the Cronbach’s alpha was 0.95 for the global score; 0.82 for
restraint; 0.87 for eating concern; 0.91 for shape concern; and 0.81 for weight concern. 2.3. Statistical Analyses All statistical analyses were conducted in SAS version 9.4 [32]. Results with p < 0.05
were considered significant. The normality of the EREC score, the CCWS score, and the
EDE-Q global and subscale scores were examined by plotting the histograms (Figure S1). The EREC and CCWS scores were normally distributed. The EDE-Q global and subscale
scores were all right skewed. We did not conduct transformations for the EDE-Q scores
because the inclusion criteria for the sensitivity analysis were based on the EDE-Q global
score. Furthermore, it facilitated interpretation of results. 2.3.4. Sensitivity Analysis among Participants with EDE-Q Global Score below Clinical
Cut-Off Values All analyses were replicated in a subset of the sample, which included participants
whose EDE-Q global scores were below clinical cut-off values, to examine whether results
differ in individuals without a potential eating disorder. A cut-off value of 1.68 was used
for males [41], whereas 4.0 was used for females [23,42]. Seven male participants and
eight female participants met the respective clinical thresholds, and 31 participants had
missing data on EDE-Q global scores. Thus, a total of 178 participants were included in the
sensitivity analysis. 3. Results 3.1. Participant Characteristics 2.3.1. Psychometric Properties of the Eating-Related Eco-Concern Scale Bland–Altman plots were visualized, where the X axis was the average of two mea-
sures, and the Y axis was the difference between two measures. For each comparison,
the mean difference and standard deviation were calculated, and 95% limits of agreement
(LOA) were calculated using the equation (Mean difference ± 1.96 * standard deviation). Higher mean differences and wider LOA indicate lower agreement between two measures. The Bland–Altman index, defined as percentage of the difference between two measure
falling beyond the LOA, was also calculated for each comparison. A Bland–Altman index
below 5% suggests good agreement between two measures [39]. Nutrients 2022, 14, 4517 5 of 14 Internal consistency, as measured by Cronbach’s alpha, was evaluated using all
items of the EREC scale. An alpha ≥0.80 was considered evidence of adequate inter-
nal consistency [40]. 2.3.2. Differences in Climate Change Worry and Eating-Related Eco-Concern by Sex
Assigned at Birth 2.3.2. Differences in Climate Change Worry and Eating-Related Eco-Concern by Sex
Assigned at Birth The differences in the CCWS score and the EREC score between male and female (sex
assigned at birth) participants was evaluated using independent samples t-tests. 2.3.3. Associations among Climate Change Worry, Eating-Related Eco-Concern, and
Disordered Eating 2.3.3. Associations among Climate Change Worry, Eating-Related Eco-Concern, and
Disordered Eating Multiple linear regression models were conducted to examine associations between:
(1) climate change worry (predictor) with eating-related eco-concern (outcome); (2) EDE-Q
global score and each subscale score (i.e., restraint, eating concern, shape concern, weight
concern; predictors) with climate change worry (outcome); and (3) EDE-Q global score
and each subscale score (predictors) with eating-related eco-concern (outcome). Age was
included as a covariate in all models. 2.3.4. Sensitivity Analysis among Participants with EDE-Q Global Score below Clinical
Cut-Off Values 3.1. Participant Characteristics I avoid eating any animal products due to my concerns about climate change. 2.03 (1.17)
0.71
4. I try not to waste food due to concerns about climate change. 3.19 (1.29)
0.63
5. I actively encourage others to change their behaviors to slow climate change. 2.63 (1.11)
0.66
6. I try to eat less because of my concerns about climate change. 1.62 (0.92)
0.59
7. I avoid genetically modified foods due to concerns about biodiversity loss. 1.85 (1.13)
0.64
8. I try to only eat organic foods or food produced without pesticides. 2.49 (1.21)
0.55
9. I avoid foods that come with excess or non-recyclable packaging. 2.97 (1.06)
0.67
10. I pay close attention to information on the impact that certain foods have on
the environment (e.g., overfishing, greenhouse gasses, irrigation). 2.89 (1.08)
0.79
Note: SD = standard deviation. The score for each item ranges from 1 = Never to 5 = Always. 3.2. Psychometric Properties of the Eating-Related Eco-Concern Scale Table 2 summarizes the mean score (standard deviation) and factor loading for each
item of the EREC scale. Results of the factor analysis (including the parallel analysis,
Kaiser’s rule, and scree plot) indicated that the EREC scale was comprised of a single factor. All items loaded adequately (factor loading ≥0.40) onto the single factor. Table 2. Descriptive statistics and factor loadings for items included in the Eating-Related Eco-
Concern Scale (n = 224). Table 2. Descriptive statistics and factor loadings for items included in the Eating-Related Eco-
Concern Scale (n = 224). Item
Mean (SD)
Factor Loading
1. I spend more time than other people searching for sustainable food. 2.44 (1.06)
0.71
2. I avoid eating meat due to concerns about climate change. 2.77 (1.40)
0.72
3. I avoid eating any animal products due to my concerns about climate change. 2.03 (1.17)
0.71
4. I try not to waste food due to concerns about climate change. 3.19 (1.29)
0.63
5. I actively encourage others to change their behaviors to slow climate change. 2.63 (1.11)
0.66
6. I try to eat less because of my concerns about climate change. 1.62 (0.92)
0.59
7. I avoid genetically modified foods due to concerns about biodiversity loss. 1.85 (1.13)
0.64
8. I try to only eat organic foods or food produced without pesticides. 2.49 (1.21)
0.55
9. I avoid foods that come with excess or non-recyclable packaging. 2.97 (1.06)
0.67
10. 3.2.2. Convergent Validity The EREC score had a large and significant correlation with the CCWS score (r = 0.57,
p < 0.0001), but a weak correlation of 0.14 with the EDE-Q global score (p = 0.0455). Bland–
Altman analyses demonstrated the agreement between the EREC scale with the CCWS
and EDE-Q global score (Figure 1). On average, participants scored 4.84 units lower on
the EREC scale than the CCWS, with a moderate 95% LOA of (−19.81, 10.14). Eight out of
224 participants had a difference between the EREC and CCWS scores falling beyond the
LOA, resulting in a Bland–Altman index of 3.57%, suggesting a good agreement between
these two measures. For the EREC and EDE-Q percentages, the mean difference was
24.75, with a wide LOA of (−21.12, 70.62). The Bland–Altman index was 6.22% (12/193),
suggesting that the EREC scale had a low agreement with the EDE-Q global score and the
EREC scale captured a distinct construct from disordered eating. 3.1. Participant Characteristics I pay close attention to information on the impact that certain foods have on
the environment (e.g., overfishing, greenhouse gasses, irrigation). 2.89 (1.08)
0.79
Note: SD = standard deviation. The score for each item ranges from 1 = Never to 5 = Always. Item 3.2.2. Convergent Validity 3.1. Participant Characteristics The sample (N = 224) had a mean age of 37.43 years (SD = 16.78, range 18–89), and 79%
(n = 177) of the sample reported being assigned female sex at birth. No participant reported
being intersex at birth. Participants identified their gender as 77% (n = 173) women, 20%
(n = 44) men, and 3% (n = 7) gender non-conforming, gender fluid, questioning or unsure,
or other. The racial composition was 82% (n = 184) White or Caucasian, 3% (n = 6) Black or
African American, 11% (n = 24) Asian, and 4% (n = 10) more than one race or other. The
majority (94%; n = 210) of the sample was non-Hispanic. Among participants who reported
their current height and weight (n = 175), mean current body mass index was 25.62 kg/m2
(SD = 5.76, range = 13.81–45.70). Table 1 summarizes the descriptive statistics for climate
change worry, eating-related eco-concern, and disordered eating. Table 1. Descriptive statistics for climate change worry and eating-related eco-concern scores, and
for the EDE-Q global and subscale scores. Measure
n
Mean (SD)
Range
Climate change worry
224
29.72 (8.39)
10–50
Eating-related eco-concern
224
24.88 (8.03)
10–50
EDE-Q Global
193
1.48 (1.20)
0.00–5.45
Restraint
207
1.33 (1.46)
0.00–6.00
Eating concern
193
0.71 (1.11)
0.00–5.40
Weight concern
194
1.85 (1.48)
0.00–6.00
Shape concern
194
2.11 (1.57)
0.00–6.00
Note: EDE-Q = Eating Disorder Examination-Questionnaire; SD = standard deviation. Higher scores indicate
higher levels of climate change worry, eating-related eco-concern, and disordered eating. Table 1. Descriptive statistics for climate change worry and eating-related eco-concern scores, and
for the EDE-Q global and subscale scores. Nutrients 2022, 14, 4517 6 of 14 3.2. Psychometric Properties of the Eating-Related Eco-Concern Scale
3.2.1. Factor Structure
Table 2 summarizes the mean score (standard deviation) and factor loading for each
item of the EREC scale. Results of the factor analysis (including the parallel analysis,
Kaiser’s rule, and scree plot) indicated that the EREC scale was comprised of a single factor. All items loaded adequately (factor loading ≥0.40) onto the single factor. Table 2. Descriptive statistics and factor loadings for items included in the Eating-Related Eco-
Concern Scale (n = 224). Item
Mean (SD)
Factor Loading
1. I spend more time than other people searching for sustainable food. 2.44 (1.06)
0.71
2. I avoid eating meat due to concerns about climate change. 2.77 (1.40)
0.72
3. 3.2.3. Internal Consistency The EREC scale demonstrated good internal consistency: Cronbach’s alpha = 0.88. The EREC scale demonstrated good internal consistency: Cronbach’s alpha = 0.88. 3.3. Examining Differences in Climate Change Worry and Eating-Related Eco-Concern by Sex
at Birth A significant difference was observed in climate change worry between female and
male participants (t (221) = −2.90, p = 0.0042), where female participants demonstrated a
significantly higher level of climate change worry (Mean = 30.53, SD = 8.06) compared with
males (Mean = 26.57, SD = 9.06). Mean scores on eating-related eco-concerns did not differ
significantly by sex at birth (t (221) = −1.73, p = 0.0852, Meanfemales = 25.31, SDfemales = 7.84,
Meanmales = 23.02, SDmales = 8.49). 7 of 14
7 of 15 Nutrients 2022, 14, 4517
Nutrients 2022 14 x FOR Figure 1. Bland-Atman plot for (a) the EREC scale and the CCWS and (b) the EREC
the EDEQ global score (%). Figure 1. Bland-Atman plot for (a) the EREC scale and the CCWS and (b) the EREC scale
EDEQ global score (%) Figure 1. Bland-Atman plot for (a) the EREC scale and the CCWS and (b) the EREC scale (%) and
the EDEQ global score (%). 3 2 3 Internal Consistency
Figure 1. Bland-Atman plot for (a) the EREC scale and the CCWS and (b) the EREC scale (%) and the
EDEQ global score (%). Figure 1. Bland-Atman plot for (a) the EREC scale and the CCWS and (b) the EREC scale (%) and
the EDEQ global score (%). Figure 1. Bland-Atman plot for (a) the EREC scale and the CCWS and (b) the EREC scale (%) and the
EDEQ global score (%). Nutrients 2022, 14, 4517 8 of 14 3.4. Associations among Climate Change Worry, Eating-Related Eco-Concern, and
Disordered Eating Table 3 shows the results from the linear regression models. Climate change worry
was positively significantly associated with eating-related eco-concern after adjusting for
age, indicating that participants with higher CCWS scores also had higher EREC scores. Climate change worry was not significantly associated with any of the examined disordered
eating characteristics (i.e., global score, restraint, eating concern, weight concern, and shape
concern). Eating-related eco-concern was positively significantly associated with EDE-Q
global score and shape concern after adjusting for age. Eating-related eco-concern was not
significantly associated with restraint, eating concern, or weight concern. 3.2.3. Internal Consistency Figure 2 presents
the regression between (1) the CCWS and the EREC scores; (2) the EDE-Q global score and
the EREC score; and (3) the EDE-Q shape concern score and the EREC score. Table 3. Associations among climate change worry, eating-related eco-concern, and disordered eating. Table 3. Associations among climate change worry, eating-related eco-concern, and disordered eating. Association between Climate Change Worry and Eating-Related Eco-Concern
Predictor
β (SE)
t (df)
p
Climate change worry
0.56 (0.05)
10.76 (1)
<0.0001
Associations between Each Disordered Eating Characteristic and Climate Change Worry
Predictor
β (SE)
t (df)
p
EDE-Q Global
0.51 (0.48)
1.06 (1)
0.2904
Restraint
0.22 (0.39)
0.57 (1)
0.5670
Eating concern
0.65 (0.53)
1.23 (1)
0.2206
Weight concern
0.36 (0.39)
0.93 (1)
0.3535
Shape concern
0.40 (0.37)
1.09 (1)
0.2760
Associations between Each Disordered Eating Characteristic and Eating-Related Eco-Concern
Predictor
β (SE)
t (df)
p
EDE-Q Global
0.93 (0.45)
2.05 (1)
0.0414
Restraint
0.50 (0.37)
1.36 (1)
0.1745
Eating concern
0.89 (0.50)
1.79 (1)
0.0751
Weight concern
0.71 (0.37)
1.95 (1)
0.0531
Shape concern
0.71 (0.35)
2.07 (1)
0.0402
Note: df = degree of freedom; SE = standard error. Age was adjusted in all models. Significant results (p < 0.05)
are bolded. S
i i i
A
l
i i
i i
h
Q Gl b l S
b l
Cli i
l Association between Climate Change Worry and Eating-Related Eco-Concern 3.5. Sensitivity Analysis in Participants Whose EDE-Q Global Scores Were below Clinical
Cut-Off Values 3.5. Sensitivity Analysis in Participants Whose EDE-Q Global Scores Were below Clinical
Cut-Off Values All analysis were replicated only in participants who did not meet the clinical cut-off
values for the EDE-Q global score (n = 178). Compared with the whole sample, this subset
did not differ significantly in age (Mean = 37.43 years, SD = 16.78 in the whole sample vs. Mean = 39.04 years, SD = 16.90 in the subset), sex at birth (79% vs. 81% female), gender iden-
tity (77% vs. 80% women), race (82% vs. 84% White or Caucasian), ethnicity (94% vs. 94%
non-Hispanic), and current BMI (Mean = 25.62 kg/m2, SD = 5.76 vs. Mean = 25.67 kg/m2,
SD = 5.71). The CCWS score, EREC score, EDE-Q global and subscale scores did not differ
significantly between the whole sample and the subset used in the sensitivity analysis
(Table S1). 4. Discussion The current study created and validated a screening tool to assess eating-related
concerns and behaviors related to climate change. The 10-item EREC scale has shown good
validity and internal consistency. First, results from factor analysis, a tool for establishing
construct validity, support the single factor structure, indicating that a single latent variable
fits the data well. This result is consistent with the CCWS, which was also represented
well by a single factor [21]. Second, the EREC score showed a strong correlation with the
CCWS score, and the Bland–Altman analysis demonstrated acceptable agreement between
these two scales. This may be because both scales were designed to measure eco-concern,
although the CCWS assesses eco-concern in general and the EREC scale focuses specifically
on eating-related eco-concern. On average, participants scored lower on the EREC scale
than the CCWS, which suggests that all individuals who worry about climate change
in general do not necessarily alter their eating behaviors as a personal contribution to
slowing climate change. In contrast, correlation of the EREC score with the global EDE-Q
score was small and the Bland–Altman analysis demonstrated low agreement between the
EREC score and EDE-Q global score, suggesting that these two measures captured distinct
constructs. This is the desired outcome since the EREC scale was designed to capture a
novel dimension (i.e., eating-related eco-concern) that is not assessed by the EDE-Q, as the
latter represents a broader general scale of eating disorder cognitions and behaviors. Female participants showed a significantly higher level of climate change worry than
male participants, corroborating the original CCWS study [21]. Evidence on sex/gender
difference in general eco-concern is well established [43]. Compared with men, women are
more likely to believe that climate change is happening [44], perceive more risks of climate
change [45], and have greater levels of concern about climate change [46]. Contrary to the
hypothesis, we did not observe a significant sex difference in eating-related eco-concern,
suggesting that although women show a higher level of concern about climate change than
men, their eating behaviors do not change more due to eco-concern. After adjusting for age, eating-related eco-concern was positively associated with
climate change worry, which is understandable as individuals who are generally worried
about climate change might change their eating behaviors due to eco-concern. 3.2.3. Internal Consistency The psychometric properties of the EREC scale remain consistent in the sensitivity
analysis. Results from the factor analysis still support a single-factor structure (Table S2). Pearson’s correlation analysis demonstrates large correlation between the EREC and CCWS
scores (r = 0.53, p < 0.0001), but weak correlation between the EREC score and the EDE-Q
global score (r = 0.09, p = 0.2282). From the Bland–Altman analysis, the EREC scale had
a better agreement with the CCWS, but a low agreement with the EDE-Q global score
(Figure S2). Cronbach’s alpha was 0.87 in the subset, suggesting good internal consistency. 9 of 14
of 15 Nutrients 2022, 14, 4517
Nutrients 2022, 14, x FOR Figure 2. Scatter plots for the association between (a) climate change worry with eating-related
concern; (b) EDE-Q global score with eating-related eco-concern; and (c) shape concern with ea
related eco-concern. Grey area represents 95% confidence interval. Figure 2. Scatter plots for the association between (a) climate change worry with eating-r
eco-concern; (b) EDE-Q global score with eating-related eco-concern; and (c) shape concern
eating-related eco-concern. Grey area represents 95% confidence interval. Figure 2. Scatter plots for the association between (a) climate change worry with eating-related eco-
concern; (b) EDE-Q global score with eating-related eco-concern; and (c) shape concern with eating-
related eco-concern. Grey area represents 95% confidence interval. Figure 2. Scatter plots for the association between (a) climate change worry with eating-related
eco-concern; (b) EDE-Q global score with eating-related eco-concern; and (c) shape concern with
eating-related eco-concern. Grey area represents 95% confidence interval. Figure 2. Scatter plots for the association between (a) climate change worry with eating-related eco-
concern; (b) EDE-Q global score with eating-related eco-concern; and (c) shape concern with eating-
related eco-concern. Grey area represents 95% confidence interval. Figure 2. Scatter plots for the association between (a) climate change worry with eating-related
eco-concern; (b) EDE-Q global score with eating-related eco-concern; and (c) shape concern with
eating-related eco-concern. Grey area represents 95% confidence interval. 10 of 14 10 of 14 Nutrients 2022, 14, 4517 Female participants demonstrated a significantly higher level of climate change worry
(Mean = 30.26, SD = 7.72) compared with males [Mean = 25.94, SD = 8.75; t (176) = −2.86,
p = 0.0047] in the subset. 3.2.3. Internal Consistency In contrast to the results for the whole sample, female partic-
ipants in the subset had higher levels of eating-related eco-concern than male partici-
pants (t (176) = −2.13, p = 0.0348, Meanfemales = 25.01, SDfemales = 7.49, Meanmales = 21.94,
SDmales = 7.86). Table S3 presents the results from the linear regression models. In the subset, the
only significant association observed was between the CCWS and the EREC scores, where
participants with higher climate change worry also had higher eating-related eco-concern. 4. Discussion Climate
change worry was not significantly associated with the EDE-Q global score or any disor-
dered eating characteristics, indicating that a general worry about climate change might
not be directly associated with one’s level of disordered eating. Further, eating-related
eco-concern was positively associated with the EDE-Q global score and shape concern
while adjusting for age. One possible mechanism underlying these associations is self-
lessness (i.e., the tendency to relinquish one’s own needs for others’ needs), which has
been associated with elevated risk of disordered eating [47]. Selflessness might also im-
pact one’s food choices: individuals with a higher degree of selflessness are more likely
to consume fruits, vegetables, and grains compared with those with a lower degree of
selflessness [48,49]. Thus, selflessness may be an important mediating factor underlying
the association between eating-related eco-concern and disordered eating, particularly
shape concern. Indeed, previous studies have examined the association between certain
types of diet, such as veganism, vegetarianism, and semi-vegetarianism, with disordered
eating, yielding mixed results [50–54]. The inconsistencies might be due to various motiva- Nutrients 2022, 14, 4517 11 of 14 11 of 14 tions underpinning vegetarianism, such as ethical, religious, environmental, and health
concerns [52,55,56]. Thus, future studies with sufficient statistical power should examine
the association between diet choices specifically due to eco-concern with disordered eating. The current findings also raise the question of whether eating-related eco-concern con-
tributes to subsequent disordered eating, specifically shape concern, or whether individuals
with existing shape concern are more likely to change eating behaviors due to eco-concern. The cross-sectional design used in this study was unable to determine the direction of
the effect. A sensitivity analysis among individuals whose EDE-Q global scores were below
clinical cut-off values was also conducted. Several differences between the sensitivity
analysis and analyses in the full sample were observed. First, the EREC score did not differ
significantly by sex assigned at birth in the full sample. In contrast, female participants
scored significantly higher on the EREC scale than male participants in the sensitivity
analysis. Indeed, the mean EREC score of females did not change significantly after
excluding those who exceeded the EDE-Q cut-off value, yet the mean EREC score of males
was significantly lower after excluding those who exceeded EDE-Q cut-off value. 4. Discussion This
suggest that male participants who had EDE-Q scores indicative of eating disorders were
more likely to have high levels of eating-related eco-concern than female participants who
meet EDE-Q cut-off values. Due to limited statistical power, we were unable to compare the
strength of associations between disordered eating and eating-related eco-concern in male
versus female participants. Furthermore, EDE-Q global score and shape concern subscale
score were significantly associated with eating-related eco-concern, yet these associations
became nonsignificant after excluding those who exceeded EDE-Q cut-off values. These
results suggest that the associations between eating-related eco-concern and disordered
eating were more pronounced in those whose scores on the EDE-Q were indicative of
possible eating disorders. These findings raise the question of direction of causality. Do
individuals with eating disorders further modify their eating behaviors due to eco-concern,
and/or do individuals who change their eating behaviors due to eco-concern may develop
subsequent eating disorders? A longitudinal study is needed to elucidate the causality of
these relationships to provide more evidence on the prevention and/or intervention of
disordered eating and eating disorders. Importantly, all questions in the EREC scale do not necessarily represent disordered
eating. The goal of this study was to explore a range of behaviors that may be associated
with modifications in eating behavior and attitudes related to climate change. Behaviors
assessed in the EREC scale, like many healthful behaviors (e.g., eating fewer highly pro-
cessed foods, reducing consumption of red meat, eating more local foods), when taken
to an extreme, or when seen together with a more concerning degree of restrictive eat-
ing, could represent a component of a concerning behavioral pattern. Indeed, one of the
challenges of treating individuals with eating disorders who have concerns about climate
change is differentiating between truly health-damaging behaviors and behaviors that are
sensible and deserve to remain in their eating repertoire because they are sustainable and
climate-friendly while being compatible with recovery from an eating disorder. In summary, this initial study developed and validated the first scale to assess eating-
related eco-concern, and is the first study to examine the association between climate change
worry, eating-related eco-concern, and disordered eating. Findings from this study will
raise awareness of this new topic within the field of disordered eating and eating disorders. Several limitations need to be taken into consideration. 4. Discussion First, due to limited time and
funding, we only recruited one sample where participants only completed the survey once;
thus, it was not possible to examine the test–retest validity using multiple samples. Second,
the sample was predominately comprised of White/Caucasian participants in the United
States. This limits the generalizability of the findings. Future research should validate this
scale in individuals who live in different geographical or climatic regions and in a more
diverse sample. Additionally, disordered eating was evaluated via a single scale. Studies
using other measures and populations (i.e., individuals with eating disorders) will expand Nutrients 2022, 14, 4517 12 of 14 12 of 14 understanding of the association between eco-concern with disordered eating or eating
disorders. Lastly, it was not possible to examine the temporal sequence of the associations
among climate change worry, eating-related eco-concern, and disordered eating due to the
cross-sectional design of this study; thus, causality could not be determined. Longitudinal
studies are needed to establish a potential relationship. 5. Conclusions The current study developed a 10-item scale to assess eating-related eco-concern
and explored its association with disordered eating based on clinical observations in
eating disorder treatment. These findings can bring awareness to the potentially new
area within the fields of disordered eating, eating disorders, and climate change, which
is important from both a research and clinical perspective. Healthcare providers should
consider whether individuals endorse eating behaviors due to eco-concern while screening
for or assessing other disordered eating behaviors. Prevention strategies, including further
education about the impact of climate change and eco-concern on disordered eating, eating
disorders, and mental health in general, should be incorporated into activities such as
climate action clubs at schools or universities. Future studies should examine psychometric
properties of the EREC scale in diverse samples, evaluate associations between eating-
related eco-concern with eating disorder diagnosis and other mental health conditions, and
further explore sex differences and temporal sequence of these associations. Supplementary Materials: The following supporting information can be downloaded at: https://
www.mdpi.com/article/10.3390/nu14214517/s1. Supplementary Material (S1): Eating-Related Eco-
Concern Questionnaire. Table S1. Descriptive statistics for climate change worry and eating-related
eco-concern scores, and for the EDE-Q global and subscale scores in participants whose EDE-Q
global scores were below clinical cut-off values (n = 178). Table S2. Descriptive statistics and factor
loadings for items included in the Eating-Related Eco-Concern Scale in participants whose EDE-Q
global scores were below clinical cut-off values (n = 178). Table S3. Associations among climate
change worry, eating-related eco-concern, and disordered eating in participants whose EDE-Q global
scores were below clinical cut-off values (n = 178). Figure S1. Histograms for (a) the Eating-Related
Eco-Concern (EREC) score; (b) the Climate Change Worry Scale (CCWS) score; (c) Eating Disorder
Examination-Questionnaire (EDE-Q) global score; (d) EDE-Q restraint subscale score; (e) EDE-Q
eating concern subscale score; (f) EDE-Q weight concern subscale score; and (g) EDE-Q shape concern
subscale score. Figure S2. Bland-Atman plot for (a) the Eating-Related Eco-Concern (EREC) scale
and the Climate Change Worry Scale (CCWS) and (b) the EREC scale (%) and the Eating Disorder
Examination-Questionnaire (EDE-Q) global score (%) in participants whose EDE-Q global scores
were below clinical cut-off values (n = 178). Author Contributions: Conceptualization, B.Q., E.C.B.-S., E.F.v.F., L.M.T., C.M.B. and M.A.M.-C.;
Methodology, B.Q., E.K.P., L.M.T., C.M.B. and M.A.M.-C.; Software, B.Q. and E.K.P.; Validation,
B.Q., E.K.P., G.E.C., A.K., A.S.D., S.M.J., E.C.B.-S., E.F.v.F., L.M.T., C.M.B. and M.A.M.-C.; Formal
Analysis, B.Q. 5. Conclusions and E.K.P.; Investigation, B.Q., E.K.P., G.E.C., A.K., A.S.D., S.M.J., E.C.B.-S., E.F.v.F.,
L.M.T., C.M.B. and M.A.M.-C.; Resources, C.M.B.; Data Curation, E.K.P.; Writing—Original Draft
Preparation, B.Q., E.K.P., G.E.C., A.K., A.S.D. and S.M.J.; Writing—Review and Editing, E.C.B.-S.,
E.F.v.F., L.M.T., C.M.B. and M.A.M.-C.; Visualization, B.Q. and E.K.P.; Supervision, L.M.T., C.M.B. and M.A.M.-C.; Project Administration, M.A.M.-C.; Funding Acquisition, C.M.B. All authors have
read and agreed to the published version of the manuscript. Author Contributions: Conceptualization, B.Q., E.C.B.-S., E.F.v.F., L.M.T., C.M.B. and M.A.M.-C.;
Methodology, B.Q., E.K.P., L.M.T., C.M.B. and M.A.M.-C.; Software, B.Q. and E.K.P.; Validation,
B.Q., E.K.P., G.E.C., A.K., A.S.D., S.M.J., E.C.B.-S., E.F.v.F., L.M.T., C.M.B. and M.A.M.-C.; Formal
Analysis, B.Q. and E.K.P.; Investigation, B.Q., E.K.P., G.E.C., A.K., A.S.D., S.M.J., E.C.B.-S., E.F.v.F.,
L.M.T., C.M.B. and M.A.M.-C.; Resources, C.M.B.; Data Curation, E.K.P.; Writing—Original Draft
Preparation, B.Q., E.K.P., G.E.C., A.K., A.S.D. and S.M.J.; Writing—Review and Editing, E.C.B.-S.,
E.F.v.F., L.M.T., C.M.B. and M.A.M.-C.; Visualization, B.Q. and E.K.P.; Supervision, L.M.T., C.M.B. and M.A.M.-C.; Project Administration, M.A.M.-C.; Funding Acquisition, C.M.B. All authors have
read and agreed to the published version of the manuscript. Funding: C.M.B. is supported by the National Institute of Mental Health (R01 MH120170; R01
MH124871; R01 MH119084; R01 MH118278); Brain and Behavior Research Foundation Distinguished
Investigator Grant; Swedish Research Council (Vetenskapsrådet, award: 538-2013-8864); Lundbeck
Foundation (Grant no. R276-2018-4581). M.A.M-C acknowledges funding from the National Institute
on Alcohol Abuse and Alcoholism (K01 AA025113). Institutional Review Board Statement: The study was conducted in accordance with the Declaration
of Helsinki, and approved by The University of North Carolina at Chapel Hill Biomedical Institutional
Review Board (reference number 22-0134, approval letter dated 03 February 2022). Nutrients 2022, 14, 4517 13 of 14 13 of 14 Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Data Availability Statement: Not applicable. Conflicts of Interest: C.M.B. reports: Shire (grant recipient, Scientific Advisory Board member);
Lundbeckfonden (grant recipient); Pearson (author, royalty recipient); Equip Health Inc. (Clinical
Advisory Board). All other authors report no conflict of interest. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Data Availability Statement: Not applicable. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Data Availability Statement: Not applicable. Conflicts of Interest: C.M.B. reports: Shire (grant recipient, Scientific Advisory Board member);
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disorders. Eat. Weight Disord. 2006, 11, 195–200. [CrossRef] [PubMed] 54. Fatima, W.; Fatima, R.; Anwar, N. Subclinical eating disorders and association with vegetarianism in female students of Saudi
Arabia: A crosssectional study. J. Nurs. Health Sci. 2018, 7, 62–67. 55. Aravena, J.; Zubarew, T.; Bedregal, P.; Zuz
2020, 91, 705–710. [CrossRef] [PubMed] 55. Aravena, J.; Zubarew, T.; Bedregal, P.; Zuzulich, S.; Urrejola, P. Vegetarian diets in first year university students. Rev. Chil. Pediatr. 2020, 91, 705–710. [CrossRef] [PubMed] 56. Rosenfeld, D.L.; Burrow, A.L. Vegetarian on purpose: Understanding the motivations of plant-based dieters. Appetite 2017,
116, 456–463. [CrossRef] [PubMed]
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English
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The Writings of Matthew Prior. I. Poems on Several Occasions
|
Modern language review/The modern language review
| 1,906
|
public-domain
| 1,424
|
There was therefore a clear opportunity for Mr Waller
to supply an accurate text for ordinary men at an ordinary price. This he has done admirably. He professes to give no more than a
good text, and he modestly confines his remarks to a short prefatory
bibliographical 'note.'
The text reproduces all the 'originalities'
of the folio-spelling,
punctuation, italics, brackets-to
the great
delight, I am sure, of Mr Dobson. It calls for no criticism beyond the
test of its editorial care. That it stands that test is its best praise. The second, and concluding, volume will contain, in addition to the
poems which do not appear in the folio, the new matter of the Prose
Dialogues from Lord Bath's Ais. at Longleat. as its author has said of a certain monarch. But the folio, which
would make a better monument to Prior the ambassador at Utrecht
than to Prior the best of England's epigrammatists, is difficult to get
and to handle. There was therefore a clear opportunity for Mr Waller
to supply an accurate text for ordinary men at an ordinary price. This he has done admirably. He professes to give no more than a
good text, and he modestly confines his remarks to a short prefatory
bibliographical 'note.'
The text reproduces all the 'originalities'
of the folio-spelling,
punctuation, italics, brackets-to
the great
delight, I am sure, of Mr Dobson. It calls for no criticism beyond the
test of its editorial care. That it stands that test is its best praise. The second, and concluding, volume will contain, in addition to the
poems which do not appear in the folio, the new matter of the Prose
Dialogues from Lord Bath's Ais. at Longleat. G. GREGORY SMITH. G. GREGORY SMITH. The Poetical Works of William Blake. A new and Verbatim Text from
the Manuscript, Engraved and Letterpress Originals. Edited by
JOHN SAMPSON. Oxford: Clarendon Press. 1905. 8vo. xxxvi+
384 pp. The Poetical Works of William Blake. A new and Verbatim Text from
the Manuscript, Engraved and Letterpress Originals. Edited by
JOHN SAMPSON. Oxford: Clarendon Press. 1905. 8vo. xxxvi+
384 pp. pp. The Lyrical Poems of William Blake. Text by JOHN SAMPSON. With
an Introduction
by WALTER RALEIGH. Oxford: Clarendon Press. 1905. 8vo. li+ 196 pp. pp. The Lyrical Poems of William Blake. Text by JOHN SAMPSON. With
an Introduction
by WALTER RALEIGH. Oxford: Clarendon Press. 1905. 8vo. li+ 196 pp. Probably nothing will take its place, for Nothing went before so Great
And nothing Greater can succeed
Nothing went before so Great
And nothing Greater can succeed as its author has said of a certain monarch. But the folio, which
would make a better monument to Prior the ambassador at Utrecht
than to Prior the best of England's epigrammatists, is difficult to get
and to handle. There was therefore a clear opportunity for Mr Waller
to supply an accurate text for ordinary men at an ordinary price. This he has done admirably. He professes to give no more than a
good text, and he modestly confines his remarks to a short prefatory
bibliographical 'note.'
The text reproduces all the 'originalities'
of the folio-spelling,
punctuation, italics, brackets-to
the great
delight, I am sure, of Mr Dobson. It calls for no criticism beyond the
test of its editorial care. That it stands that test is its best praise. The second, and concluding, volume will contain, in addition to the
poems which do not appear in the folio, the new matter of the Prose
Dialogues from Lord Bath's Ais. at Longleat. as its author has said of a certain monarch. But the folio, which
would make a better monument to Prior the ambassador at Utrecht
than to Prior the best of England's epigrammatists, is difficult to get
and to handle. There was therefore a clear opportunity for Mr Waller
to supply an accurate text for ordinary men at an ordinary price. This he has done admirably. He professes to give no more than a
good text, and he modestly confines his remarks to a short prefatory
bibliographical 'note.'
The text reproduces all the 'originalities'
of the folio-spelling,
punctuation, italics, brackets-to
the great
delight, I am sure, of Mr Dobson. It calls for no criticism beyond the
test of its editorial care. That it stands that test is its best praise. The second, and concluding, volume will contain, in addition to the
poems which do not appear in the folio, the new matter of the Prose
Dialogues from Lord Bath's Ais. at Longleat. as its author has said of a certain monarch. But the folio, which
would make a better monument to Prior the ambassador at Utrecht
than to Prior the best of England's epigrammatists, is difficult to get
and to handle. This content downloaded from 195.34.78.242 on Wed, 25 Jun 2014 03:30:35 AM
All use subject to JSTOR Terms and Conditions The Writings of Matthew Prior. I. Poems on Several Occasions by A. R. Waller
Review by: G. Gregory Smith
The Modern Language Review, Vol. 1, No. 4 (Jul., 1906), p. 343
Published by: Modern Humanities Research Association
Stable URL: http://www.jstor.org/stable/3713465 . Accessed: 25/06/2014 03:30 Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at
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All use subject to JSTOR Terms and Conditions Reviews
Reviews 343
343 The Writings of Matthew Prior. I. Poems on Several Occasions. The
Text edited by A. R. WALLER (Cambridge English Classics). Cambridge: University Press. 1905. 8vo. xxvi +268 pp. The Writings of Matthew Prior. I. Poems on Several Occasions. The
Text edited by A. R. WALLER (Cambridge English Classics). Cambridge: University Press. 1905. 8vo. xxvi +268 pp. There has been no lack of critical interest in Mat. Prior's work, from
Johnson's Life down to Mr Dobson's sympathetic 'vignette,' but of
critical texts it would be hard to say that 'nothing remained to be
done.' Mr Dobson has given us a reprint of'Selected
Poems,' and
Mr Brimley Johnson a modernized version in the 'Aldine Poets.'
Neither of these can be accepted as substitutes for Mat.'s stupendous
folio. Probably nothing will take its place, for
There has been no lack of critical interest in Mat. Prior's work, from
Johnson's Life down to Mr Dobson's sympathetic 'vignette,' but of
critical texts it would be hard to say that 'nothing remained to be
done.' Mr Dobson has given us a reprint of'Selected
Poems,' and
Mr Brimley Johnson a modernized version in the 'Aldine Poets.'
Neither of these can be accepted as substitutes for Mat.'s stupendous
folio. Though Mr Sampson and Mr Raleigh admit that the inner mysteries
of prophet Blake have yet to be unravelled, it must be said that they
have done more than all their predecessors to help criticism in its dis-
tracting task. Mr Sampson's work is mainly, if not exclusively, textual
and bibliographical. He argues for the use of "d' in some of Blake's
participles and for the advantage of preserving the poet's ' tyger' and
'desart'-niceties
which Gilchrist and his fellows despised, and which
the aesthetic brethren may refuse to be of moment to the message of
the mystic. But Mr Sampson has confidence in his accuracy, and he
shows that it is not an impertinence. For, apart from the general
Though Mr Sampson and Mr Raleigh admit that the inner mysteries
of prophet Blake have yet to be unravelled, it must be said that they
have done more than all their predecessors to help criticism in its dis-
tracting task. Mr Sampson's work is mainly, if not exclusively, textual
and bibliographical. He argues for the use of "d' in some of Blake's
participles and for the advantage of preserving the poet's ' tyger' and
'desart'-niceties
which Gilchrist and his fellows despised, and which
the aesthetic brethren may refuse to be of moment to the message of
the mystic. But Mr Sampson has confidence in his accuracy, and he
shows that it is not an impertinence. For, apart from the general
|
https://openalex.org/W2236658294
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https://hal-riip.archives-ouvertes.fr/pasteur-01351206/document
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English
| null |
Workplace exposure to diesel and gasoline engine exhausts and the risk of colorectal cancer in Canadian men
|
Environmental health
| 2,016
|
cc-by
| 10,732
|
Workplace exposure to diesel and gasoline engine
exhausts and the risk of colorectal cancer in Canadian
men. Linda Kachuri, Paul J Villeneuve, Marie-Élise Parent, Kenneth C Johnson,
Shelley A Harris To cite this version: Linda Kachuri, Paul J Villeneuve, Marie-Élise Parent, Kenneth C Johnson, Shelley A Harris. Work-
place exposure to diesel and gasoline engine exhausts and the risk of colorectal cancer in Canadian
men.. Environmental health : a global access science source [electronic resource], 2016, 15 (4), pp.1-12. 10.1186/s12940-016-0088-1. pasteur-01351206 Workplace exposure to diesel and gasoline engine
exhausts and the risk of colorectal cancer in Canadian
men. Distributed under a Creative Commons Attribution 4.0 International License © 2016 Kachuri et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. RESEARCH Open Access Open Access Abstract Conclusions: Our findings suggest that sustained high-level exposure diesel emissions may increase the risk
of rectal cancer. Keywords: Diesel emissions, Gasoline emissions, Colon cancer, Rectal cancer, Colorectal cancer, Occupational
cancer, Case–control study Keywords: Diesel emissions, Gasoline emissions, Colon cancer, Rectal cancer, Colorectal cancer, Occupational
cancer, Case–control study Workplace exposure to diesel and gasoline
engine exhausts and the risk of colorectal
cancer in Canadian men Linda Kachuri1,2,3*
, Paul J. Villeneuve1,2,4, Marie-Élise Parent5, Kenneth C. Johnson6, the Canadian Cancer Registries
Epidemiology Research Group and Shelley A. Harris1,2,3 * Correspondence: linda.kachuri@mail.utoronto.ca
1Dalla Lana School of Public Health, University of Toronto, 155 College Street,
6th Floor, Toronto, ON M5T 3 M7, Canada
2Occupational Cancer Research Centre, Cancer Care Ontario, 525 University
Avenue, 3rd Floor, Toronto, ON M5G 2 L3, Canada
Full list of author information is available at the end of the article HAL Id: pasteur-01351206
https://riip.hal.science/pasteur-01351206v1
Submitted on 2 Aug 2016 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 Kachuri et al. Environmental Health (2016) 15:4
DOI 10.1186/s12940-016-0088-1 Abstract Background: The International Agency for Research on Cancer (IARC) classified diesel exhaust as carcinogenic to
humans (Group 1) and gasoline exhaust as a possible carcinogen (Group 2B) based studies of lung cancer, however
the evidence for other sites is limited. We addressed this question by investigating exposure to diesel and gasoline
emissions with respect to risk of colorectal cancer in men. Methods: We used data from a population-based case–control study with incident cases of colon (n = 931) and
rectal (n = 840) cancer and 1360 controls from 7 Canadian provinces conducted in 1994–1997. Lifetime occupational
history and information on other risk factors was collected. Occupational hygienists, blinded to case–control status,
assigned exposures to each job for 3 dimensions: concentration, frequency, and reliability. Logistic regression
was used to estimate odds ratios (OR) and their 95 % confidence intervals (CI), adjusted for age, province, use
of proxy respondents, smoking, body-mass index, physical activity, intake of alcohol, processed meats, and
occupational exposure to asbestos and aromatic amines. Results: Among CRC cases, 638 (36 %) were exposed to diesel and 814 (46 %) were exposed to gasoline
emissions. Relative to the unexposed, elevated risks were observed among subjects ever exposed to high
concentration levels of diesel emissions for colorectal cancer (OR = 1.65, 95 % CI = 0.98–2.80) and rectal cancer
(OR = 1.98, 95 % CI = 1.09–3.60), but not colon cancer. Prolonged (>10 years) exposure at high concentrations
was also associated with high risks of rectal cancer (OR = 2.33 95 % CI = 0.94–5.78; p-trend = 0.02). No
statistically significant associations were observed for gasoline emissions. Results: Among CRC cases, 638 (36 %) were exposed to diesel and 814 (46 %) were exposed to gasoline
emissions. Relative to the unexposed, elevated risks were observed among subjects ever exposed to high
concentration levels of diesel emissions for colorectal cancer (OR = 1.65, 95 % CI = 0.98–2.80) and rectal cancer
(OR = 1.98, 95 % CI = 1.09–3.60), but not colon cancer. Prolonged (>10 years) exposure at high concentrations
was also associated with high risks of rectal cancer (OR = 2.33 95 % CI = 0.94–5.78; p-trend = 0.02). No
statistically significant associations were observed for gasoline emissions. Conclusions: Our findings suggest that sustained high-level exposure diesel emissions may increase the risk
of rectal cancer. Background Although in most developed coun-
tries exposures to gasoline emissions have surpassed
those from diesel, few studies have examined its effects
on cancer risk, especially for sites other than the lung. y
g g
p
y
Of the total cases ascertained, physician consent was
obtained for 89 % of rectal (n = 1169) and 85 % (n =
1375) colon cases. Deceased cases were excluded (76
rectal; 138 colon), as well as patients whose physician re-
fused consent (71 rectal; 98 colon). The NECSS collected
detailed information for a number of modifiable cancer
risk factors, including: sociodemographic characteristics,
anthropometry, diet, smoking, alcohol use, exposure to
environmental tobacco smoke, and physical activity. Individuals were also asked to provide details on their
lifetime residential and occupational history. Information
on diet from 2 years prior to the interview was collected
using a modified 60-item food-frequency diet question-
naire based on instruments that have been extensively vali-
dated [17, 18]. Completed questionnaire were returned by
830 rectal and 959 male colon cases, representing 71 and
70 % of those contacted, respectively. Questionnaires were
mailed to 4270 men who were identified as eligible con-
trols. Approximately 7 % (n = 287) were returned due to an
incorrect address. Completed questionnaires were received
from 2547 male controls, representing 59.6 % of those
ascertained and 64.0 % of those contacted. Colorectal cancer (CRC), affecting the colon and/or
the rectal areas, is a major contributor to the worldwide
cancer burden [6]. CRC has been linked to several modi-
fiable risk factors including obesity, physical inactivity,
consumption of red and processed meat and smoking
[7–9]. In addition to physical activity, a potential role for
other occupational risk factors has been suggested [10, 11]. To date, few studies have investigated the relationship
between exposure to diesel and gasoline emissions and
CRC risk, but they did report modest positive associations
[12–14]. However, only two case–control studies used a
systematic and validated approach to exposure assessment
[12, 13], and many of these studies lacked detailed infor-
mation on several major lifestyle-related CRC risk factors,
such as body-mass index (BMI), diet and physical activity. Currently, the overall evidence linking diesel and gasoline
emissions to CRC is considered to be weak [3]. Further
investigating the carcinogenic potential of these prevalent
exposures has been identified as a high priority by IARC
[15]. Background engine exhaust [1, 2]. The two largest exposed occupa-
tional groups are truck drivers and heavy equipment op-
erators [1, 2]. Diesel and gasoline are the most widely
used fuels in combustion engines, and their emissions
are comprised of a complex mixture of chemicals, in-
cluding polycyclic aromatic hydrocarbons (PAHs) and
nitroarenes, carbon monoxide, and volatile organic com-
pounds, such as benzene and formaldehyde [3, 4]. While similar particles are emitted from both gasoline
and diesel powered engines, the distribution and surface
properties of the particles are different, which suggests Motor vehicle exhaust emissions are ubiquitous, with
exposure occurring through indoor and outdoor air, as
well as in many occupational settings. An estimated
781,000 workers (92% of employed males), or 4.6 % of
the working Canadian population, are exposed to diesel * Correspondence: linda.kachuri@mail.utoronto.ca
1Dalla Lana School of Public Health, University of Toronto, 155 College Street,
6th Floor, Toronto, ON M5T 3 M7, Canada
2Occupational Cancer Research Centre, Cancer Care Ontario, 525 University
Avenue, 3rd Floor, Toronto, ON M5G 2 L3, Canada
Full list of author information is available at the end of the article Kachuri et al. Environmental Health (2016) 15:4 Page 2 of 12 Page 2 of 12 potential differences in the health effects associated
with these exposures [5]. collected in Ontario, therefore this analysis is based on
male participants from 7 provinces. Cancer-free controls
were recruited using a random sample of the provincial
population obtained from health insurance plans in 5
provinces (Prince Edward Island, Nova Scotia, Manitoba,
Saskatchewan and British Columbia) and random-digit
dialing in Newfoundland and Alberta [16]. Frequency-
matching to the overall case grouping (19 cancer sites)
was used to select controls for every case within each sex
and 5-year age group for any cancer site. The International Agency for Research on Cancer
(IARC) recently classified diesel exhaust as carcinogenic
to humans (Group 1), based on studies of lung cancer,
an update from the 1988 classification of probably car-
cinogenic to humans (Group 2A) [3]. However, the evi-
dence for other cancers remains limited. Gasoline was
classified as a possible human carcinogen (Group 2B) by
IARC in 1989 and again in 2012, based on inadequate
evidence in humans and sufficient evidence in experi-
mental animals [3]. Study population The data used in this analysis are from the colon and
rectal cancer case–control components of the Canadian
National Enhanced Cancer Surveillance System (NECSS),
a collaborative effort between Health Canada and the
cancer registries in 8 Canadian provinces. The NECSS
collected data from a population-based sample of 21022
cases of 19 different cancers and 5039 controls between
1994 and 1997. Data for rectal and colon cases were not Background To our knowledge, this is the largest population-
based case–control study of the effects of diesel and
gasoline emissions on the risk of colon and rectal cancer,
with a comprehensive exposure assessment strategy, along
with detailed information on important confounders. This work addresses an important gap in our under-
standing of the extensive health effects of exposure to
diesel and gasoline emissions, and has implications
for preventive efforts. The study sample was restricted to men because we
expected few women to be occupationally exposed to
diesel and gasoline emissions during the relevant expos-
ure periods (between the 1960s–1990s). We excluded
individuals under the age of 40, and those who had not
worked for at least one year, since individuals with shorter
employment histories would have had fewer opportunities
for exposure, and their CRC diagnoses would be more
likely attributable to genetic conditions, such as familial ad-
enomatous polyposis and hereditary non-polyposis colorec-
tal cancer, which confer lifetime risks as high as 70–80 %
[19, 20], with a mid-40s average age at diagnosis [21]. In
the NECSS, 2.9 % (n = 28) of incident colon cancer and
2.1 % (n = 18) rectal cancer cases were diagnosed before
the age of 40; the corresponding number of controls
excluded to meet the age requirement was 282. After
applying the exclusion criteria there remained a total of
931 colon and 840 rectal cancer cases, and 1360 controls. Kachuri et al. Environmental Health (2016) 15:4 In addition, jobs were not coded for diesel or gasoline
emissions exposure if they were self-reported as retire-
ment (n = 87), student (n = 35), disability (n = 5), institu-
tionalized (n = 1), and unemployed (n = 10). Exposure to diesel and gasoline emissions
The effects of occupational exposure to diesel and
gasoline emissions were modeled using 5 exposure met-
rics: ever exposure, highest attained exposure concentra-
tion, duration of exposure, frequency of exposure, and
cumulative exposure. Ever exposure was modeled as a
binary variable indicating that an individual had worked
in a job that was classified as exposed across any of the
dimensions evaluated in the exposure assessment. The
highest attained exposure concentration corresponded
to the maximum concentration value assigned to jobs
with an individual’s employment history. Duration of
exposure corresponded to years of employment in occu-
pations classified as exposed. Duration was modeled
continuously, as well as using ordinal categories based
on tertiles of exposure duration among the controls. Each set of cut-points was determined separately for
diesel and gasoline emissions. y
The occupational exposure assessment was based on
the expert-approach, where a team of industrial hygien-
ists assigned individual exposures based on details pro-
vided for each job [26]. This approach is considered to
be reliable and recognized as the reference method for
such a study design [27]. It has been described in detail
elsewhere [28], and has been successfully applied to
other case–control studies [28, 29] and previously pub-
lished analyses of NECSS data [22–24]. When assigning
exposures, hygienists had access to previous NECSS
expert-based codings, summarized as a job exposure
matrix, which they could modify to reflect circumstances
specific to each job description. Exposure coding was re-
peated for a random subset of 96 participants with 385
jobs to estimate the reliability of the exposure coding. Our analysis of this subset suggests excellent inter-rater
agreement between exposure coders (weighted kappa =
0.81, 95 % CI: 0.78-0.85; manuscript in preparation). The cumulative measure of diesel and gasoline exposure
was defined as: CE ¼
X
k
i¼1
CiFiDi The assignment of exposure to diesel and gasoline
emissions was carried out across three dimensions: con-
centration, frequency, and reliability. Each of these vari-
ables was defined using a semi-quantitative scale: none
(unexposed), low, medium, or high. Non-exposure was
defined as exposure up to background levels found in
the general environment. Kachuri et al. Environmental Health (2016) 15:4 Kachuri et al. Environmental Health (2016) 15:4 Page 3 of 12 Page 3 of 12 they were 18 years old to the time of the interview. The
information for each occupation included the job title,
main tasks performed, type of industry, location, and
period of employment. Using these job descriptions, a
team of chemists and industrial hygienists, who were
blinded to case or control status, carried out a comprehen-
sive exposure assessment and determined the exposure
status of each job with respect to diesel emissions, gasoline
emissions, aromatic amines, asbestos and crystalline silica. Concentration was assessed on a relative scale with
respect to established benchmarks. Low concentration
was assigned to subjects with the lowest possible concen-
tration that could still be considered above the background
level of the general population. The same exposure assess-
ment approach was also applied to a number of other
exposures of interest and potential confounders, including
crystalline silica, asbestos and aromatic amines. Concentration was assessed on a relative scale with
respect to established benchmarks. Low concentration
was assigned to subjects with the lowest possible concen-
tration that could still be considered above the background
level of the general population. The same exposure assess-
ment approach was also applied to a number of other
exposures of interest and potential confounders, including
crystalline silica, asbestos and aromatic amines. The reliability metric was used to measure the confi-
dence that the exposure was actually present in the job
that was evaluated. Low reliability refers to “possible” ex-
posure, medium reliability was interpreted as “probable”
exposure, and high reliability corresponded to “definite”
exposure. In order to reduce the potential for exposure
misclassification, all exposure metrics that used in this
analysis classified estimates with a low reliability score
corresponding to “possible” exposure (n = 613, 30.4 %
for diesel and n = 342, 19.7 % for gasoline) as unexposed. y
As in our previously published studies of lung cancer
[22–24], the occupation and industry coding was reviewed
and upgraded to the more precise 7-digit Canadian
Classification and Dictionary of Occupation (CCDO)
codes [25] which were used in the subsequent steps of the
exposure assessment. A total of 16,009 jobs contained suffi-
cient occupational information for the exposure assess-
ment. Exposure metrics were not assigned to jobs with
insufficient information (n = 199), including those missing
full-time, seasonal or part-time employment status (n = 74). Kachuri et al. Environmental Health (2016) 15:4 The frequency of exposure was
determined based on the proportion of time in a typical
workweek that the participant was exposed to a substance. Low frequency of exposure corresponded to <5 %,
5 %–30 % was classified as medium, and high fre-
quency was defined as >30 % of work time. This metric
also accounted for part-time or seasonal employment. where CE was the cumulative exposure, i represented the
ith job held, k was the total number of jobs held, C was the
concentration of diesel or gasoline exposure (low, medium,
high), F was the frequency of exposure (low, medium,
high), and D was the duration of employment in years. Assignment of occupational exposures Participants were asked to provide information for each
job held, in Canada, for at least 12 months from the time Kachuri et al. Environmental Health (2016) 15:4 Kachuri et al. Environmental Health (2016) 15:4 Kachuri et al. Environmental Health (2016) 15:4 Page 4 of 12 Page 4 of 12 diesel and gasoline exposure were treated as continuous
in the regression model in order to obtain an estimate of
the slope and associated p-values (p-trend). Descriptive
analyses were conducted and the influence of possible
risk factors suggested by the literature and by previous
analyses of NECSS data was investigated. These included
the characteristics presented in Table 1, as well as
additional nutritional factors, including consumption of
red meat and intake of added fat. Exposure metrics for
diesel and gasoline emissions were entered into the
statistical models together in order to account for the
simultaneous exposure to both of these constituents of
motor vehicle exhaust. Additional occupational con-
founders and potential co-exposures included asbestos
and aromatic amines, which were entered into the
models as dichotomous variables. diesel and gasoline exposure were treated as continuous
in the regression model in order to obtain an estimate of
the slope and associated p-values (p-trend). Descriptive
analyses were conducted and the influence of possible
risk factors suggested by the literature and by previous
analyses of NECSS data was investigated. These included
the characteristics presented in Table 1, as well as
additional nutritional factors, including consumption of
red meat and intake of added fat. Exposure metrics for
diesel and gasoline emissions were entered into the
statistical models together in order to account for the
simultaneous exposure to both of these constituents of
motor vehicle exhaust. Additional occupational con-
founders and potential co-exposures included asbestos
and aromatic amines, which were entered into the
models as dichotomous variables. A total of 638 (36.0) CRC cases and 491 (36.1 %)
controls were exposed to diesel emissions at some point
during their occupational history (Table 2). Exposure to
gasoline emissions was more prevalent than diesel emis-
sions, with 814 (46.0) cases and 577 (57.6 %) controls
exposed (Table 3). Ever exposure to either diesel (OR =
0.88, 95 % CI = 0.74–1.06) or gasoline (OR = 1.10, 95 %
CI = 0.93–1.31) was not associated with statistically sig-
nificant increases in the odds of CRC. Similar results
were observed when colon and rectal cancers were
considered separately (Tables 4 and 5). Kachuri et al. Environmental Health (2016) 15:4 A positive association with CRC was observed for
individuals who had been exposed to diesel emissions at
high levels of concentration compared to unexposed indi-
viduals (OR = 1.65, 95 % CI = 0.98–2.80; p-trend = 0.78). Examining colon and rectal cancer separately (Table 4) re-
vealed that the highest attained concentration of diesel
emissions was more strongly associated with rectal
(OR = 1.98, 95 % CI = 1.09–3.60; p-trend = 0.56) than colon
cancer (OR = 1.35, 95 % CI = 0.72–2.54; p-trend = 0.91). The tests for trend did not reach statistical significance,
which suggests that the increase in rectal cancer risk is not
linear, and appears to sharply rise only for exposure to high
concentrations of diesel emissions. Adjustments were also made for age at cancer diagnosis
or interview, province of residence, use of proxy respon-
dents; BMI categories (<18.5 (underweight), 18.5 to <25.0
(normal), 25.0 to <30.0 (overweight), and ≥30 (obese));
percent (%) change from maximum lifetime BMI; cigarette
pack-years (number of years smoking on average 20
cigarettes per day); consumption of processed meat; and
physical activity. In order to comprehensively adjust for
different types of physical activity, a composite index
was created, which included hours per month of moderate
and strenuous activity [30, 31]. Results for minimally ad-
justed models with age, province of residence, and use of
proxy respondents as the only covariates are provided
as Additional file 1: Table S1, Additional file 2: Table S2,
Additional file 3: Table S3, Additional file 4: Table S4. Duration of exposure to diesel emissions did not ap-
pear to be statistically significantly associated with any
cancer. However, statistically significant positive trends
in cancer risk where revealed when analyses were re-
stricted duration of exposure at high concentrations
only. Compared to the unexposed, men with >10 years
of diesel exposure at high concentrations had an ele-
vated risk of colorectal (OR = 1.90, 95 % CI = 0.85–4.23;
p-trend = 0.02) and rectal cancer (OR = 2.33, 95 % CI =
0.94–5.78; p-trend = 0.02) compared to the unexposed. All analyses were conducted using SAS version 9.4
(Cary, N.C.). The participating cancer registries obtained approval
of the NECSS study protocol from their respective ethics
review boards. The study was also approved by Research
Ethics Boards at Health Canada and the University of
Toronto. Results A summary of socio-demographic and occupational
characteristics is presented in Table 1. Compared to con-
trols, CRC cases were older and more likely to require
the use of proxy respondents. A statistically significant
association emerged between increasing BMI and CRC
risk. Consistent with previous studies, increased con-
sumption of processed meat was positively associated
with CRC (p < 0.05). However, there was no association
with consumption of fruits and vegetables, or physical ac-
tivity. Among the additional occupational agents that were
evaluated, ever exposure to aromatic amines (OR = 1.73, 95
% CI = 0.84–3.45), asbestos (OR = 1.43, 95 % CI = 1.15–
1.78), and crystalline silica (OR = 1.09, 95 % CI = 0.93–
1.28) were associated with increased risk of CRC. Kachuri et al. Environmental Health (2016) 15:4 Exposure to gasoline emissions was not associated
with a marked increase in the risk of CRC, colon or rec-
tal cancers for any of the metrics tested. Notably, cumu-
lative exposure to either diesel or gasoline emissions was
not associated with an appreciable increase in the odds
of CRC, colon, or rectal cancer. Statistical analysis Odds ratios (OR) and 95 % confidence intervals (CI)
were estimated using unconditional logistic regression
for each of the exposure metrics. Trends were also
examined using logistic regression. Ordinal metrics of Discussion Exposure to diesel and gasoline emissions is widespread,
and accurately assessing its impact on multiple health
outcomes continues to be a public health priority. In this
study, our aim was to conduct a comprehensive investi-
gation of occupational exposure to diesel and gasoline
emissions on colon and rectal cancer risk, using data
from a large population-based case–control study. Des-
pite the robust associations previously observed for lung
cancer in these data [22] and other studies, the increases
in colon and rectal cancer risk appeared to be attenu-
ated. Statistically significant excesses in rectal cancer risk
were observed for exposure to diesel emissions at high Kachuri et al. Discussion Environmental Health (2016) 15:4 Page 5 of 12 Table 1 Selected demographic characteristics of incident colon and rectal cancer cases and controls in the Canadian National
Enhanced Cancer Surveillance System 1994–97
Colon (n = 931)
Rectum (n = 840)
Controls (n = 1360)
ORa
95 % CI
N
(%)
N
(%)
N
(%)
Age groups (years)
40 to <50
67
(7.2)
79
(9.4)
137
(10.1)
50 to <60
174
(18.7)
179
(21.3)
239
(17.6)
60 to <70
445
(47.9)
380
(45.2)
581
(45.7)
70≤
244
(26.2)
202
(24.1)
403
(29.6)
Province of residence
Newfoundland
42
(4.5)
46
(5.5)
105
(7.7)
Prince Edward Island
20
(2.2)
17
(2.0)
63
(4.6)
Nova Scotia
77
(8.3)
71
(8.5)
307
(22.6)
Manitoba
97
(10.4)
85
(10.1)
126
(9.3)
Saskatchewan
90
(9.7)
108
(12.9)
120
(8.8)
Alberta
283
(30.4)
184
(21.9)
265
(19.5)
British Columbia
322
(34.6)
329
(39.2)
374
(27.5)
Use of proxy respondents
None (self)
579
(62.2)
485
(57.7)
903
(66.4)
1.00
Other
352
(37.8)
355
(42.3)
457
(33.6)
1.35
(1.16,
1.57)
Education (total years)
< 9
219
(23.5)
189
(22.5)
308
(22.7)
1.00
9 to 11
295
(31.7)
273
(32.5)
395
(29.0)
0.99
(0.81,
1.21)
12 to 14
241
(25.9)
233
(27.7)
350
(25.7)
0.80
(0.64,
1.00)
14≤
176
(18.9)
145
(17.3)
307
(22.6)
0.61
(0.48,
0.77)
BMI (kg/m2)
Normal: 18.5 to <25
251
(27.0)
254
(30.2)
490
(36.0)
1.00
Underweight: <18.5
14
(1.5)
6
(0.7)
20
(1.5)
0.99
(0.52,
1.91)
Overweight: 25 to <30
464
(49.9)
418
(49.8)
641
(47.1)
1.44
(1.22,
1.70)
Obese: 30≤
202
(21.7)
162
(19.3)
209
(15.4)
1.89
(1.52,
2.35)
Percent change (%) from maximum lifetime weight in kg
Q1: <2.4
287
(30.8)
233
(27.7)
280
(20.6)
1.00
Q2: 2.4 to <4.9
226
(24.3)
202
(24.1)
359
(26.4)
0.60
(0.49,
0.74)
Q3: 4.9 to <8.8
222
(23.9)
202
(24.1)
336
(24.7)
0.68
(0.55,
0.83)
Q4: 8.8 ≤
196
(21.1)
203
(24.2)
385
(28.3)
0.56
(0.46,
0.69)
Cigarette pack-years
Never smokers
178
(19.4)
161
(19.5)
302
(22.6)
1.00
< 10
145
(15.8)
153
(18.6)
223
(16.7)
1.19
(0.94,
1.52)
10 to <20
173
(18.8)
136
(16.5)
233
(17.4)
1.19
(0.94,
1.50)
20 to <30
155
(16.9)
143
(17.3)
214
(16.0)
1.38
(1.08,
1.75)
30 to <40
107
(11.6)
104
(12.6)
147
(11.0)
1.43
(1.09,
1.88)
40 to <50
101
(11.0)
79
(9.6)
108
(8.1)
1.61
(1.20,
2.17)
50 to <60
25
(2.7)
16
(1.9)
53
(4.0)
0.86
(0.55,
1.36)
60≤
35
(3.8)
33
(4.0)
56
(4.2)
1.29
(0.86,
1.94)
Quartiles of alcohol intake (servings/week)
None
259
(27.8)
227
(27.0)
451
(33.2)
1.00 Table 1 Selected demographic characteristics of incident colon and rectal cancer cases and controls in the Canadian National
Enhanced Cancer Surveillance System 1994–97 Kachuri et al. bComposite index of physical activity was derived y summing the hours/month of moderate and strenuous activity Discussion Environmental Health (2016) 15:4 Page 7 of 12 levels of concentration, as well as for long durations of
exposure at high levels of concentration. Exposure to
gasoline emissions was not associated with elevated can-
cer risks in our data
Table 2 Adjusted odds ratios (OR) and corresponding 95 %
confidence intervals (CI) for colorectal cancer in relation to
occupational exposure to diesel emissions
Exposure Metrica
Cases (%)
Controls (%)
ORb (95 % CI)
Ever exposed
No
1133
(64.0)
869
(63.9)
1.00
Yes
638
(36.0)
491
(36.1)
0.88
(0.74,
1.06)
Highest attained exposure concentration
Unexposed
1133
(64.0)
869
(63.9)
1.00
Low
450
(25.4)
377
(27.7)
0.79
(0.65,
0.96)
Medium
136
(7.7)
89
(6.5)
1.00
(0.73,
1.36)
High
52
(2.9)
25
(1.8)
1.65
(0.98,
2.80)
p-trend
0.78
Duration of exposure (years)
Unexposed
1133
(64.5)
869
(64.4)
1.00
> 0 to <11
208
(11.9)
157
(11.6)
0.90
(0.70,
1.16)
≥11 to ≤31
220
(12.5)
166
(12.3)
0.91
(0.71,
1.18)
> 31
195
(11.1)
157
(11.6)
0.80
(0.60,
1.08)
p-trend
0.11
Duration of exposure at high concentration (years)
Unexposed
1719
(97.2)
1335
(98.2)
1.00
> 0 to ≤10
29
(1.6)
13
(1.0)
1.60
(0.80,
3.21)
> 10
21
(1.2)
11
(0.8)
1.90
(0.85,
4.23)
p-trend
0.04
Frequency of exposure
Unexposed
1191
(68.8)
910
(71.1)
1.00
Low: 5 %
76
(4.4)
60
(4.7)
1.21
(0.80,
1.82)
Medium: 6–30 %
351
(20.3)
229
(17.9)
1.10
(0.88,
1.38)
High: >30 %
113
(6.5)
81
(6.3)
1.06
(0.76,
1.48)
p-trend
0.37
Cumulative occupational exposurec
Unexposed
1133
(64.5)
869
(64.6)
1.00
Lowest tertile
174
(9.9)
139
(10.3)
0.86
(0.65,
1.14)
Middle tertile
246
(14.0)
183
(13.6)
0.85
(0.66,
1.09)
Highest tertile
158
(11.6)
158
(11.7)
0.89
(0.69,
1.15)
p-trend
0.24
Total
1771
(100.0)
1360
(100.0)
aExposures were restricted to estimates with reliability > possible; estimates
with low reliability were classified as unexposed
bAdjusted for age, province of residence, use of proxy respondents, BMI
categories, percent change in weight from maximum lifetime weight, cigarette
pack years, combined physical activity index (hours/month), alcohol consumption
(servings/week), consumption of processed meat (servings/week), occupational
exposure to asbestos (yes/no), occupational exposure to aromatic amines (yes/no)
cCumulative metric of exposure to diesel emissions was derived from estimates of
concentration of exposure, frequency of exposure and duration of employment An important finding of this analysis was the 2-fold
increase in the odds of rectal cancer with exposure to
diesel exhaust. Discussion Environmental Health (2016) 15:4 Page 6 of 12 Table 1 Selected demographic characteristics of incident colon and rectal cancer cases and controls in the Canadian National
Enhanced Cancer Surveillance System 1994–97 (Continued)
Q1: ≤2.0
168
(18.1)
145
(17.3)
253
(18.6)
1.04
(0.84,
1.29)
Q2: >2.0 to ≤6
157
(16.9)
131
(15.6)
247
(18.2)
1.03
(0.82,
1.28)
Q3: >6 to ≤14.5
186
(20.0)
152
(18.1)
216
(15.8)
1.29
(1.03,
1.61)
Q4: >14.5
161
(17.3)
185
(22.0)
193
(14.2)
1.50
(1.19,
1.87)
Quartiles of processed meat intake (servings/week)
Q1: ≤1.4
216
(23.2)
192
(22.9)
427
(31.4)
1.00
Q2: >1.4 to ≤3.5
224
(24.1)
232
(27.7)
368
(27.1)
1.32
(1.08,
1.61)
Q3: >3.5 to ≤6.5
245
(26.3)
212
(25.2)
267
(19.6)
1.81
(1.46,
2.23)
Q4: >6.5
246
(26.4)
204
(24.3)
298
(21.9)
1.49
(1.21,
1.83)
Quartiles of total vegetable intake (servings/week)
Q1: ≤12.5
225
(24.2)
217
(25.8)
343
(25.2)
1.00
Q2: >12.5 to ≤18.4
247
(26.5)
204
(24.3)
338
(24.9)
1.05
(0.86,
1.29)
Q3: >18.4 to ≤25.0
227
(24.4)
215
(25.6)
336
(24.7)
1.01
(0.83,
1.25)
Q4: >25.0
232
(24.9)
204
(24.3)
343
(25.2)
1.03
(0.83,
1.26)
Quartiles of total fruit intake (servings/week)
Q1: ≤4.4
229
(24.6)
211
(25.1)
347
(25.5)
1.00
Q2: >4.4 to ≤8.9
236
(25.4)
208
(24.8)
345
(25.4)
0.99
(0.80,
1.21)
Q3: >8.9 to ≤14
239
(26.7)
218
(26.0)
341
(25.1)
1.01
(0.82,
1.24)
Q4: >14
227
(24.4)
203
(24.2)
327
(24.0)
0.99
(0.80,
1.22)
Quartiles of moderate physical activity (hours/month)
Q1: ≤3.8
222
(23.9)
204
(24.3)
355
(26.1)
1.00
Q2: >3.8 to ≤12.2
240
(25.8)
206
(24.5)
339
(24.9)
1.00
(0.81,
1.23)
Q3: >12.2 to ≤25.3
244
(26.2)
196
(23.3)
343
(25.2)
0.95
(0.77,
1.17)
Q4: >25.3
225
(24.2)
234
(27.9)
323
(23.8)
1.05
(0.85,
1.29)
Quartiles of strenuous physical activity (hours/month)
Q1: 0
460
(49.4)
397
(47.3)
673
(49.5)
1.00
Q2: >0 to ≤1.2
151
(16.2)
151
(18.0)
221
(16.3)
0.97
(0.79,
1.20)
Q3: >1.2 to ≤7.3
167
(17.9)
136
(16.2)
230
(16.9)
0.96
(0.78,
1.18)
Q4: >7.3
153
(16.4)
156
(18.6)
236
(17.4)
0.89
(0.72,
1.10)
Composite index of strenuous and moderate physical activityb (quartiles)
Q1: Least active
203
(21.8)
186
(22.1)
340
(25.0)
1.00
Q2
257
(27.6)
217
(25.8)
340
(25.0)
1.10
(0.89,
1.35)
Q3
221
(23.7)
190
(22.6)
340
(25.0)
0.94
(0.76,
1.16)
Q4: Most active
250
(26.9)
247
(29.4)
340
(25.0)
1.09
(0.88,
1.34)
Ever exposed to aromatic amines
No
914
(98.2)
830
(98.8)
1348
(99.1)
1.00
Yes
17
(1.8)
10
(1.2)
12
(0.9)
1.71
(0.84,
3.45)
Ever exposed to asbestos
No
781
(83.9)
719
(85.6)
1209
(88.9)
1.00
Yes
150
(16.1)
121
(14.4)
151
(11.1)
1.43
(1.15,
1.78)
Ever exposed to crystalline silica
No
597
(35.8)
293
(34.9)
929
(68.3)
1.00
Yes
334
(64.1)
547
(65.1)
431
(31.7)
1.09
(0.93,
1.28)
aOR estimated for colon and rectal cancers combined
bComposite index of physical activity was derived y summing the hours/month of moderate and strenuous activity Table 1 Selected demographic characteristics of incident colon and rectal cancer cases and controls in the Canadian National
Enhanced Cancer Surveillance System 1994–97 (Continued) Table 1 Selected demographic characteristics of incident colon and rectal cancer cases and controls in the Canadian National
Enhanced Cancer Surveillance System 1994–97 (Continued) Kachuri et al. Discussion However, the association observed for the
hi h
t
tt i
d
t
ti
f di
l
d
Table 3 Adjusted odds ratios (OR) and corresponding 95 %
confidence intervals (CI) for colorectal cancer in relation to
occupational exposure to gasoline emissions
Exposure metrica
Cases (%)
Controls (%)
ORb (95 % CI)
Ever exposed
No
957
(54.0)
783
(42.4)
1.00
Yes
814
(46.0)
577
(57.6)
1.10
(0.93,
1.31)
Highest attained exposure concentration
Unexposed
957
(54.0)
783
(57.7)
1.00
Low
647
(36.5)
462
(34.0)
1.17
(0.97,
1.41)
Medium
109
(6.2)
71
(5.2)
1.04
(0.73,
1.49)
High
58
(3.3)
44
(3.2)
1.04
(0.67,
1.63)
p-trend
0.77
Duration of exposure (years)
Unexposed
957
(54.7)
783
(58.1)
1.00
> 0 to <7
245
(14.0)
179
(13.3)
1.07
(0.84,
1.36)
≥7 to ≤26
285
(16.3)
196
(14.5)
1.11
(0.88,
1.40)
> 26
264
(15.1)
190
(14.1)
1.11
(0.84,
1.45)
p-trend
0.32
Duration of exposure at high concentration (years)
Unexposed
1713
(96.9)
1316
(97.1)
1.00
> 0 to ≤5
30
(1.7)
21
(1.6)
0.95
(0.51,
1.75)
> 5
25
(1.4)
18
(1.3)
1.11
(0.57,
2.17)
p-trend
0.83
Frequency of exposure
Unexposed
1110
(64.1)
852
(66.6)
1.00
Low: 5 %
46
(2.7)
56
(4.4)
0.68
(0.43,
1.07)
Medium: 6–30 %
437
(25.3)
282
(22.0)
1.10
(0.89,
1.36)
High: >30 %
138
(8.0)
90
(7.0)
1.09
(0.79,
1.49)
p-trend
0.33
Cumulative occupational exposurec
Unexposed
957
(54.6)
783
(58.0)
1.00
Lowest tertile
225
(12.8)
175
(13.0)
0.95
(0.73,
1.24)
Middle tertile
308
(17.6)
204
(15.1)
1.16
(0.93,
1.45)
Highest tertile
262
(15.0)
187
(13.9)
1.13
(0.88,
1.45)
p-trend
0.27
Total
1771
(100.0)
1360
(100.0)
aExposures were restricted to estimates with reliability > possible; estimates
with low reliability were classified as unexposed
bAdjusted for age, province of residence, use of proxy respondents, BMI
categories, percent change in weight from maximum lifetime weight, cigarette
pack years, combined physical activity index (hours/month), alcohol consumption
(servings/week), consumption of processed meat (servings/week), occupational
exposure to asbestos (yes/no), occupational exposure to aromatic amines (yes/no)
cCumulative metric of exposure to diesel emissions was derived from estimates of
concentration of exposure, frequency of exposure and duration of employment levels of concentration, as well as for long durations of
exposure at high levels of concentration. Exposure to
gasoline emissions was not associated with elevated can-
cer risks in our data. levels of concentration, as well as for long durations of
exposure at high levels of concentration. Discussion Exposure to
gasoline emissions was not associated with elevated can-
cer risks in our data. An important finding of this analysis was the 2-fold
increase in the odds of rectal cancer with exposure to
diesel exhaust. However, the association observed for the
highest attained concentration of diesel exposure and Kachuri et al. Discussion Environmental Health (2016) 15:4 Page 8 of 12 Table 4 Adjusted odds ratios (OR) and corresponding 95 % confidence intervals (CI) for rectal cancer and colon cancer in relation to
occupational exposure to diesel emissions
Rectal cancer (n = 840)
Colon cancer (n = 931)
Exposure metrica
Cases (%)
Controls (%)
ORb
(95 % CI)
Cases (%)
Controls (%)
ORb
(95 % CI)
Ever exposed
No
538
(64.0)
869
(63.9)
1.00
595
(63.9)
869
(63.9)
1.00
Yes
302
(36.0)
491
(36.1)
0.89
(0.72,
1.10)
336
(36.1)
491
(36.1)
0.90
(0.73,
1.11)
Highest attained exposure concentration
Unexposed
538
(64.1)
869
(63.9)
1.00
595
(63.9)
869
(63.9)
1.00
Low
212
(25.2)
377
(27.7)
0.78
(0.61,
0.99)
238
(25.6)
377
(27.7)
0.81
(0.64,
1.03)
Medium
61
(7.3)
89
(6.5)
0.99
(0.68,
1.44)
75
(8.1)
89
(6.5)
1.09
(0.76,
1.56)
High
29
(3.5)
25
(1.8)
1.98
(1.09,
3.60)
23
(2.5)
25
(1.8)
1.35
(0.72,
2.54)
p-trend
0.56
p-trend
0.91
Duration of exposure (years)
Unexposed
538
(64.5)
869
(64.4)
1.00
595
(64.5)
869
(64.4)
1.00
> 0 to <11
99
(11.9)
157
(11.6)
0.91
(0.67,
1.24)
109
(11.8)
157
(11.6)
0.91
(0.68,
1.24)
≥11 to ≤31
112
(13.4)
166
(12.3)
0.99
(0.73,
1.35)
108
(11.7)
166
(12.3)
0.87
(0.64,
1.18)
> 31
85
(10.2)
157
(11.6)
0.76
(0.52,
1.10)
110
(11.9)
157
(11.6)
0.86
(0.60,
1.21)
p-trend
0.16
p-trend
0.31
Duration of exposure at high concentration (years)
Unexposed
811
(96.7)
1335
(98.2)
1.00
908
(97.6)
1335
(98.2)
1.00
> 0 to ≤10
16
(1.9)
13
(1.0)
1.84
(0.84,
4.05)
13
(1.4)
13
(1.0)
1.37
(0.60,
3.14)
> 10
12
(1.4)
11
(0.8)
2.33
(0.94,
5.78)
9
(1.0)
11
(0.8)
1.34
(0.51,
3.55)
p-trend
0.02
p-trend
0.39
Frequency of exposure
Unexposed
558
(68.2)
910
(71.1)
1.00
633
(69.3)
910
(71.1)
1.00
Low: 5 %
33
(4.0)
60
(4.7)
1.27
(0.76,
2.12)
43
(4.7)
60
(4.7)
1.21
(0.75,
1.94)
Medium: 6–30 %
171
(20.9)
229
(17.9)
1.15
(0.87,
1.51)
180
(19.7)
229
(17.9)
1.10
(0.84,
1.44)
High: >30 %
56
(6.9)
81
(6.3)
1.20
(0.80,
1.79)
57
(6.2)
81
(6.3)
0.95
(0.64,
1.41)
p-trend
0.16
p-trend
0.70
Cumulative occupational exposurec
Unexposed
538
(64.5)
869
(64.4)
1.00
595
(64.5)
869
(64.4)
1.00
Lowest tertile
87
(10.4)
139
(10.3)
0.87
(0.63,
1.20)
87
(9.4)
139
(10.3)
0.85
(0.61,
1.18)
Middle tertile
116
(13.9)
183
(13.6)
0.88
(0.65,
1.18)
130
(14.1)
183
(13.6)
0.83
(0.63,
1.11)
Highest tertile
93
(11.2)
158
(11.7)
0.92
(0.66,
1.28)
111
(12.0)
158
(11.7)
0.98
(0.71,
1.35)
p-trend
0.38
p-trend
0.42
aExposures were restricted to estimates with reliability > possible; estimates with low reliability were classified as unexposed
bAdjusted for age, province of residence, use of proxy respondents, BMI categories, percent change in weight from maximum lifetime weight, cigarette pack years,
combined physical activity index (hours/month), alcohol consumption (servings/week), consumption of processed meat (servings/week), occupational exposure to
asbestos (yes/no), occupational exposure to aromatic amines (yes/no)
cCumulative metric of exposure to diesel emissions was derived from estimates of concentration of exposure, frequency of exposure and duration of employment aExposures were restricted to estimates with reliability > possible; estimates with low reliability were classified as unexposed
bAdjusted for age, province of residence, use of proxy respondents, BMI categories, percent change in weight from maximum lifetime weight, cigarette pack years,
combined physical activity index (hours/month), alcohol consumption (servings/week), consumption of processed meat (servings/week), occupational exposure to
asbestos (yes/no), occupational exposure to aromatic amines (yes/no)
cCumulative metric of exposure to diesel emissions was derived from estimates of concentration of exposure, frequency of exposure and duration of employmen (>10 years) exposure at very high concentrations,
such as underground miners, which represent the
benchmark for high concentration of diesel exposure
in the NECSS. Discussion Our results point to concentration as a key
dimension of exposure influencing the association with
cancer risk. However, given the inconsistent overall pat-
tern of results and the modest prior evidence for a causal duration of exposure at high concentration should be
viewed in the context of weak and null associations
observed for other exposure metrics. The lack of
monotonically increasing trends in risk for frequency
and duration of exposure, suggests that diesel emis-
sions may be associated with rectal cancer risk only
in specific occupational sub-groups with prolonged Kachuri et al. Discussion Environmental Health (2016) 15:4 Page 9 of 12 Table 5 Adjusted odds ratios (OR) and corresponding 95 % confidence intervals (CI) for rectal cancer and colon cancer in relation to
occupational exposure to gasoline emissions
Rectal cancer (n = 840)
Colon cancer (n = 931)
Exposure metrica
Cases (%)
Controls (%)
ORb
(95 % CI)
Cases (%)
Controls (%)
ORb
(95 % CI)
Ever exposed
No
452
(53.8)
783
(57.6)
1.00
505
(54.2)
783
(57.6)
1.00
Yes
388
(46.2)
577
(42.4)
1.12
(0.91,
1.38)
426
(45.8)
577
(42.4)
1.04
(0.85,
1.27)
Highest attained exposure concentration
Unexposed
452
(53.8)
783
(57.6)
1.00
505
(54.2)
783
(57.6)
1.00
Low
305
(36.3)
462
(34.0)
1.20
(0.96,
1.50)
342
(36.7)
462
(34)
1.10
(0.88,
1.37)
Medium
55
(6.6)
71
(5.2)
1.12
(0.73,
1.73)
54
(5.8)
71
(5.2)
0.92
(0.60,
1.42)
High
28
(3.3)
44
(3.2)
1.04
(0.60,
1.79)
30
(3.2)
44
(3.2)
1.04
(0.61,
1.76)
p-trend
0.74
p-trend
0.91
Duration of exposure (years)
Unexposed
452
(54.3)
783
(58.1)
1.00
505
(55.0)
783
(58.1)
1.00
> 0 to <7
117
(14.1)
179
(13.3)
1.09
(0.82,
1.46)
128
(13.9)
161
(11.9)
1.00
(0.76,
1.34)
≥7 to ≤26
144
(17.3)
196
(14.5)
1.20
(0.91,
1.59)
141
(15.4)
185
(13.7)
0.96
(0.73,
1.27)
> 26
120
(14.4)
190
(14.1)
1.08
(0.77,
1.51)
144
(15.7)
219
(16.3)
1.10
(0.79,
1.51)
p-trend
0.33
p-trend
0.77
Duration of exposure at high concentration (years)
Unexposed
812
(96.8)
1316
(97.1)
1.00
901
(97.0)
1316
(97.1)
1.00
> 0 to <5
15
(1.8)
21
(1.6)
1.02
(0.48,
2.16)
15
(1.6)
21
(1.6)
0.94
(0.46,
1.94)
≥5
12
(1.4)
18
(1.3)
1.08
(0.48,
2.39)
13
(1.4)
18
(1.3)
1.13
(0.51,
2.52)
p-trend
0.84
p-trend
0.84
Frequency of exposure
Unexposed
528
(64.6)
852
(66.6)
1.00
582
(63.8)
852
(66.6)
1.00
Low: 5 %
14
(1.7)
56
(4.4)
0.46
(0.24,
0.89)
32
(3.5)
56
(4.4)
0.85
(0.51,
1.42)
Medium: 6–30 %
213
(26.0)
282
(22.0)
1.08
(0.84,
1.40)
224
(24.5)
282
(22.0)
1.08
(0.84,
1.38)
High: >30 %
63
(7.7)
90
(7.0)
1.00
(0.68,
1.48)
75
(8.2)
90
(7.0)
1.16
(0.80,
1.66)
p-trend
0.72
p-trend
0.30
Cumulative occupational exposurec
Unexposed
452
(54.3)
783
(58.0)
1.00
505
(55.0)
783
(58.0)
1.00
Lowest tertile
117
(14.1)
175
(13.0)
1.13
(0.84,
1.51)
108
(11.8)
144
(10.7)
0.85
(0.63,
1.15)
Middle tertile
140
(16.8)
204
(15.1)
1.14
(0.86,
1.51)
168
(18.3)
217
(16.1)
1.13
(0.87,
1.48)
Highest tertile
124
(14.9)
187
(13.9)
1.11
(0.81,
1.51)
138
(15.0)
205
(15.2)
1.06
(0.78,
1.43)
p-trend
0.38
p-trend
0.55
aExposures were restricted to estimates with reliability > possible; estimates with low reliability were classified as unexposed
bAdjusted for age, province of residence, use of proxy respondents, BMI categories, percent change in weight from maximum lifetime weight, cigarette pack
years, combined physical activity index (hours/month), alcohol consumption (servings/week), consumption of processed meat (servings/week), occupational
exposure to asbestos (yes/no), occupational exposure to aromatic amines (yes/no)
cCumulative metric of exposure to diesel emissions was derived from estimates of concentration of exposure, frequency of exposure and duration of employment Table 5 Adjusted odds ratios (OR) and corresponding 95 % confidence intervals (CI) for rectal cancer and colon cancer in relation to
occupational exposure to gasoline emissions aExposures were restricted to estimates with reliability > possible; estimates with low reliability were classified as unexposed
bAdjusted for age, province of residence, use of proxy respondents, BMI categories, percent change in weight from maximum lifetime weight, cigarette pack
years, combined physical activity index (hours/month), alcohol consumption (servings/week), consumption of processed meat (servings/week), occupational
exposure to asbestos (yes/no), occupational exposure to aromatic amines (yes/no)
cCumulative metric of exposure to diesel emissions was derived from estimates of concentration of exposure, frequency of exposure and duration of employmen capillaries is likely the pathway by which these particles
translocate to other organs [32]. Discussion Although the carcinogenic
mechanism of PAHs has been attributed to genotoxic
effects, such as the formation of bulky DNA adducts
[33–35], the underlying process is less clear for other
constituents of motor vehicle exhaust, such as elemental
carbon [35, 36]. Toxicology data suggest that generation relationship, the observed increases in rectal cancer risk
should be viewed with caution, and it is possible that some
of these associations may be due to chance. The hypothesis linking diesel emissions to increased
risk of colon and rectal cancer postulates that clear-
ance of inhaled the gases and particulate matter by
mucocilliary transport and diffusion into the pulmonary Page 10 of 12 Page 10 of 12 Kachuri et al. Environmental Health (2016) 15:4 Page 10 of 12 of reactive oxygen species (ROS) and the resulting
mutations represent another potential carcinogenic mech-
anism. Dietary exposure to diesel exhaust particles has
been linked with ROS-mediated protein oxidation, DNA
breaks, and increased formation of DNA-adducts in
colon mucosa cells [35]. Studies in experimental models
also indicate that a large single dose of exposure to
diesel exhaust leads to more pronounced DNA damage,
compared to the effects of the same total dose adminis-
tered in smaller increments over time [37]. These observa-
tions suggest that an exposure threshold may exist for the
genotoxic effects of diesel exhaust particles, and this is
consistent with the results of our analysis, where concen-
tration of exposure, but not duration alone, was associated
with cancer risk. risk factors. One of the key strengths of our study
was the availability of extensive information on anthropo-
metric measures, diet, and physical activity, which allowed
us to adjust for these important determinants of CRC
risk in a more comprehensive way, compared to pre-
vious studies. Another key advantage of our study is our rigorous
and extensive exposure assessment. Compared to studies
of job title or industry alone, the expert review enhances
our ability to take into consideration idiosyncrasies within
each job that can influence exposure dimensions. Experts
could take into consideration the time-varying nature of
motor vehicle exhaust emissions, resulting from changes
in engine technology, use of protective equipment, and
policy regulations [39]. Discussion The resulting semi-quantitative in-
dices have been shown to be a credible way of assessing
exposure, and given the blinded nature of our assessment,
this also serves to mitigate the potential for recall bias that
is often introduced in self-reported case–control data [40]. Furthermore, our exposure assessment provided informa-
tion on other occupational agents, such as aromatic
amines, asbestos and crystalline silica, which have been
taken into account in this analysis. Previous epidemiological studies of diesel and gasoline
emissions and CRC risk have reported a somewhat dif-
ferent pattern of results to the one we observed in our
data. In a large Canadian population-based case–control
study in the 1980’s, exposure to diesel engine exhaust
was linked to increased risks of colon cancer (OR = 1.7
for long-term, high-level exposure) [12]. Contrary to our
findings, gasoline engine exhaust was associated with
elevated risks of rectal cancer (OR = 1.6 for long-term,
high-level exposure) [12]. However, this analysis was
based on small case series (364 colon and 190 rectal
cancers), and lacked information on BMI, diet and phys-
ical activity. In another analysis of the same dataset,
Goldberg et al. [13] assessed the association of colon
cancer with diesel engine emissions in 497 male cases
compared with 1514 other cancer patients (excluding
cancers of the lung, peritoneum, esophagus, stomach
and rectum) and 533 population controls. When the
pooled control group was used, the resulting OR for
colon cancer was 1.6, whereas the risk increased to 2.1
when only the population-based controls were used [13]. It is plausible that the levels of exposure experienced by
population-based controls in our study were higher,
which would attenuate the observed associations. Despite its many advantages, our exposure assessment
also has some limitations. The response rates were mod-
est for cases and controls in our study. Although selec-
tion bias resulting from the lower response rate in
controls may be a concern, we do not believe that the
magnitude of this bias would be large enough to lead to
seriously distort the odds ratios for diesel exposure that
have been reported. Firstly, it is unlikely that differences
in study participation were directly related to diesel and
gasoline exposure, since this was not explicitly identified
as a study hypothesis during participant recruitment. Conclusions
f
d Our findings are suggestive of a modest association
between occupational exposure to high concentrations
of diesel emissions and risk of rectal cancer. Received: 10 July 2015 Accepted: 10 January 2016 Received: 10 July 2015 Accepted: 10 January 2016 Author details
1 1Dalla Lana School of Public Health, University of Toronto, 155 College Street,
6th Floor, Toronto, ON M5T 3 M7, Canada. 2Occupational Cancer Research
Centre, Cancer Care Ontario, 525 University Avenue, 3rd Floor, Toronto, ON
M5G 2 L3, Canada. 3Prevention and Cancer Control, Cancer Care Ontario, 620
University Ave, Toronto, ON M5G 2 L7, Canada. 4CHAIM Research Centre,
Carleton University, 5435 Herzberg Laboratories, 1125 Colonel By Drive,
Ottawa, ON K1S 5B6, Canada. 5INRS-Institut Armand-Frappier, Institut national
de la recherche scientifique, University of Quebec, 531 boul. des Prairies,
Édifice 12, Laval, QC H7V 1B7, Canada. 6Department of Epidemiology and
Community Medicine, University of Ottawa, 451 Smyth Rd, Ottawa, ON K1H
8 M5, Canada. Discussion hygienists at INRS Institute Armand- Frappier, Louise Nadon, Benoit Latreille,
Ramzan Lakhani, and Mounia Rhazi for their contribution to this work. We also
acknowledge the support of the Ontario Occupational Cancer Research Center
(OCRC) and Health Canada. Marie- Élise Parent is the recipient of career awards
from Fonds de recherche du Québec- Santé (FRQS). The reliance on self-reported job histories makes our
data vulnerable to measurement error resulting from in-
accuracies in recall, which can decrease statistical power
and attenuate the observed associations. Misclassification
of exposure likely occurred, although this would have been
non-differential, thereby attenuating the observed associa-
tions. Moreover, in the absence of direct measurements of
exposure, our semi-quantitative indices cannot be used for
quantitative risk assessment. References
d Parkin DM, Bray F, Ferlay J, Pisani P. Estimating the world cancer burden:
Globocan 2000. Int J Cancer. 2001;94(2):153–6. 7. World Cancer Research Fund. American Institute for Cancer Research. Continuous Update Project Interim Report Summary Washington, DC: AICR. 2011 8. Wolin KY, Yan Y, Colditz GA, Lee IM. Physical activity and colon
cancer prevention: a meta-analysis. Br J Cancer. 2009;100(4):611–6. doi:10.1038/sj.bjc.6604917. Abbreviations CRC: colorectal cancer; OR: odds ratio; CI: confidence intervals; IARC: International
Agency for Research on Cancer; NECSS: National Enhanced Cancer
Surveillance System. CRC: colorectal cancer; OR: odds ratio; CI: confidence intervals; IARC: International
Agency for Research on Cancer; NECSS: National Enhanced Cancer
Surveillance System. j j
9. Sandhu MS, White IR, McPherson K. Systematic review of the prospective cohort
studies on meat consumption and colorectal cancer risk: a meta-analytical
approach. Cancer Epidemiol Biomarkers Prev. 2001;10(5):439–46. Competing interests 10. Arbman G, Axelson O, Fredriksson M, Nilsson E, Sjodahl R. Do occupational
factors influence the risk of colon and rectal cancer in different ways? Cancer. 1993;72(9):2543–9. The authors declare no conflicts of interest. No payment or services received
from a third party. No relevant financial activities outside the submitted work. No patents (planned, pending, or issued) broadly relevant to this work. No other relationships or conditions or circumstances that present a
potential conflict of interest. 11. Dumas S, Parent ME, Siemiatycki J, Brisson J. Rectal cancer and occupational
risk factors: a hypothesis-generating, exposure-based case–control study. Int J Cancer. 2000;87(6):874–9. 11. Dumas S, Parent ME, Siemiatycki J, Brisson J. Rectal cancer and occupational
risk factors: a hypothesis-generating, exposure-based case–control study. Int J Cancer. 2000;87(6):874–9. 12. Siemiatycki J, Gerin M, Stewart P, Nadon L, Dewar R, Richardson L. Associations between several sites of cancer and ten types of exhaust and
combustion products. Results from a case-referent study in Montreal. Scand
J Work Environ Health. 1988;14(2):79–90. Authors’ contribution Conception and design of the NECSS study and data collection were carried
out by KCJ and members of the Canadian Cancer Registries Epidemiology
Research Group. Conception and design of the secondary analysis of NECSS
data, along with data acquisition, were conducted by PJV, MEP, SAH and
KCJ. Exposure assessment was lead by MEP. Data analysis and drafting of
manuscript was carried out by LK. Data interpretation was lead by LK. All authors participated in revising the manuscript critically for important
intellectual content. All authors read and approved the final manuscript 13. Goldberg MS, Parent ME, Siemiatycki J, Desy M, Nadon L, Richardson L, et al. A case–control study of the relationship between the risk of colon cancer in
men and exposures to occupational agents. Am J Ind Med. 2001;39(6):531–46. 14. Fang R, Le N, Band P. Identification of occupational cancer risks in British
Columbia, Canada: a population-based case–control study of 1,155 cases of
colon cancer. International journal of environmental research and public
health. 2011;8(10):3821–43. doi:10.3390/ijerph8103821. 15. International Agency for Research on Cancer. Report of the Advisory Group
to Recommend Priorities for IARC Monographs during 2010–2014. Lyon,
France:IARC;2008. Discussion The potential for selection bias is further reduced since
only 1.2 % (n = 199) of eligible jobs were excluded from
the exposure assessment due to insufficient information. The impact of non-participation bias is also likely to
be minimal because adjustment for socioeconomic fac-
tors that are expected to be related to participation, such
as education, do not have an appreciable effect on the
observed associations with diesel and gasoline exposure. For instance, after adjusting for total years of schooling,
the rectal cancer association with exposure to the
highest concentration level of diesel emissions remained
statistically significant and comparable in magnitude
(OR = 1.90 vs. OR = 1.98). A similar pattern was observed
for duration of exposure at high concentrations, and other
metrics of diesel and gasoline exposure. In interpreting the results of our study, it is important
to consider that the workplace exposure of NECSS sub-
jects may have been lower than what may be required to
produce a detectable increase in cancer risk. The effects
of exposure could also be modified by individual differ-
ences in ability to metabolize and clear PAHs, and repair
DNA damage resulting from oxidative stress generated
by other constituents of motor vehicle exhaust [38]. An
additional challenge in estimating the effects of diesel
and gasoline emissions lies in disentangling the effects of
one of these exposures from the other, while also con-
trolling for their mutual confounding. It should also be
noted that even if a causal relationship with exposure to
diesel emissions exists, it’s contribution to overall CRC
risk may be overshadowed by other lifestyle and genetic Lastly, the associations observed for known colon and
rectal cancer risk factors in earlier NECSS publications
[41, 42] and in our data, including BMI and dietary
factors (i.e.: red and processed meat consumption), also
exhibited dose–response patterns that were comparable Kachuri et al. Environmental Health (2016) 15:4 Page 11 of 12 Page 11 of 12 in magnitude and direction to those that have been
previously reported in the literature [7, 9, 43]. hygienists at INRS Institute Armand- Frappier, Louise Nadon, Benoit Latreille,
Ramzan Lakhani, and Mounia Rhazi for their contribution to this work. We also
acknowledge the support of the Ontario Occupational Cancer Research Center
(OCRC) and Health Canada. Marie- Élise Parent is the recipient of career awards
from Fonds de recherche du Québec- Santé (FRQS). References
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and Insurance Board (Ontario) - WSIB# 10011. We thank the chemists/industrial The Canadian Cancer Registries Epidemiology Research Group comprised a
principal investigator from some of the provincial cancer registries involved
in the National Enhanced Cancer Surveillance System: Farah McCrate, Eastern
Health, Newfoundland; Ron Dewar, Nova Scotia Cancer Registry; Nancy Kreiger,
Cancer Care Ontario; Donna Turner, Cancer Care Manitoba. This project was funded by a research grant provided by the Workplace Safety
and Insurance Board (Ontario) - WSIB# 10011. We thank the chemists/industrial The Canadian Cancer Registries Epidemiology Research Group comprised a
principal investigator from some of the provincial cancer registries involved
in the National Enhanced Cancer Surveillance System: Farah McCrate, Eastern
Health, Newfoundland; Ron Dewar, Nova Scotia Cancer Registry; Nancy Kreiger,
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diesel exhaust and carbon black in F344 rats. Fundamental and applied
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• Our selector tool helps you to find the most relevant journal
• We provide round the clock customer support
• Convenient online submission
• Thorough peer review
• Inclusion in PubMed and all major indexing services
• Maximum visibility for your research
Submit your manuscript at
www.biomedcentral.com/submit
and we will help you at every step: 37. Dybdahl M, Risom L, Bornholdt J, Autrup H, Loft S, Wallin H. Inflammatory
and genotoxic effects of diesel particles in vitro and in vivo. Mutat Res. 2004;562(1–2):119–31. doi:10.1016/j.mrgentox.2004.05.010. 38. Wang H, Yamamoto JF, Caberto C, Saltzman B, Decker R, Vogt TM, et al. Genetic variation in the bioactivation pathway for polycyclic hydrocarbons
and heterocyclic amines in relation to risk of colorectal neoplasia. Carcinogenesis. 2011;32(2):203–9. doi:10.1093/carcin/bgq237. 38. Wang H, Yamamoto JF, Caberto C, Saltzman B, Decker R, Vogt TM, et al. Genetic variation in the bioactivation pathway for polycyclic hydrocarbons
and heterocyclic amines in relation to risk of colorectal neoplasia. Carcinogenesis. 2011;32(2):203–9. doi:10.1093/carcin/bgq237. 39. Pronk A, Coble J, Stewart PA. Occupational exposure to diesel engine
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exhaust: a literature review. Journal of exposure science & environmental
epidemiology. 2009;19(5):443–57. doi:10.1038/jes.2009.21.
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SPAFACON2021
Papers from the SEAMEO SPAFA International Conference on
SOUTHEAST ASIAN ARCHAEOLOGY AND FINE ARTS
13 - 17 December 2021 SPAFACON2021
Papers from the SEAMEO SPAFA International Conference on
SOUTHEAST ASIAN ARCHAEOLOGY AND FINE ARTS
13 - 17 December 2021 SPAFACON2021
Papers from the SEAMEO SPAFA International Conference on
SOUTHEAST ASIAN ARCHAEOLOGY AND FINE ARTS
13 - 17 December 2021 Editor: Noel Hidalgo Tan Editor: Noel Hidalgo Tan SPAFACON2021 is published by SEAMEO SPAFA,
the Regional Centre for Archaeology and Fine Arts
established by the Southeast Asian Ministers of
Education Organization. SEAMEO SPAFA focusses on
archaeology and fine arts in Southeast Asia, and
promotes awareness and appreciation of the cultural
heritage of the region. Its member-countries are Brunei,
Cambodia, Indonesia, Lao PDR, Malaysia, Myanmar,
the Philippines, Singapore, Thailand, Timor-Leste,
and Vietnam; and its associate member-countries are
Australia, Canada, France, Germany, the Netherlands,
New Zealand, Spain and the United Kingdom. 2021 SEAMEO SPAFA
ISBN: 978-616-7961-55-2
ISBN (e-book): 978-616-7961-54-5
DOI 10.26721/spafa.pqcnu8815a
Publisher
SEAMEO SPAFA Regional Centre for Archaeology and Fine Arts
81/1 Sri Ayutthaya Road, Dusit
Bangkok 10300, Thailand
Tel: +66 (0) 2280 4022 to 9
Fax: +66 (0) 2280 4030
www.seameo-spafa.org
E-mail: spafa@seameo-spafa.org
Editor
Dr Noel Hidalgo Tan
Organizing Committee
Mrs Somlak Charoenpot
Ms Vassana Kerdsupap
Mr Kanal Khiev
Mr Ean Lee
Ms Gabrielle Anne Mangaser
Dr Hatthaya Siriphatthanakun
Ms Ratchaporn Tesjeeb
Mr Patrick Xia Linhao
Dr Noel Hidalgo Tan
Copyright in the volume as a whole is vested in SEAMEO SPAFA, and
individual chapters also belong to their respective authors and licensed
Commons CC BY-SA 4.0. To view a copy of this license, visit https://cr
org/licenses/by sa/4 0/ 2021 SEAMEO SPAFA
ISBN: 978-616-7961-55-2
ISBN (e-book): 978-616-7961-54-5
DOI 10.26721/spafa.pqcnu8815a Publisher
SEAMEO SPAFA Regional Centre for Archaeology and Fine Arts
81/1 Sri Ayutthaya Road, Dusit
Bangkok 10300, Thailand
Tel: +66 (0) 2280 4022 to 9
Fax: +66 (0) 2280 4030 Organizing Committee
Mrs Somlak Charoenpot
Ms Vassana Kerdsupap
Mr Kanal Khiev
Mr Ean Lee
Ms Gabrielle Anne Mangaser
Dr Hatthaya Siriphatthanakun
Ms Ratchaporn Tesjeeb
Mr Patrick Xia Linhao
Dr Noel Hidalgo Tan Copyright in the volume as a whole is vested in SEAMEO SPAFA, and copyright in the
individual chapters also belong to their respective authors and licensed under Creative
Commons CC BY-SA 4.0. To view a copy of this license, visit https://creativecommons. org/licenses/by-sa/4.0/ The papers in this publication have not been peer-reviewed and may thus contain
preliminary findings, errors, or information that has yet to be endorsed by the relevant
academic community. The views expressed in the publication do not necessarily reflect
the opinions or policies of SEAMEO SPAFA. CONTENT
Title
Author
Page
Introduction
A Historiography of Settlement Archaeology in Southeast
Asia, with Emphasis on the Pre-industrial State Formations
Gyles Iannone
1
Ocean Imperatives: analysing shipping infrastructure for the
study of maritime networks in Southeast Asia
Veronica Walker Vadillo
17
Forms of government and local community participation in
the management of cultural World Heritage sites in Southeast
Asia
Vithaya Arporn
24
Cultural interaction between Việt Nam and Southeast Asian
nations in the 15th-16th centuries: An overview of pottery
items from ancient shipwrecks on display at the Museum of
History in Hồ Chí Minh City
Phạm Ngọc Uyên,
Nguyễn Thị Tú Anh
29
Sequential Least-Cost Path Sailing Model for Early 17th
Century South China Sea: Digitally Navigating the Selden
Map of China
Wesa Perttola
40
The Prevailing Art and Tradition of Intentional Dental
Modification in Prehistoric Southeast Asia
Maria Kathryn N. Purnell
56
The Still Unexplored Parts of Southeast Asian Archaeology:
Colonial Archaeology Singapore
Sxuann Sim
74
Khao San Dam: The Archaeological Evidence of Burnt Rice
Festival in Southern Thailand
Pakpadee Yukongdi
83
Before Bagan: Using Archaeological Data Sets to Assess the
Traditional Historical Narrative
Scott Macrae,Gyles
Iannone, Kong Cheong,
Pyiet Phyo Kyaw
96
The Rock Art in Kinta Valley, West Malaysia: A synthesis
Chaw Yeh Saw
Hsiao Mei Goh
114
New Archaeological Discoveries: Gates and Turrets of 16th
Burmese Royal Capital of Haṁsāvatī
Thaw Zin Latt
131
A preliminary survey of Chinese ceramics in Champa
archaeological sites
Do Truong Giang
148
A Study on the Structure and Significance of the North
Sanctuary at Western Prasat Top
SATO Yuni, TAMURA
Tomomi, SUGIYAMA
Hiroshi, LAM Sopheak,
SOK Keo Sovannara,
LOEUNG Ravattey, ROS
Visoth
166
The funeral cave of Laang Spean
Valéry Zeitoun,
Heng Sophady, Hubert
Forestier
173 Introduction Title
Author
Page
The Bronze Age People of Ban Kao: A Preliminary Analysis
of the Human Remains from Ban Ta Po Archaeological Site,
Western Thailand
Naruphol
Wangthongchaicharoen,
Supamas Duangsakul,
Pira Venunan, Sukanya
Lertwinitnun, Siriyupon
Tubpenthai
187
The Mt. Popa Watershed and Bagan’s Bronze-Iron Age
Elizabeth Moore
195
After 30 Years and During a Pandemic: Pottery Production
and Distribution in Bagacay, Talibon in the Island of Bohol in
the Philippines
Rhayan Gatbonton
Melendres
205
Heritage Education in Myanmar – developing resilience and
sustainability through community engagement
Su Su, Win Thant Win
Shwin, Ohnmar Myo,
Charlotte Galloway,
Elizabeth Moore
220
As my father said: Traditional boatbuilding in Pasuruan, East
Java
Agni Mochtar, Putri
Taniardi, R. Ahmad
Ginanjar Purnawibawa
234
Tangibility-Intangibility on UNESCO World Heritage
Baroque Philippine Churches: the Spirit of Place and Its
Collective Memory
Hee Sook Lee-Niinioja
241
Myinkaba village Bagan: The Resilience of Traditional
Knowledge and Culture
Theint Aung
254
Thai Carpentry Knowledge Transmission: Development of
Traditional Apprenticeships in a New Context
Nichamon Hiranpruek
262
The Factors of Market Success and Failure of Contemporary
Artists from ASEAN countries
Dr Rémy JARRY
268
The Forgotten Women: Investigating the Absence of the
Female Artist from Traditionally Male-Centric Southeast
Asian Contemporary Art Historical Narratives
Vasanth Narayanan
279
As One With Nature: Southeast Asian Aesthetic Expressions
Victor R. Savage
289
The Series Of Archaeological Dances: A Historical Study and
Dance Moves Recording With Labanotation
Dharakorn Chandnasaro
309
Musical Instruments on the 16th century bas reliefs in
the North Gallery-East Wing of Angkor Wat : Dating and
Significance
Arsenio Nicolas
324
Photography in Indonesian Archaeology of the 19th to the
Early 20th Century
Ahmad Kholdun Ibnu
Sholah
356
Old Burmese weights were not opium weights. They were
weights. What else do we know about them? Bob Hudson
369
Religious Object” Exhibition in the Context of Cultural
Change and Covid-19 Social Distancing (Case studies of
Khmer’s Nagar boat in the South of Vietnam)
Phạm Thị Thủy Chung
381 Title
Author
Page
Creativity and Innovation in Cultural Heritage Management
in Plunturan Village, Pulung District, Ponorogo Regency,
East Java Province of Indonesia Towards Tourism Village
Ria Kusuma Wardani,
S.Pd. Introduction 392
Beyond the artefact : promoting technology
Cécile de Francquen
407
Indonesian Museum after New Order Regime: The
Representation that Never Disappears
Ayu Dipta Kirana, Fajar
Aji Jiwandono
416
Geological Museum Innovations to Dealing with Covid-19
Pandemic
Ifan Yoga Pratama
Suharyogi, Agustina
Djafar, Rahajeng Ayu
Permana Sari, Paradita
Kenyo Arum Dewantoro
424 Title
Author
Page
Creativity and Innovation in Cultural Heritage Management
in Plunturan Village, Pulung District, Ponorogo Regency,
East Java Province of Indonesia Towards Tourism Village
Ria Kusuma Wardani,
S.Pd. 392
Beyond the artefact : promoting technology
Cécile de Francquen
407
Indonesian Museum after New Order Regime: The
Representation that Never Disappears
Ayu Dipta Kirana, Fajar
Aji Jiwandono
416
Geological Museum Innovations to Dealing with Covid-19
Pandemic
Ifan Yoga Pratama
Suharyogi, Agustina
Djafar, Rahajeng Ayu
Permana Sari, Paradita
Kenyo Arum Dewantoro
424 INTRODUCTION This volume contains the extended abstracts from the papers presented at the SEAMEO
SPAFA International Conference on Southeast Asian Archaeology and Fine Arts, which
was held online from 13 to 17 December 2021. Also known as the SPAFACON2021, this
conference was organised online due to the pandemic. Despite the disruption brought
about by Covid-19 to our in-person events, training programmes and field research, it is
heartening to see that archaeology and cultural heritage has continued under new modes
of communication and collaboration. This fourth iteration of the SPAFACON is also scheduled a year earlier than our usual
triennial cycle to commemorate the 50th anniversary of SEAMEO initiating a centre
dedicated towards archaeology and the fine arts. Over the past year, SPAFA has also been
highlighting this legacy of international cooperation and capacity-building by sharing our
photographic archives on our social media. I am delighted by the high level of enthusiasm and intellectual curiosity brought by
the participants to the conference. During our call for papers we received close to 90
submissions, but owing to the pressures of time and the online format, we were only able
to accept 34 papers for the conference. The variety of papers present here, although a
small set compared with our usual proceedings, reflects the breadth of the centre’s ambit –
covering not just archaeology, but also performing arts, visual arts, museum studies, and
other aspects of Southeast Asian cultural heritage. I would like to thank all the participants, without whom this conference would not be
possible in its present form, in particular, our Governing Board members who represent
every country in Southeast Asia, and to the Ministry of Culture, Thailand and the Ministry
of Education, Thailand for their long-standing support of SEAMEO SPAFA and its
activities. Mrs Somlak Charoenpot
Centre Director
SEAMEO SPAFA Mrs Somlak Charoenpot
Centre Director
SEAMEO SPAFA Mrs Somlak Charoenpot
Centre Director
SEAMEO SPAFA SPAFACON2021 74 he SEAMEO SPAFA International Conference on Southeast Asian Archaeology and Fine Arts Abstract Existing archaeological studies have focused predominantly on 14th century Singapore
while colonial archaeology in Singapore remains understudied. With most archaeological
sites in Singapore also yielding artifacts from the 19th to early 20th century, there is an
enormous potential for the development of the field (Miksic 2013, p.419). Although
colonial records can provide information on colonial Singapore, more mundane daily
activities and lives of the people are under-documented. This paper seeks to identify
the potential and importance of studying Singapore and Southeast Asian’s colonial
archaeological record. The Still Unexplored Parts of Southeast Asian Archaeology:
Colonial Archaeology Singapore 10792/pqcnu8815a-07
Sxuann Sim
Nanyang Technological University
sxuannme001@e.ntu.edu.sg Archaeology; Singapore archaeology; Colonial archaeology; Archaeological ceramics
studies Introduction Archaeology in Singapore has come a long way since the first systematic dig conducted
at Fort Canning in 1984. The excavation, led by Emeritus Professor John N. Miksic,
unearthed numerous artifacts from the 19th century layer and the 14th century layer. Inorganic materials such as ceramics preserve well in the archaeological record, which
may explain why ceramics continue to dominate the assemblage of finds in Singapore
and Southeast Asia. Existing archaeological studies have focused predominantly on
14th-century Singapore while colonial archaeology in Singapore remains understudied. With most of the archaeological sites in Singapore also yielding artifacts from the 19th
to early 20th century, there is an enormous potential for the development of the field
(Miksic 2013: 419). Although colonial records can provide a rich source of information
on colonial Singapore, more mundane daily activities and lives of the people continue
to be under-documented. Colonial archaeology thus, can supplement existing colonial
records to illuminate under-documented daily activities of the 19th to the early 20th century
inhabitants in Singapore. Colonial archaeology can be defined as “the study of time and place of European rule of
non-European lands claimed by Europeans” (Muckle 2006: 51). This paper follows the
definition proposed by archaeologist Miriam T. Stark that the historic period in Southeast
Asia “is associated with the earliest European penetration in the region,” stretching from
the late 19th century to the early 20th century (2018: 1). Generally, there are four main
occupation layers in Singapore. Layer I dates to the contemporary period. Layer II dates
from the 19th to 20th century and is marked by a reddish-brown soil. Layer III dates from
the 14th to the 16th century and is marked by a dark brown soil. Layer IV is a sterile
yellowish-brown soil (Miksic 2013: 228-234). Keywords Archaeology; Singapore archaeology; Colonial archaeology; Archaeological ceramics
studies SPAFACON2021 The SEAMEO SPAFA International Conference on Southeast Asian Archaeology and Fine Arts he SEAMEO SPAFA International Conference on Southeast Asian Archaeology and Fine Arts 75 Preceding Literature i There is a significant lack of research and evaluation on the archaeological research
conducted during the historical period in Southeast Asia (Stark 2018: 1-3). Most
archaeological research in Southeast Asia has concentrated either on the prehistoric
period (especially on the Paleolithic, Bronze, and Iron Ages) or are part of the national
historic preservation effort (Stark 2018: 3). Efforts concentrated on preservation and
reconstruction of archaeological structures tend to focus on remains excavated from the
9th to 14th century. Stark argues that this can be attributed to how archaeological remains
of this period resonated “with nationalist claims and also attract cultural tourism” (Stark
2018: 3). SPAFACON2021 The SEAMEO SPAFA International Conference on Southeast Asian Archaeology and Fine Arts The SEAMEO SPAFA International Conference on Southeast Asian Archaeology and Fine Arts 76 he SEAMEO SPAFA International Conference on Southeast Asian Archaeology and Fine Arts Recent archaeological ceramic studies have shown that colonisation in the region had a
differential impact across Southeast Asia (Stark 2018: 5). This appeared to be surprising to
some as the region is generally considered as a homogenous area (Stark 2018: 5). To date,
there are two main studies conducted on SEA ceramics during the colonial period. The first
being Ueda et al.’s “Paths to Power in the Early Stage of Colonialism: An Archaeological
Study of the Sultanate of Banten, Java, Indonesia, the Seventeenth to Early Nineteenth
Century” (2016). Previous studies on Banten’s sultanate have focused solely on Banten’s
change in monumental architecture to identify and determine when the decline of the
Banten’s sultanate occurred (Ueda et al.: 112). Ueda et al.’s petrographic studies showed
that the Banten court continued to import large numbers of Chinese porcelain and Southeast
Asian produced kendi even after becoming a vassal state to the Dutch East India Company
(Ueda et al.: 110-111). This shows that the sultan’s influence during Banten’s vassal
state status remains considerable. Ueda et al.’s study has demonstrated the importance
of studying the archaeological ceramic records in understanding the colonial period. Through the record, it was noted that the decline of the sultanate of Banten happened
gradually and not immediately, which contradicted the historical records and previous
assumptions (Ueda et al.: 111). Preceding Literature i In another study conducted in 2014, Professor Li Min found that by comparing the
ceramic assemblages from both the pre-colonial and colonial period in Philippines, she
was able to uncover how political power and economic status of indigenous chiefs and
elites were transformed during the Spanish colonialization (p. 67). She noted that prior to
being colonized, Philippines was importing significant amounts of Chinese porcelain (Li
2014: 45). The eventual loss of control of their ports to the Spanish resulted in a heavy
loss of money and power for the local elites (Li 2014: 45). Li’s study exhibited how the
study of material culture in the Philippines can help understand the several ways in which
the indigenous chiefdoms negotiated and competed with other rising maritime powers of
that period. Similarly in the field of Spanish Colonial Archaeology, Gilda Hernández Sánchez noted
the importance of studying artifacts, buildings, and material culture in the reconstruction
of the ‘indigenous’ history as most historical records are documented by the colonisers
(2012: 23). Hence, history based on historical records alone cannot provide an accurate
representation of the history of the communities who were colonised (Sánchez 2012: 23). It is, therefore, vital for researchers to consider alternative records other than the historical
documents such as the archaeological record as they can offer “day-to-day domestic
activities, common industries such as ceramic-making or consume patterns” (Sánchez SPAFACON2021 The SEAMEO SPAFA International Conference on Southeast Asian Archaeology and Fine Arts The SEAMEO SPAFA International Conference on Southeast Asian Archaeology and Fine Arts he SEAMEO SPAFA International Conference on Southeast Asian Archaeology and Fine Arts 77 2012: 24). The trend of studying the colonial objects is reflective of the highlighting
the dualistic representation of colonial reality by contrasting the colonial records to the
archaeological records as it was regarded as “both central experiences of people living
in colonial situations” (Sánchez 2012: 22). Hispanic Archaeology has tended to focus its
narrative on the contribution of the Spanish during colonisation while there are only a few
studies dedicated towards understanding the locals’ experiences and responses towards
colonisation. Amanda D. Roberts Thompson’s analysis on the ceramics excavated at Santa Maria de
Galve have revealed the various economic alternatives that Spanish colonists engaged in
(Thompson 2012: 48). According to the Spanish colonial records, all trading activities
between the colonists and foreigners are forbidden (Thompson 2012: 48). Preceding Literature i However, this
was highly unsustainable, which may have encouraged these colonists to look for economic
alternatives in order to support themselves (Thompson 2012: 48). This was supplemented
by the archaeological records as ceramics were found in abundance at Santa Maria de
Galve despite historical records showing that the settlement only received five shipments
of ceramics (Thompson 2012: 48). Hence, by studying the diversity of ceramics excavated at the Officers’ Barracks and the
Hospital/Warehouse at Santa Maria de Galve, Thompson was able to deduce that the
colonists such as the military officers engaged in the illicit trade of ceramics. (Thompson
2012: 62- 63). Locals and lower rank barracks however, did not have access to these
ceramics as their diversity index of the ceramics were significantly lower compared
to the areas where “higher class individuals” have access to (Thompson 2012: 62-
63). Thompson’s study has demonstrated an alternative way of approaching colonial
archaeological ceramic analysis while also analysing the significance of looking at the
archaeological record. Alternative economic activities would have never been revealed if
we relied solely on historical records of Santa Maria de Galve. Similarly, in Singapore, little is known about the archaeological record from the 19th to
20th century. Archaeological studies have mostly focused on 14th to 16th century Singapore. In Singapore and the Silk Road of the Sea, 1300-1800, Miksic noted that there is simply
not enough trained manpower to carry out archaeological research (2013: 431). As a
result, most of the archaeological materials from the 19th to 20th century, except for a
selected few from Pulau Saigon (PSG) site and Colombo Court (CCT), have not been
sorted and entered into a database. The number of Singapore archaeological sites that
yielded 19th to 20th century artefacts are as follows: Fort Canning (FTC), Colombo Court SPAFACON2021 78 he SEAMEO SPAFA International Conference on Southeast Asian Archaeology and Fine Arts (CCT), Empress Place (EMP), Parliament House Complex (PHC), Singapore Cricket
Club (SCC), St. Andrew’s Cathedral (STA), Pulau Saigon (PSG), Duxton Hill (DXT),
and Istana Kampong Gelam (IKG) (Miksic 2013: 405-431). (CCT), Empress Place (EMP), Parliament House Complex (PHC), Singapore Cricket
Club (SCC), St. Andrew’s Cathedral (STA), Pulau Saigon (PSG), Duxton Hill (DXT),
and Istana Kampong Gelam (IKG) (Miksic 2013: 405-431). Despite the number of archaeological materials excavated from the 19th to 20th century,
there are only three studies carried out to date, all conducted by Jennifer Barry. Preceding Literature i Additionally, Barry observed patterns of vertical distribution, finding that there
is a general increase in artifacts frequencies with depth (until spit 7) followed by a gradual SPAFACON2021 The SEAMEO SPAFA International Conference on Southeast Asian Archaeology and Fine Arts The SEAMEO SPAFA International Conference on Southeast Asian Archaeology and Fine Arts 79 he SEAMEO SPAFA International Conference on Southeast Asian Archaeology and Fine Arts decrease (Barry 2007: 56). However, no further analyses or hypotheses were made and
tested in Barry’s first paper. It was only towards the end of the paper when Barry made
several broad general statements concerning the site. Barry’s second edition provided a more comprehensive study on the European ceramics
excavated at IKG. 24 colonial era coins were also included in this paper to provide a
better terminus post quem and a terminus ante quem date of the spits within Square I to
IV (Barry 2009: 112). Notably, the second edition’s appendix, titled statistical analysis,
was far more comprehensive than the previous edition. It was divided into 1) total finds,
2) densities of artefacts per spit, 3) densities of artefacts within IKG, and 4) spit dating
and conjoining artifacts (maker’s mark on European ceramics and colonial era coins) in
order to determine the terminus post quem and a terminus ante quem of the spits (Barry
2009: 126-135). Through Barry’s statistical analysis section in the second edition, she observed certain
patterns and correlations within the archaeological record. For example, she noted
subsquare IVB2 and spit 4 has the highest concentration of Asian stoneware across all
51 subsquares at IKG (Barry 2009: .130). One of the biggest limitations of her statistical
analysis is that, given that different classes of materials weigh differently, basing an entire
analysis solely on weight can be problematic. Naturally, organic materials like bone, seed,
and shells are lighter than ceramics. Similarly, within the ceramics category, earthenware
is typically lighter than porcelain while stoneware is generally heavier than porcelain. Hence, Asian stoneware may be overrepresented in the data since it is typically heavier than
the rest of the artefacts in the assemblage. Nevertheless, this particular act of data mining
by Barry has provided extremely useful information regarding the archaeological data in
IKG, which can provide direction for future research. This includes noting the density of
the distribution of artifacts across IKG. Preceding Literature i In her
first report, Barry proposed a typology that relied on general observations on the clay
and paste colour, which gave her a clue on the provenance (i.e. European. Chinese,
Japanese), before separating them further based on their decorative design (Barry 2000:
14). However, Barry’s research has mainly focused on European artefacts, specifically the
European porcelain that were excavated at PSG. Because of the extensive disturbance of
the site, it was impossible to reconstruct the stratigraphy of the site. Using site maps and
notes, she listed five possible ideas regarding how the artefacts were deposited at PSG
(Barry 2000: 20-21). She contended that prior to 1889, the primary area for refuse disposal was most likely
located at the foot of Pearl’s Hill (Barry 2000: 18). Despite the introduction of incineration
in 1889, large finds of glass and stoneware in the river indicate the potential of continued
use of PSG as a disposal area (Barry 2000: 19). The presence of popular Chinese exports
of blue and white porcelain further suggest that PSG was potentially used as a dumping
area for the warehouses located around the area (Barry 2000: 19). These ideas, however,
have never been deductively tested according to the hypothesis-deductive method. The
hypo-deductive method would increase our confidence in the hypotheses and further
inferences we develop on PSG. Barry’s second study focused primarily on the site named Istana Kampong Gelam (IKG)
where she focused on European ceramics and coins. In the first edition, she recorded the
overall number, weight, and distribution of European ceramics excavated (Barry 2007:
54). Barry also attempted to date the different spits based on the maker’s mark found on
either on the vessel base or on the underside of the rims, which allowed her to establish the
terminus post quem and terminus ante quem of the spits within Square I to IV (Barry 2007:
59). Barry’s first edition is particularly useful in guiding the identification of the European
wares excavated throughout 19th to early 20th century sites across Singapore. However, her
sorting of European ceramics is still very much in the preliminary stage as it was based
simply on the decorative techniques and motifs, and/or the maker’s mark that she was able
to identify. Preceding Literature i This includes having most of the ceramic locating
in Square IV and most of the other materials such as metal, glass and organic material
locating in Square II. It is important to note that Barry’s papers were never meant to serve
as archaeological reports and Barry, herself, acknowledges this. Based on the existing literature both in Southeast Asia and Singapore, much of Singapore’s
colonial archaeology is left unexplored. It is distressing to note that there are practically
no comprehensive studies on Singapore colonial archaeology despite the number of
archaeological sites and artefacts available. One of the biggest advantages of conducting
an archaeological ceramic analysis lies in the fact that these ceramics can provide us
information about the inhabitants at the site such as the potential social stratification within SPAFACON2021 The SEAMEO SPAFA International Conference on Southeast Asian Archaeology and Fine Arts The SEAMEO SPAFA International Conference on Southeast Asian Archaeology and Fine Arts The SEAMEO SPAFA International Conference on Southeast Asian Archaeology and Fine Arts 80 he SEAMEO SPAFA International Conference on Southeast Asian Archaeology and Fine Arts the site and the utilitarian functions of the ceramics. Archaeologists studying 19th to 20th
century Singapore have a distinct advantage over 14th to 16th century scholars due to the
addition of the historical records. For example, from the historical records, we are aware
that FTC (Keramat site) was previously occupied by Malay lighthouse keepers while the
other parts of the FTC site (near the Fire Director’s Residence, Sculpture Garden and
Telecoms Terrace) was occupied by colonial officials and their families (Miksic 2013: 416-
418). This is also reflected in the archaeological records where we see a higher frequency
of local and Southeast Asian artefacts excavated at the Keramat site (Miksic 2013: 418). On the other hand, more European porcelain and children’s toys were excavated at other
parts of the FTC site (Miksic 2013: 418). Possible future direction in research The call for the standardisation of terminologies and
the application of the hypothetico-deductive method for 19th to 20th century Singapore
archaeological materials allow research to move forward beyond Barry’s proposal. Standardisation of terminologies allows for reproducibility of data beyond colonial
archaeological sites in Singapore. The hypothetico-deductive method allows researchers
to test hypotheses created about the site. A strong methodology and framework provide
more rigor and confidence in the hypotheses generated. It is important to focus on creating a more in-depth spatial analysis of the entire 19th to
early 20th century assemblage either based on systematically collected samples or the
entire assemblage. Barry’s research on 19th to 20th century Singapore has provided certain
questions that could be used as a starting point of inquiry for the archaeological materials
excavated from PSG and IKG. The call for the standardisation of terminologies and
the application of the hypothetico-deductive method for 19th to 20th century Singapore
archaeological materials allow research to move forward beyond Barry’s proposal. Standardisation of terminologies allows for reproducibility of data beyond colonial
archaeological sites in Singapore. The hypothetico-deductive method allows researchers
to test hypotheses created about the site. A strong methodology and framework provide
more rigor and confidence in the hypotheses generated. The paper therefore proposes the importance of a standardisation of approach to
archaeological ceramics in 19th to early 20th century Singapore and in SEA. Till date, there
has not been any establishment of ceramic typology for 19th to 20th century Singapore
and SEA despite the fact that ceramic sherds make up the bulk of the assemblages found
in the region. Additionally, existing ceramics catalogs have also been dedicated largely
towards collectables that often do not reflect local inhabitants’ ceramic consumption. As
demonstrated in this paper, there is a relative lack of colonial archaeological studies in
Singapore and SEA and a general lack in study of consumption centers. Mass production
pottery factories increasingly took over the production of ceramics, studying consumption
centers would thus, allow for a better understanding of the production, distribution,
and consumption of ceramics. Possible future direction in research Due to the lack of research in archaeological analysis
of ceramics, a great deal of discussion in this paper has centered upon the potential of
colonial archaeological studies of ceramics and the importance of creating a systematic
approach towards the 19th to early 20th century ceramics in both Singapore and Southeast
Asia, which include the standardisation of terminologies and the importance of using a
hypothetico-deductive method. Possible future direction in research Due to the lack of research in colonial Singapore archaeology, there is an absence of
comparable and standardised terminology, typology, and methodology. A standardised
typology is crucial to archaeology as they enable archaeologists to compare similar sites
to one another. Typologies and classifications systems are essentially grouping techniques
created to aid the ordering and analysis of the archaeological data. With standardisation
of these terminologies, archaeologists are able to use the same criterion to describe 19th
to 20th century artifacts excavated not only in Singapore but also potentially across 19th to
early 20th century sites in SEA. As seen above, archaeological ceramics conducted in SEA and in Spanish colonial
archaeology have demonstrated how archaeological studies can not only contextualise
aspects in historical documents but also provide significant information regarding past
inhabitants and economies that are often absent from the historical records. The only 19th
to early 20th century classification that exists today for Singapore archaeological sites
was created by Barry. Majority of her classification focused solely on European produced
ceramics. Her initial classification of the materials excavated at IKG were also limited to
Asian stoneware, Asian porcelain, Asian earthenware, European ceramic, Metal, Glass,
Organic, and Miscellaneous. Further sorting is necessary for a more complex and nuanced
analysis of the site. For example, her category of Asian porcelain includes both Chinese
and Japanese porcelain. The frequency of occurrence between the two types of porcelain
can be vastly different. Preliminary studies on other 19th to early 20th century sites in
Singapore have shown that the occurrence of Japanese porcelain was significantly lower
than Chinese porcelain. Grouping the two under the same category will cause the Japanese
porcelain to be overrepresented in the data, which can skew the archaeological data. SPAFACON2021 The SEAMEO SPAFA International Conference on Southeast Asian Archaeology and Fine Arts he SEAMEO SPAFA International Conference on Southeast Asian Archaeology and Fine Arts 81 It is important to focus on creating a more in-depth spatial analysis of the entire 19th to
early 20th century assemblage either based on systematically collected samples or the
entire assemblage. Barry’s research on 19th to 20th century Singapore has provided certain
questions that could be used as a starting point of inquiry for the archaeological materials
excavated from PSG and IKG. Acknowledgments I would like to thank SPAFA for giving me the opportunity to write about something
that’s close to my heart. I would like to include a special note of thanks to Professor
Goh Geok Yian and Professor John N. Miksic for inspiring me to proceed with this
research and their encouragement throughout my M.A. studies. I would also like to thank
Nanyang Technological University (NTU) of Singapore and the National Heritage Board
of Singapore for their continued support. SPAFACON2021 The SEAMEO SPAFA International Conference on Southeast Asian Archaeology and Fine Arts 82 he SEAMEO SPAFA International Conference on Southeast Asian Archaeology and Fine Arts References Barry, J (2009) Istana Kampong Glam: archaeological excavations at a nineteenth
century Malay Palace in Singapore. Stamford: Rheidol Press. Barry, J (2007) Istana Kampong Glam: European ceramic from archaeological
excavations at a nineteenth century Malay palace in Singapore. Stamford:
Rheidol Press. Barry, J (2000) Pulau Saigon: a post-eighteenth century archaeological assemblage
recovered from a former island in the Singapore River. Stamford: Rheidol Press. Li, M (2013) Fragments of Globalization: Archaeological Porcelain and the Early
Colonial Dynamics in the Philippines. Asian Perspectives, 52(1): 43-74. Miksic, JN (2013) Singapore and the Silk Road of the Sea, 1300-1800. Singapore: NUS
Press and National Museum of Singapore. Muckle, R (2006) Introducing Archaeology. Toronto: University of Toronto Sánchez, GH (2012) Archaeology of Colonialism. In: GH Sánchez (ed.) Ceramics and
the Spanish Conquest: Response and Continuity of Indigenous Pottery
Technology in Central Mexico. Leiden: Brill. Stark, MT (2014) South and Southeast Asia: Historical Archaeology. In: C Smith (ed.)
Encyclopedia of Global Archaeology. New York: Springer. Thompson, ADR (2012) Evaluating Spanish Colonial Alternative Economies in the
Archaeological Record. Historical Archaeology, 46(4): 48-69. Ueda, K, Wibisono, SC, Harkantiningsih, N and Lim, CS (2016) Paths to Power in the
Early Stage of Colonialism: An Archaeological Study of the Sultanate of Banten,
Java, Indonesia, the Seventeenth to Early Nineteenth Century. Asian
Perspectives, 55(1): 89-119. SPAFACON2021
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Bisphenol A and di-ethylhexyl phthalate analysis in gallon water distributed in Makassar
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DOI: 10.33830/fsj.v3i1.4739.2023 DOI: 10.33830/fsj.v3i1.4739.2023
Diterima: 23 Januari 2023, Diperbaiki: 21 Mei 2023, Disetujui: 9 Juni 2023 ABSTRACT Bisphenol A (BPA) and Di-Ethylhexyl Phthalate (DEHP) are chemical compounds that are
used as raw materials for making disposable bottles or PET, including gallons. BPA can
migrate into food or drink if the temperature is raised due to heating processes. This research
aimed to identify BPA and DEHP compounds in various brands of gallon water distributed
in Makassar and the effect of sun exposure towards BPA and DEHP migration. The sampling
method was done based on an early analysis from field survey data in the location that
represents all gallon water refills distributed in Makassar. There were 4 selected samples from
2 most marketed brands of gallon water refill. Research method included sample preparation
with two different treatments, namely with exposure and without exposure to sunlight by letting
them settle inside and outside the room for 6 days, afterwards, BPA and DEHP were analyzed
using Gas Chromatography - Mass Spectrometry (GC-MS). The results of the study show
that in various brands gallon water packages, both with sun exposure or without, no BPA
and DEHP compounds were detected. Keywords : gallon water, BPA, DEHP, GC-MS. Keywords : gallon water, BPA, DEHP, GC-MS. Copyright © 2023, Food Scientia Journal of Food Science and Technology ANALISIS BISPHENOL A DAN DI-ETHYLHEXYL PHTHALATE
DALAM AIR GALON YANG BEREDAR DI KOTA MAKASSAR
Bisphenol A and Di-ethylhexyl Phthalate Analysis in Gallon Water
Distributed in Makassar Endah Dwijayanti1, Rachim Munadi2, Sry Wahyuningsih3, Iffana Dani Maulida4
1,2,3 Chemistry Study Program of Islam University of Makassar
Jl. Perintis Kemerdekaan Km.9, Makassar, South Sulawesi 90245, Indonesia
4Program Studi Teknologi Pangan, Fakultas Sains dan Teknologi, Universitas Terbuka
e-mail: endahdwijayanti.dty@uim-makassar.ac.id DOI: 10.33830/fsj.v3i1.4739.2023
Diterima: 23 Januari 2023, Diperbaiki: 21 Mei 2023, Disetujui: 9 Juni 2023 Food Scientia Journal of Food Science and Technology 3(1) 2023, 92-110 Food Scientia Journal of Food Science and Technology 3(1) 2023, 92-110 Food Scientia Journal of Food Science and Technology 3(1) 2023, 92-110 Makassar serta pengaruh paparan cahaya matahari terhadap migrasi BPA dan DEHP. Metode pengambilan sampel dilakukan berdasarkan analisis data awal yang diperoleh dari
hasil survei lapangan di tempat yang mewakili semua air galon isi ulang yang beredar di Kota
Makassar. Sampel air galon yang diambil sebanyak 4 sampel, dari 2 merek sampel air galon
isi ulang yang paling banyak dipasarkan. Metode penelitian meliputi preparasi sampel
dengan 2 perlakuan berbeda yaitu dengan paparan dan tanpa paparan cahaya matahari
dengan cara mendiamkan di dalam dan di luar ruangan selama 6 hari. Selanjutnya BPA dan
DEHP dianalisis dengan menggunakan Gas Chromatography- Mass Spectrometry (GC-MS). Hasil penelitian yang telah dilakukan menunjukkan bahwa air galon berbagai merek yang
beredar di Kota Makassar baik yang terpapar maupun yang tidak terpapar cahaya matahari
tidak terdeteksi mengandung BPA dan DEHP. Kata Kunci : air galon, BPA, DEHP, GC-MS. Kata Kunci : air galon, BPA, DEHP, GC-MS. Copyright © 2023, Food Scientia Journal of Food Science and Technology 93
Copyright © 2023, Food Scientia Journal of Food Science and Technology ABSTRAK Bisphenol A (BPA) dan Di-Ethylhexyl Phthalate (DEHP) merupakan senyawa kimia yang
menjadi bahan pembuatan botol sekali pakai atau PET, tak terkecuali galon. BPA dan DEHP
dapat berpindah atau bermigrasi ke dalam makanan atau minuman jika suhunya dinaikkan
karena pemanasan. Penelitian ini telah dilakukan dengan tujuan untuk mengidentifikasi
senyawa BPA dan DEHP pada beberapa air galon berbagai merek yang beredar di Kota Copyright © 2023, Food Scientia Journal of Food Science and Technology Food Scientia Journal of Food Science and Technology 3(1) 2023, 92-110 Food Scientia Journal of Food Science and Technology 3(1) 2023, 92-110 PENDAHULUAN Bertambahnya jumlah penduduk menyebabkan bertambahnya penggunaan air
untuk berbagai kebutuhan. Perkembangan kemajuan teknologi dan kesibukan
aktivitas masyarakat mengakibatkan masyarakat cenderung memilih cara yang lebih
praktis dengan biaya yang relatif murah dalam memenuhi kebutuhan penggunaan air,
khususnya air minum di daerah perkotaan seperti kota Makassar. Berdasarkan data
Badan Pusat Statistik Sulawesi Selatan (2023), persentase rumah tangga yang
menggunakan sumber air minum utama dari air kemasan bermerk dan air isi ulang di
provinsi Sulawesi Selatan pada tahun 2018, 2019 dan 2020 secara berturut-turut adalah
33,63%, 34,16% dan 35,75% yang menunjukkan peningkatan penggunaan air minum
utama dari kemasan bermerek dan air isi ulang. Air minum isi ulang pada depot air isi ulang menjadi salah satu pilihan yang
paling sering digunakan oleh sebagian masyarakat. Air minum isi ulang dianggap
sebagai alternatif air minum yang praktis, efisien dan harganya yang terjangkau. Hal
ini membuat banyak pelaku usaha memilih untuk memproduksi air galon isi ulang,
karena menjadi suatu peluang usaha yang sangat menjanjikan. Banyak pelaku usaha
yang hanya memikirkan tujuan mencari keuntungan (money-oriented), dan tidak
memperhatikan keamanan serta keselamatan bagi para konsumen air minum isi ulang
yang akan mengkonsumsinya (Purwanti, 2020). Penggunaan galon isi ulang dapat memberikan dampak terhadap kesehatan dan
lingkungan. Dampak negatif yang ditimbulkan dapat berasal kandungan kimia yang
terdapat pada galon yang cukup membahayakan bagi kesehatan diantaranya adalah
Bisphenol A (BPA) yang berpotensi menimbulkan gangguan sistem (endokrin) 93 Food Scientia Journal of Food Science and Technology 3(1) 2023, 92-110 hormon atau endocrine-disrupting chemicals (EDC), kerusakan pada organ kelamin
jantan pria, menurunkan produksi sperma, kanker pada testis, pubertas dini pada
wanita, peningkatan berat badan, komplikasi kehamilan, memberi efek pada organ
prostat dan malignasi dan menyebabkan sampah plastik tidak dapat terurai (Ilmiawati,
et al., 2017; Bahagia et al., 2019; Girsang, et al., 2020). Menurut Peraturan Menteri Kesehatan nomor 492/MENKES/PER/IV/2010,
persyaratan kualitas air minum untuk seluruh penyelenggara air minum wajib
memenuhi persyaratan fisika, mikrobiologis, kimia dan radioaktif. Batas maksimum
BPA menurut BPOM (2011) pada peraturan kepala Badan POM No. HK
03.1.23.07.11.6664 ditetapkan pada peralatan makan-minum adalah 0,6 ppm. Selain BPA, Di-Ethylhexyl Phthalate (DEHP) yang merupakan bahan
tambahan pembuatan botol Polietilena Tereftalat (PET) yang umumnya digunakan
sebagai pengemas air mineral, dapat mengakibatkan penyakit seperti diare, muntah-
muntah dan tukak lambung (Kurniawan & Pusfitasari, 2017). DEHP bersifat
karsinogenik dan ambang batas DEHP dalam air minum yang ditetapkan oleh
Peraturan Menteri Kesehatan nomor 492/MENKES/PER/IV/2010, yaitu sebesar 0.008
mg/L. Penelitian yang dilakukan oleh Girsang, et al. Copyright © 2023, Food Scientia Journal of Food Science and Technology 94
Copyright © 2023, Food Scientia Journal of Food Science and Technology Alat dan Bahan Peralatan yang digunakan dalam penelitian ini adalah sentrifuge, gelas kimia,
pipet mikro, pipet volume, pipet tetes dan Gas Chromatography-Mass Spectrometry
(GC-MS) QP2010 Ultra Shimadzu. Bahan-bahan yang digunakan adalah air galon isi
ulang, aquades (H2O) p.a., asam klorida (HCl) p.a., gas nitrogen (N2), heksana (C6H14),
N,O-bis(trymethylsilyl)-trifluororoacetamide (BSTFA), metanol (CH3OH) p.a.,
Trymethylchlorosilane (TMCS) 1 %, natrium hidroksida (NaOH) dan piridin (C5H5N). Pengambilan Sampel g
p
Sampel dipilih berdasarkan hasil analisis data awal yang diperoleh dari survei
lapangan di lokasi yang mewakili rata-rata persebaran semua merek air galon isi ulang
yang beredar di Kota Makassar. Pengambilan sampel menggunakan metode purposive
sampling dengan kriteria sampel adalah air galon isi ulang yang paling banyak diminati
atau dikonsumsi masyarakat (Syamsul et al., 2020). Survei lapangan dilakukan di
beberapa lokasi yang tersebar di Kota Makassar (Kecamatan Biringkanaya,
Kecamatan Panakukang, Kecamatan Tamalate, Kecamatan Mariso, dan Kecamatan
Manggala). Pada setiap kecamatan, 3 minimarket yang berbeda dipilih secara acak
sebagai lokasi survei lapangan. Survei lapangan dilakukan dengan tujuan untuk
mengetahui galon merek apa saja yang paling banyak diminati oleh masyarakat Kota
Makassar dan untuk mengetahui tempat penyimpanan galon bermerek di minimarket
tersebut. Hasil dari survei lapangan menunjukan 2 merek galon isi ulang dengan
peminat terbanyak yaitu merek A dan merek B, yang dijadikan sampel pada penelitian
ini. Hasil survei lapangan juga menunjukkan bahwa terdapat 2 cara penyimpanan
untuk air galon bermerek yang beredar di kota Makassar, sehingga sampel air galon
yang diambil juga diberi 2 perlakuan yaitu dengan paparan cahaya matahari yang
diberi tambahan kode “1” dan tanpa paparan cahaya matahari yang diberi kode “2”. PENDAHULUAN (2020) yang berjudul deteksi
migrasi material pembungkus makanan ke air karena pemanasan mendapatkan hasil
bahwa terjadi migrasi material pembungkus makanan ke dalam air akuades,
kemungkinan merupakan material dari bahan aditif ftalat, yaitu dimetil ftalat. Berdasarkan penelitian tersebut, migrasi senyawa kimia BPA dan DEHP dapat
menyebabkan kontaminasi air. Selain itu, hasil penelitian Kristiyanto (2013)
menunjukkan adanya pengaruh kadar BPA terhadap paparan sinar matahari. Begitu
juga dengan penelitian yang dilakukan oleh Lubis, et al. (2021) yang menunjukkan
sampel air botol kemasan yang disimpan pada suhu paparan sinar matahari
mengandung BPA dibandingkan sampel tanpa paparan sinar matahari. Penelitian ini bertujuan untuk mengidentifikasi keberadaan BPA dan DEHP
pada sampel air galon di kota Makassar. Selain itu juga untuk menentukan pengaruh
paparan sinar matahari terhadap kandungan BPA dan DEHP pada air galon berbagai
merek di Kota Makassar. Hasil penelitian ini diharapkan dapat menjadi sumber
informasi bagi masyarakat terkait timbulnya senyawa-senyawa berbahaya yang 94 Food Scientia Journal of Food Science and Technology 3(1) 2023, 92-110 kemungkinan ada di dalam air minum galon akibat pengaruh penempatan galon yang
terpapar sinar matahari. kemungkinan ada di dalam air minum galon akibat pengaruh penempatan galon yang
terpapar sinar matahari. 95
Copyright © 2023, Food Scientia Journal of Food Science and Technology Copyright © 2023, Food Scientia Journal of Food Science and Technology Identifikasi Senyawa Bisphenol A (BPA) Identifikasi Senyawa Bisphenol A (BPA)
Nilai pH masing-masing sampel disesuaikan pada rentang pH 5,5 dan 6,5
dengan menggunakan larutan HCl. Kolom Solid Phase Extraction (SPE) dikondisikan
dengan metanol dan ditambahkan dengan air Milli-Q sebagai buffer sebelum sampel
dimasukkan ke dalamnya. Kolom kemudian dicuci dengan metanol 5% dan
dikeringkan dengan menarik udara secara vakum. Selanjutnya, analit dielusi dengan
metanol ke dalam botol bersih dan dikeringkan di bawah aliran gas nitrogen. BPA
kemudian diderivatisasi dengan menambahkan 70 mL BSTFA dan 1 % TMCS dan 10
L piridin ke dalam vial dan ditempatkan ke dalam oven pada suhu 70oC selama 15
menit. Selanjutnya larutan dikeringkan di bawah gas nitrogen dan ditambahkan 1 mL
heksana. Analisis dilakukan dengan GC-MS menggunakan kolom DB-5 (Mead &
Seaton, 2011). Copyright © 2023, Food Scientia Journal of Food Science and Technology Preparasi Sampel Preparasi Sampel
Sampel galon bermerek yang berisi air minum isi ulang (A1, A2, B1 dan B2)
didiamkan selama 6 hari, dengan penyimpanan sampel air galon (A1 dan B1) di luar 95 Food Scientia Journal of Food Science and Technology 3(1) 2023, 92-110 ruangan atau terpapar cahaya matahari langsung dan sampel air galon (A2 dan B2) di
dalam ruangan yang tidak terpapar cahaya matahari. Setelah 6 hari, masing-masing
sampel air galon di ambil sebanyak 100 mL, kemudian disentrifugasi dengan
kecepatan 250 rpm selama 10 menit. Hasil sentrifugasi dimasukkan ke dalam wadah
kaca sebanyak 75 mL, selanjutnya hasil pemisahan digunakan untuk mengidentifikasi
senyawa BPA dan DEHP yang terdapat dalam sampel. Identifikasi Senyawa Di-Ethylhexyl Phthalate (DEHP) y
y
y
(
)
Masing-masing sampel diambil sebanyak 25 mL dan diekstraksi dengan
heksana menggunakan metode ekstraksi cair-cair. Selanjutnya, ekstrak dianalisis
menggunakan GC-MS dengan metode ion selektif. Laju alir helium, hidrogen, dan
udara secara berturut-turut adalah 4, 30, dan 300 mL/menit. Suhu sisi injeksi dan
detektor dijaga pada 250ºC dan 280ºC. Suhu oven terprogram dimulai dari 50ºC
selama 3 menit dan meningkat dengan kecepatan 15ºC/menit sampai dengan 260ºC. Selanjutnya analisa dilakukan secara isotermal sampai kromatogram diperoleh. Identifikasi senyawa dilakukan dengan membandingkan waktu retensi dengan larutan
standar DEHP menggunakan dua ion dan perbandingannya pada setiap analit (Amin
et al., 2018). Penentuan Bisphenol A (BPA) dan Di-Ethylhexyl Phthalate (DEHP)
dilakukan dengan menggunakan data yang diperoleh dari penelitian ini berupa
kromatogram dengan puncak (peak), waktu retensi (tR) dan luas puncak. 96 Copyright © 2023, Food Scientia Journal of Food Science and Technology Food Scientia Journal of Food Science and Technology 3(1) 2023, 92-110 Copyright © 2023, Food Scientia Journal of Food Science and Technology 97
Copyright © 2023, Food Scientia Journal of Food Science and Technology HASIL PEMBAHASAN HASIL PEMBAHASAN
Identifikasi BPA dan DEHP dilakukan dengan tujuan untuk mengetahui ada
atau tidaknya kontaminasi BPA pada sampel air galon bermerek yang beredar di Kota
Makassar serta pengaruh paparan sinar matahari terhadap kandungan BPA dan DEHP. Pengaruh panas matahari merupakan faktor eksternal yang dapat mengakibatkan
kerusakan pada bahan kemasan galon. Panas dari matahari akan mengakibatkan
terjadinya reaksi kimia pada bahan plastik galon. Akibat reaksi kimia yang terjadi akan
membuat ikatan kimia pada plastik akan bereaksi membuat bahan aditif kimia plastik
terlepas ke air. Reaksi ini tidak dapat dihindarkan karena adanya panas yang berasal
dari cahaya matahari (Girsang et al., 2020). Identifikasi BPA dan DEHP yang dilakukan pada semua merek menggunakan
GC-MS QP2010 Ultra Shimadzu. Informasi dari spektra GC adalah waktu retensi
untuk tiap senyawa dalam sampel. Sedangkan untuk MS, bisa diperoleh informasi
mengenai massa molekul relatif dari tiap senyawa dalam sampel tersebut (Setyowati,
et al., 2013). Data yang diperoleh dari GC-MS ini berupa kromatogram dengan puncak
(peak), waktu retensi (tR) dan luas puncak (% area). Peak kromatogram yang
diharapkan sesimetris mungkin dan seruncing mungkin untuk menjamin bahwa
pemisahan efisien (Darmapatni et al., 2016). Waktu retensi adalah waktu analit dideteksi setelah terelusi pada fase diam. Waktu retensi biasanya digunakan dalam analisis kualitatif dengan membandingkan
waktu retensi antara standar dengan analit. Luas puncak adalah jumlah total analit yang
dihitung dari awal puncak hingga titik akhir puncak. Konsentrasi analit dapat dihitung
melalui area puncak yang terukur pada kromatogram dibandingkan dengan plot grafik
kurva kalibrasi standar eksternal (Kristiyanto, 2013). Pada Gambar 3, menurut Mead
& Seaton (2011), jika suatu sampel mengandung BPA maka waktu retensi senyawa
tersebut adalah 3,4320 menit (peak ke II) dengan fragmentasi mass peak tertinggi pada
MS yang dihasilkan adalah 357 m/z dan 358 m/z, sedangkan jika suatu sampel
mengandung DEHP maka waktu retensi senyawa tersebut adalah 7,668 menit dengan
fragmentasi mass peak tertinggi pada MS yang dihasilkan adalah 149 m/z dan 167 m/z
yang terlihat pada Gambar 4 (Kurniawan & Pusfitasari, 2018). Hasil pada GC-MS yang diperoleh pada penelitian ini tidak sesuai dengan teori
tersebut, yaitu ternyata pada sampel A1, A2, B1, dan B2 tidak terdeteksi adanya BPA
dan DEHP. Dari Kromatogram yang diperoleh untuk sampel A1 terdeteksi 20 peak 97 Food Scientia Journal of Food Science and Technology 3(1) 2023, 92-110 Copyright © 2023, Food Scientia Journal of Food Science and Technology Food Scientia Journal of Food Science and Technology 3(1) 2023, 92-110 yang mengindikasikan ada 20 jenis senyawa. Namun beberapa peak menunjukkan
hasil deteksi senyawa yang sama. Hal ini bisa terjadi karena pengaruh fase gerak dan
fase diam yang ada di dalam alat GC serta interaksi-interaksi fisik yang terjadi antara
sistem dalam kromatografi. Kromatogram sampel A1 menunjukkan persen area
tertinggi adalah senyawa piridin pada peak 17 (Gambar 5 (a)) dan pada kromatogram
sampel B1 juga diperoleh senyawa piridin sebagai persen area tertinggi, yaitu pada
peak 20 (Gambar 5 (b)). Gambar 6 menunjukkan spektra MS dari senyawa piridin
yang terdeteksi sebagai kandungan tertinggi dalam sampel A1 dan B1. Senyawa yang
terdeteksi dalam sampel A2 dan B2 pada kondisi tidak terpapar sinar matahari
ditunjukkan pada kromatogram pada Gambar 7. Persen area tertinggi masing-masing
yaitu senyawa disiloxane dan senyawa piridin. Gambar 8 menunjukkan spektra MS
senyawa disiloxane dan piridin sebagai dua senyawa tertinggi kandungannya di dalam
sampel yang terdeteksi di kromatogram. Senyawa piridin merupakan senyawa
aromatik yang digunakan sebagai bahan baku di industri kimia salah satunya sebagai
pelarut pada proses pembuatan polikarbonat. Tipe polikarbonat yang paling umum
adalah bisphenol A (BPA) (Mitchell, 2001). Adanya senyawa tersebut pada dua merek
sampel yang dianalisis menunjukkan bahwa wadah galon tersebut terbuat dari jenis
polikarbonat akan tetapi kadar atau konsentrasi BPA yang terkandung dalam air galon
sangat kecil sehingga tidak terdeteksi langsung pada instrumen GC. Hasil untuk pengujian DEHP kromatogram sampel merek A1 dan B1 diperoleh
data senyawa dengan persen area tertinggi masing-masing yaitu senyawa metana
(Gambar 9 (a)) dan senyawa piperazine (Gambar 9 (b)). Gambar 10 menunjukkan
spektra MS kedua senyawa tersebut. Kromatogram sampel merek A2 dan B2
menunjukkan bahwa senyawa dengan persen area tertinggi masing-masing yaitu
senyawa carbamic acid (Gambar 11 (a)) dan senyawa 3-isobutyryloxy (Gambar 11
(b)). Gambar 12 menunjukkan spektra MS masing-masing senyawa tersebut. Plastik
dapat melepaskan senyawa kimia dalam proses degradasi salah satunya gas methane
akibat radiasi sinar matahari, 98 Copyright © 2023, Food Scientia Journal of Food Science and Technology Food Scientia Journal of Food Science and Technology 3(1) 2023, 92-110 Gambar 3. Kromatogram GC dan Spektra MS BPA Gambar 3. Kromatogram GC dan Spektra MS BPA Sumber: Luo et al. (2014)
Sumber: Kurniawan & Pusfitasari (2018)
Gambar 4. Kromatogram GC dan Spektra MS DEHP Sumber: Luo et al. (2014) Sumber: Kurniawan & Pusfitasari (2018) Gambar 4. Kromatogram GC dan Spektra MS DEHP Hal ini didukung dari hasil kromatogram yang menunjukkan methane
terbentuk pada sampel yang terpapar sinar matahari (Royer et al., 2018). Copyright © 2023, Food Scientia Journal of Food Science and Technology Copyright © 2023, Food Scientia Journal of Food Science and Technology Food Scientia Journal of Food Science and Technology 3(1) 2023, 92-110 Hasil
kromatogram juga menunjukkan adanya senyawa piperazine, carbamic acid dan 3-
Isobutyryloxy. Senyawa-senyawa tersebut merupakan senyawa yang digunakan pada
proses pembuatan plastik (Material Safety Data Sheet, 2010; Kopp, et al., 1982;
SWECO, 2018). Dari data kromatogram tersebut menunjukkan bahwa senyawa-
senyawa kontaminan dari bahan plastik tersebut terlepas ke air dan terdeteksi. 99 Copyright © 2023, Food Scientia Journal of Food Science and Technology Food Scientia Journal of Food Science and Technology 3(1) 2023, 92-110
Gambar 5a. Kromatogram BPA merek A1
(a) Food Scientia Journal of Food Science and Technology 3(1) 2023, 92-110 Copyright © 2023, Food Scientia Journal of Food Science and Technology Food Scientia Journal of Food Science and Technology 3(1) 2023, 92-110 (a (a) Gambar 5a. Kromatogram BPA merek A1 100 Copyright © 2023, Food Scientia Journal of Food Science and Technology Copyright © 2023, Food Scientia Journal of Food Science and Technology 103
Copyright © 2023, Food Scientia Journal of Food Science and Technology Copyright © 2023, Food Scientia Journal of Food Science and Technology Copyright © 2023, Food Scientia Journal of Food Science and Technology Food Scientia Journal of Food Science and Technology 3(1) 2023, 92-110 Gambar 5b. Kromatogram BPA merek B1 (b) Gambar 5b. Kromatogram BPA merek B1 101 Copyright © 2023, Food Scientia Journal of Food Science and Technology Food Scientia Journal of Food Science and Technology 3(1) 2023, 92-110
Gambar 6. Spektra MS senyawa dari sampel merek A1 (a) pyridine dan merek B1
(b) pyridine
(b)
(a)
(a) Food Scientia Journal of Food Science and Technology 3(1) 2023, 92-110 Food Scientia Journal of Food Science and Technology 3(1) 2023, 92-110 Food Scientia Journal of Food Science and Technology 3(1) 2023, 92-110 Gambar 6. Spektra MS senyawa dari sampel merek A1 (a) pyridine dan merek B1
(b) pyridine
(b)
(a) Gambar 6. Spektra MS senyawa dari sampel merek A1 (a) pyridine dan merek B1
(b)
(a) Gambar 6. Spektra MS senyawa dari sampel merek A1 (a) pyridine dan merek B1
(b) pyridine Gambar 7a. Kromatogram BPA sampel merek A2
(a) (a) Gambar 7a. Kromatogram BPA sampel merek A2 Gambar 7a. Kromatogram BPA sampel merek A2 102 Copyright © 2023, Food Scientia Journal of Food Science and Technology Food Scientia Journal of Food Science and Technology 3(1) 2023, 92-110 Food Scientia Journal of Food Science and Technology 3(1) 2023, 92-110 Gambar 7b. Kromatogram BPA sampel merek B2
(b)
(a) t © 2023, Food Scientia Journal of Food Science and
Gambar 7b. Kromatogram BP
Gambar 8. Spektra MS sampel merek A (b) Gambar 7b. Kromatogram BPA sampel merek B2 Gambar 8. Spektra MS sampel merek A2 (a) Disiloxane (b) Pyridine
(a)
(b) Gambar 8. Spektra MS sampel merek A2 (a) Disiloxane (b) Pyridine
(a)
(b) (a) Gambar 8. Spektra MS sampel merek A2 (a) Disiloxane (b) Pyridine 103 Food Scientia Journal of Food Science and Technology 3(1) 2023, 92-110 104
Gambar 9. Kromatogram DEHP sampel (a) merek A1 (b) merek B1 Gambar 9. Kromatogram DEHP sampel (a) merek A1 (b) merek B1 Gambar 9. Kromatogram DEHP sampel (a) merek A1 (b) merek B1 104 Food Scientia Journal of Food Science and Technology 3(1) 2023, 92-110 Food Scientia Journal of Food Science and Technology 3(1) 2023, 92-110 Gambar 10. Spektra MS sampel merek A1 (a) Methane dan merek B1 (b) Piperazine
(b)
(a) ambar 10 Spektra MS sampel merek A1 (a) Methane dan merek B1 (b) Piperazin
(b)
(a) (a) Gambar 10. Spektra MS sampel merek A1 (a) Methane dan merek B1 (b) Piperazine Gambar 10. Copyright © 2023, Food Scientia Journal of Food Science and Technology 106
Copyright © 2023, Food Scientia Journal of Food Science and Technology Food Scientia Journal of Food Science and Technology 3(1) 2023, 92-110 Spektra MS sampel merek A1 (a) Methane dan merek B1 (b) Piperazine Gambar 10. Spektra MS sampel merek A1 (a) Methane dan merek B1 (b) Piperazine Gambar 11a. Kromatogram DEHP sampel merek A2 Gambar 11a. Kromatogram DEHP sampel merek A2 Gambar 11a. Kromatogram DEHP sampel merek A2 105 Copyright © 2023, Food Scientia Journal of Food Science and Technology Copyright © 2023, Food Scientia Journal of Food Science and Technology Food Scientia Journal of Food Science and Technology 3(1) 2023, 92-110 Food Scientia Journal of Food Science and Technology 3(1) 2023, 92-110 Gambar 11b. Kromatogram DEHP sampel merek B2 Gambar 11b. Kromatogram DEHP sampel merek B2 Gambar 12. Spektra MS sampel merek A2 (a) carbamic acid dan merek B2 (b) 3-
Isobutyryloxy
(a)
(b) (a) (b) Gambar 12. Spektra MS sampel merek A2 (a) carbamic acid dan merek B2 (b) 3-
Isobutyryloxy Hasil metode identifikasi BPA sampel A dengan dan tanpa paparan cahaya
matahari, menunjukkan sejumlah senyawa kimia yang terdeteksi. Senyawa kimia yang
banyak terdeteksi terdapat pada sampel air galon yang tidak terpapar cahaya matahari
untuk identifikasi BPA. Hal tersebut disebabkan oleh adanya perlakuan pendahuluan 106 Food Scientia Journal of Food Science and Technology 3(1) 2023, 92-110 pada metode kromatografi yang dilakukan untuk analisis BPA. Sedangkan pada
metode identifikasi DEHP pada sampel A dan B menunjukkan hasil yang berbeda. Sampel A menunjukkan sampel yang tidak terpapar sinar matahari lebih banyak
senyawa yang terdeteksi, sedangkan pada sampel B menunjukkan sampel yang
terpapar sinar matahari terdeteksi lebih banyak senyawa dibanding sampel yang tidak
terpapar. Hal ini dimungkinkan karena metode identifikasi yang menggunakan ion
selektif sangat memungkinkan banyak ion-ion yang dipertukarkan atau terlibat. Karakter DEHP dan BPA yang merupakan bahan pembentuk polimer seperti halnya
plastik sangat mudah berubah dan mengalami kondensasi maupun propagasi
membentuk senyawa lain. Akibatnya senyawa yang terdeteksi antara sampel A dan
sampel B pada metode kromatografi ini mengalami bias. Jenis senyawa yang terdeteksi di kromatogram adalah senyawa-senyawa hasil
degradasi polimer bahan kemasan galon. Masing-masing memiliki ambang batas yang
diperbolehkan oleh standar BPOM. Menurut Kepala Badan Pengawas Obat dan
Makanan (BPOM) (2019), masyarakat harus dilindungi dari penggunaan kemasan
pangan yang tidak memenuhi persyaratan keamanan pangan. Batas migrasi kemasan
pangan berdasarkaan tipe pangan dan kondisi penggunaan untuk jenis kemasan plastik
sebagaimana telah ditetapkan dalam Peraturan Kepala Badan Pengawas Obat dan
Makanan Nomor HK.03.1.23.07.11.6664 tahun 2011 tentang pengawasan kemasaan
pangan, perlu disesuaikan dengan perkembangan ilmu pengetahuan dan teknologi
terkini. Food Scientia Journal of Food Science and Technology 3(1) 2023, 92-110 Kemasan plastik memiliki potensi untuk melepaskan komponen penyusunnya
ke dalam produk. Pelepasan tersebut dapat terjadi karena suhu yang tinggi dan
penyimpanan yang lama. Hasil pengawasan dan kajian yang dilakukan BPOM
terhadap beberapa jenis kemasan plastik menunjukkan bahwa tingkat paparan
masyarakat Indonesia masih dalam taraf aman. 107
Copyright © 2023, Food Scientia Journal of Food Science and Technology Copyright © 2023, Food Scientia Journal of Food Science and Technology 107
opyright © 2023, Food Scientia Journal of Food Science and Technology 108
Copyright © 2023, Food Scientia Journal of Food Science and Technology KESIMPULAN KESIMPULAN
Berdasarkan penelitian yang telah dilakukan pada beberapa air galon bermerek
yang beredar di Kota Makassar dapat disimpulkan bahwa baik yang terpapar maupun
yang tidak terpapar cahaya matahari tidak terdeteksi mengandung Bisphenol A (BPA)
dan Di-Ethylhexyl Phthalate (DEHP). Senyawa yang terdeteksi adalah piridin,
Disiloxane, Piperazine, dan Isobutyriloxy. Saran untuk penelitian lebih lanjut dapat
menggunakan sampel air galon merek lain dan menggunakan metode instrumen 107 Food Scientia Journal of Food Science and Technology 3(1) 2023, 92-110 lainnya yang lebih reliable, seperti High Performance Liquid Chromatography
(HPLC). lainnya yang lebih reliable, seperti High Performance Liquid Chromatography
(HPLC). lainnya yang lebih reliable, seperti High Performance Liquid Chromatography
(HPLC). DAFTAR PUSTAKA DAFTAR PUSTAKA
Amin, F., Rohaeti, E., & Rafi, M. (2018). Evaluasi migrasi di-(2-etilheksil)ftalat dari
botol polietilena tereftalat menggunakan gc/ms. Jurnal ITEKIMIA, 3(1). Badan Pusat Statistik Sulawesi Selatan. (2023). Persentase rumah tangga menurut
sumber air utama yang digunakan rumah tangga untuk minum di provinsi
Sulawesi Selatan. Bahagia, Yunita, I., & Susanti, R. M. (2019). Analisis implementasi kebijakan kantong
plastik berbayar di ritel modern Banda Aceh. Serambi Engineering, IV(2), 549–
556. BPOM. (2011). Peraturan kepala badan pengawas obat dan makanan Republik
Indonesia nomor HK.03.1.23.07.11.6664 tahun 2011. Jakarta. Consumer Product Safety Commission (CPSC). (2018). Test method: CPSC-CH-
C1001-09.4. standard operating procedure for determination of phthalates. https://www.cpsc.gov/en/Business--Manufacturing/BusinessEducation/Business- Darmapatni, K. A. G., Basori, A., & Suaniti, N. M. (2016). Pengembangan metode gc-
ms untuk penetapan kadar acetaminophen pada spesimen rambut manusia. Jurnal
Biosains Pascasarjana, 18(3). Girsang, T. P., Wijaya, I. M. M., & Gunam, I. B. W. (2020). Deteksi migrasi material
pembungkus makanan ke air karena pemanasan. Jurnal Rekayasa Dan
Manajemen Agroindustri, 8(2), 310–318. Ilmiawati, C., Reza, M., Rahmatini, & Rustam, E. (2017). Edukasi pemakaian plastik
sebagai kemasan makanan dan minuman serta risikonya terhadap kesehatan pada
komunitas di kecamatan bungus teluk kabung, Padang. Logista Jurnal Ilmiah
Pengabdian Kepada Masyarakat, 1(1), 20–28. Kementrian Kesehatan Republik Indonesia. (2010). Keputusan menteri kesehatan
Republik Indonesia nomor 492/MENKES/Per/IV/2010 tentang persyaratan
kualitas air minum. Jakarta. Kepala Badan Pengawas Obat dan Makanan. (2019). Peraturan badan pengawas dan
makanan nomor 20 tahun 2019 tentang kemasan pangan. 108 10
Copyright © 2023, Food Scientia Journal of Food Science and Technology Copyright © 2023, Food Scientia Journal of Food Science and Technology Food Scientia Journal of Food Science and Technology 3(1) 2023, 92-110 Kopp, R. D., Grogler, G. D., & Mann, M. D. (1982). Use of carbonic-acid/carbamic-
acid anhydrides as CO2-cleaving blowing agents in the production of cellular or
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bisfenol a dalam botolplastik jenis polikarbonat yang ditetapkan menggunakan
kromatografi cair kinerja tinggi fase terbalik. Skripsi. Jurusan Farmasi. Universitas Sanata Dharma. Yogyakarta. Kurniawan, H. H., & Pusfitasari, E. D. (2017). Uji kualitatif bisphenol a dan
diethylhexyl phthalate menggunakan teknik gc/ms berdasarkan perhitungan
isotop rasio dan indeks retensi. Prosiding Seminar Nasional Kimia UNY. Yogyakarta. Luo, H., Sun, G., shi, Y., Shen, Y., & Xu, K. (2014). Evaluation of the Di(2-
ethylhexyl)phthalate released from polyvinyl chloride medical devices that
contact blood. SpringerPlus, 3(1), 58. https://doi.org/10.1186/2193-1801-3-58 Mead, R. N., & Seaton, P. Copyright © 2023, Food Scientia Journal of Food Science and Technology DAFTAR PUSTAKA J. (2011). GC-MS quantitation and identification of
bisphenol-A isolated from water. Journal of Chemical Education, 88(8), 1130–
1132. https://doi.org/10.1021/ed1006053 Purwanti, E. A. (2020). Air minum galon isi ulang studi tentang perlindungan hukum
bagi konsumen pada depot air minum isi ulang TIRTA QTA. Skripsi. Program
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Copyright © 2023, Food Scientia Journal of Food Science and Technology Copyright © 2023, Food Scientia Journal of Food Science and Technology Food Scientia Journal of Food Science and Technology 3(1) 2023, 92-110 Syamsul, M., Nur, N. H., & Mat, M. A. (2020). Bacteriology quality of refill drinking
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ANÁLISE DA ORIGEM E FREQUÊNCIA DE GOLS EM PARTIDAS OFICIAIS DE FUTSAL DA CATEGORIA SUB-20
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Revista Ibero-Americana de Humanidades, Ciências e Educação
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1 Acadêmico de Educação Física- Universidade Estadual de Roraima - UERR
2 Acadêmico de Educação Física- Universidade Estadual de Roraima - UERR
3Acadêmico de Educação Física- Universidade Estadual de Roraima - UERR
4 Acadêmica de Educação Física- Universidade Estadual de Roraima - UERR
5 Acadêmica de Educação Física- Universidade Estadual de Roraima - UERR
6 Doutor em Ciências do Movimento Humano- Universidade Estadual de Roraima – UERR Revista Ibero- Americana de Humanidades, Ciências e Educação- REASE Revista Ibero- Americana de Humanidades, Ciências e Educação- REASE doi.org/10.51891/rease.v9i5.10146 Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.9.n.05. mai. 2023.
ISSN - 2675 – 3375 ANÁLISE DA ORIGEM E FREQUÊNCIA DE GOLS EM PARTIDAS OFICIAIS DE
FUTSAL DA CATEGORIA SUB-20
ANALYSIS OF THE ORIGIN AND FREQUENCY OF GOALS IN OFFICIAL U-20
FUTSAL MATCHES Jeandson Costa de Aguiar1
Luciano Costa de Aquino Júnior2
Yan Almeida de Araújo3
Kethelen Ágata Costa Ventura4
Maria Isadora Marinho de Oliveira5
Ricardo Alexandre Rodrigues Santa Cruz6 Jeandson Costa de Aguiar1
Luciano Costa de Aquino Júnior2
Yan Almeida de Araújo3
Kethelen Ágata Costa Ventura4
Maria Isadora Marinho de Oliveira5
Ricardo Alexandre Rodrigues Santa Cruz6 RESUMO: O objetivo do presente estudo foi analisar a origem e o período de maior
frequência de gols em partidas oficiais de futsal da categoria sub-20. A pesquisa se caracteriza
por um estudo quantitativo, descritivo e observacional. A amostra foi composta por cinco
equipes que disputavam o Campeonato Roraimense de Futsal da categoria Sub-20 no ano de
2023. Foram filmados 10 jogos da fase classificatória da competição. Para analisar a origem
dos gols foi utilizada uma classificação com diferentes contextos táticos ofensivos das
equipes e para a análise da frequência de ocorrência dos gols dividiu-se o tempo total de jogo
em quatro períodos de 10 minutos. Os resultados evidenciaram que o ataque posicional
obteve a maior incidência na origem dos gols de toda a competição, com 43,8%, totalizando
28 gols em 10 jogos analisados. Verificando mais detalhadamente, se pode constatar também
que se for agrupado o ataque posicional com o contra-ataque juntamente com as jogadas
individuais será obtido o total de 67,3% dos gols da competição. Já para a frequência, os
resultados mostraram que foi no 3o período (20min e 1seg a 30min) que ocorreram o maior
número de gols. Este estudo concluiu que é no segundo tempo de jogo que há maior
Incidência de gols. Espera-se que os resultados encontrados possam contribuir com as
comissões técnicas e os atletas no desempenho dos treinamentos e jogos e que outros estudos
sejam realizados em competições de futsal de diferentes categorias e naipes, contribuindo
dessa forma com mais informações sobre como os gols surgem e em quais momentos dos
jogos de futsal eles ocorrem. 3454 Palavras-chave: Análise. Desempenho. Futsal. 1 Acadêmico de Educação Física- Universidade Estadual de Roraima - UERR
2 Acadêmico de Educação Física- Universidade Estadual de Roraima - UERR
3Acadêmico de Educação Física- Universidade Estadual de Roraima - UERR
4 Acadêmica de Educação Física- Universidade Estadual de Roraima - UERR
5 Acadêmica de Educação Física- Universidade Estadual de Roraima - UERR
6 Doutor em Ciências do Movimento Humano- Universidade Estadual de Roraima – UERR Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.9.n.05. mai. ANÁLISE DA ORIGEM E FREQUÊNCIA DE GOLS EM PARTIDAS OFICIAIS DE
FUTSAL DA CATEGORIA SUB-20
ANALYSIS OF THE ORIGIN AND FREQUENCY OF GOALS IN OFFICIAL U-20
FUTSAL MATCHES 2023
ISSN - 2675 – 3375 Revista Ibero- Americana de Humanidades, Ciências e Educação- REASE ABSTRACT: The objective of the present study was to analyze the origin and period of
higher frequency of goals in official futsal matches for the under-20 category. The research
is characterized by a quantitative, descriptive and observational study. The sample
consisted of five teams that competed in the Roraimense Futsal Championship of the
Under-20 category in the year 2023. 10 games of the qualifying phase of the competition were
filmed. To analyze the origin of the goals, a classification with different offensive tactical
contexts of the teams was used and for the analysis of the frequency of occurrence of the
goals, the total game time was divided into four periods of 10 minutes. The results showed
that the positional attack had the highest incidence in the origin of the goals of the entire
competition, with 43.8%, totaling 28 goals in 10 analyzed games. Checking in more detail, it
can also be seen that if the positional attack is grouped with the counterattack together with
the individual plays, a total of 67.3% of the goals of the competition will be obtained. As for
the frequency, the results showed that it was in the 3rd period (20min and 1sec to 30min)
that the highest number of goals occurred. This study concluded that it is in the second half
of the game that there is a higher incidence of goals. It is hoped that the results found can
contribute to the technical commissions and athletes in the performance of training and
games and that other studies are carried out in futsal competitions of different categories
and suits, thus contributing with more information on how goals arise and in which
moments of the futsal games they occur. Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.9.n.05. mai. 2023. INTRODUÇÃO O futsal é uma modalidade coletiva e intermitente, que apresenta como principais
características as ações simultâneas de ataque e defesa entre duas equipes. Essas
características do futsal exigem dos atletas uma elevada solicitação física, técnica e tática
(SANTA CRUZ et al., 2016). As exigências impostas pelas regras e a complexidade das
ações induzem os jogadores a uma permanente atitude técnico/tática para superarem a
imprevisibilidade das situações de jogo (MÜLLER et al., 2018). 3455 A dinâmica coletiva de cooperação com os companheiros e de oposição com os
adversários, juntamente com situações imprevisíveis, aleatórias e variáveis tornam o futsal
um esporte com características complexas e sistêmicas (UEDA et al., 2020). Dessa forma,
as interações entre os jogadores são essenciais para a continuidade do jogo e o desempenho
nas partidas é norteado pelas principais fontes de informação sobre espaço e tempo que
surgem entre atacantes e defensores (ALVES et al, 2022). A análise de desempenho no esporte vem auxiliando treinadores, atletas e
pesquisadores a desenvolverem uma compreensão objetiva do desempenho esportivo de
forma real. No esporte de alto rendimento a análise de desempenho tem se mostrado uma
técnica indispensável em diversas equipes esportivas, permitindo aos atletas e treinadores ,
ISSN - 2675 – 3375 Revista Ibero- Americana de Humanidades, Ciências e Educação- REASE Revista Ibero- Americana de Humanidades, Ciências e Educação- REASE obterem feedbacks em tempo real de estatísticas de partidas e sequências de vídeo usando
sistemas ajustáveis de internet (ALVES et al., 2021). No futsal, a análise de desempenho do jogo é uma ferramenta que auxilia os
treinadores a elaborarem seus treinos e organizarem suas equipes, qualificando assim a sua
ação pedagógica (VOSER et al., 2018). Observar os aspectos técnicos/táticos decorrentes do
jogo, torna-se um fator necessário para conhecer as ações individuais e coletivas dos atletas. Moraes et al., (2013) destacam que o processo de coleta e análise dos dados nos jogos de futsal
têm se tornado cada vez mais importantes na busca do aumento do rendimento coletivo e
individual, evidenciando que a análise sobre os jogos tem grande potencial para auxiliar os
treinadores a compreenderem o cenário atual e promover treinos mais específicos e eficazes. MÉTODOS O presente estudo se caracteriza por ser uma pesquisa quantitativa, descritiva de
cunho observacional (PEREIRA, 2018). A amostra foi composta por cinco equipes que
disputavam o Campeonato Roraimense de Futsal da categoria Sub-20 no ano de 2023. Foram
observados 10 jogos da fase classificatória da competição. Os jogos foram filmados em loco
e os dados coletados por meio das observações das imagens em vídeos, sendo analisados e
organizados em planilhas do Programa Microsoft Excel, onde utilizou- se os valores
absolutos, percentual e média. A entidade promotora da competição foi a Federação Roraimense de Futsal (FRFS)
e o seu representante legal (Presidente) assinou o Termo de Consentimento autorizando a
pesquisa, anteriormente ao processo de coleta dos dados. INTRODUÇÃO Garganta (2001), enfatiza que as informações colhidas a partir da análise de jogo são
uma das variáveis que mais colaboram com a construção metodológica dos treinamentos e
que o processo de coletar e analisar os dados a partir das observações dos jogos são aspectos
cada vez mais importantes na busca do melhor rendimento dos atletas e consequentemente
das equipes. Dessa forma, o objetivo do presente estudo foi analisar a origem e a frequência
dos gols em partidas oficiais de futsal da categoria Sub-20. 3456 Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.9.n.05. mai. 2023.
ISSN
2675
3375 Análise da Origem dos Gols Para indicar a origem dos gols foram adotados os contextos técnicos/táticos
ofensivos citados por Voser et al. (2016) e as adaptações citadas por Gonçalves e Galvão
(2019) que possibilitou classificar os gols nas seguintes categorias: Revista Ibero- Americana de Humanidades, Ciências e Educação- REASE • Ataque Posicional (AP): situação onde o ataque e a defesa já estão organizados em
suas posições e se enfrentam em 4x4; • Jogadas Individuais (JI): é a ação individual do jogador onde que, utiliza-se da sua
habilidade para realizar a possibilidade de fazer o gol; • Contra-Ataque (CA): é a transição da defesa para o ataque em velocidade com ou
sem superioridade numérica; • Faltas com Barreira (FB): é uma possibilidade clara de se marcar um gol quando na
quadra ofensiva, pois tem a oportunidade de se elaborar uma manobra através da bola
estar parada e seus adversários posicionados defensivamente a cinco metros da bola,
onde que quando as jogadas bem ensaiadas, treinadas e organizadas pode-se
possibilitar uma clara oportunidade de gol; • Escanteio (E); Lateral (L): ocorrem com muita frequência em jogos de futsal e podem
ser propícios para se elaborar manobras ensaiadas para finalizar em gol; • Pênalti (P) ou Tiro Livre sem Barreira (TLB): Pênalti é uma infração que uma equipe
comete sobre seu adversário ou ao jogo dentro da área do goleiro, dessa forma é
realizada uma oportunidade clara de se realizar o gol onde que, prevalece a
característica e a técnica de cada jogador ou do goleiro para converter ou não o gol. Análise da Origem dos Gols Já
o Tiro Livre sem Barreira (TLB) também é uma oportunidade clara de realizar o gol
porem é mais distante, sendo cobrado a 10 metros do gol, onde a infração só ocorre se
uma equipe cometer a 6a faltas em um período do jogo, da mesma forma, prevalece
a característica e a técnica de cada jogador ou goleiro para converter ou não o gol; 3457 • Expulsão Temporária com inferioridade numérica do adversário (ET): ocorre
eventualmente nos jogos de futsal, pois por dois minutos a equipe infratora fica com
inferioridade numérica e assim prevalece à calma e a tranquilidade do adversário de
propiciar a possibilidade de gol; • Goleiro Linha (GL): a equipe se utiliza de um goleiro ou jogador de linha
devidamente uniformizado de forma diferente de seus outros companheiros que
atuam na linha, para criar uma superioridade numérica a efetuar gols ou manter a
posse de bola. ANÁLISE DOS PERÍODOS DE OCORRÊNCIA DOS GOLS Para identificar os períodos de ocorrência dos gols durante as partidas de futsal Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.9.n.05. mai. 2023. ISSN - 2675 – 3375 Revista Ibero- Americana de Humanidades, Ciências e Educação- REASE Revista Ibero- Americana de Humanidades, Ciências e Educação- REASE analisadas, utilizou-se a periodicidade citada por Staudt e Voser, 2011, dividindo o tempo
total da partida em quatro períodos, da seguinte maneira: • 1o período (0 a 10min); • 2o período (10min e 01s a 20min); • 3o período (20min e 01s a 30min); • 4o período (30min e 01s a 40min). RESULTADOS E DISCUSSÃO Na tabela 1 apresentamos a origem dos gols de acordo com os contextos
técnico/táticos ocorridos nas partidas de futsal analisadas. 3458
Tabela 1 – Origem dos gols durante os jogos de futsal. Origem dos
Gols
AP
JI
CA
FB
E/L
P/TLB
ET
GL
Número de
Gols
28
06
09
02
14
03
02
00
%
43,8%
9,5%
14,0%
3,1%
21,8%
4,7%
3,1%
0,0%
Legenda: AP: ataque posicional; JI: jogada individual; CA: contra-ataque; FB: falta com
barreira; E/L: escanteio e lateral; P/TLB: pênalti e tiro livre sem barreira; ET: expulsão
temporária; GL: goleiro linha. Tabela 1 – Origem dos gols durante os jogos de futsal. Na tabela 2 apresentamos a distribuição dos gols de acordo com os períodos de
ocorrência nas partidas de futsal analisadas. Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.9.n.05. mai. 2023. ISSN - 2675 – 3375
Tabela 2 – Distribuição da ocorrência dos gols ao longo dos períodos. Períodos
1o
Período
2o
Período
3o
Período
4o
Período
Duração
0 a 10m
10m e 01s a
20m
20m e 01s a
30m
30m e 01s a
40m
Total de Gols
11
13
23
17
% Incidência
dos Gols
17,2%
20,4%
35,9%
26,5%
Legenda: m: minutos; s: segundos Tabela 2 – Distribuição da ocorrência dos gols ao longo dos períodos. Tabela 2 – Distribuição da ocorrência dos gols ao longo dos períodos. Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.9.n.05. mai. 2023. ISSN - 2675 – 3375 Revista Ibero- Americana de Humanidades, Ciências e Educação- REASE O objetivo do presente estudo foi analisar a origem e a frequência dos gols em
partidas oficiais de futsal da categoria Sub-20. Paulichen et al., (2020) indicam que o
constante estudo e análise das partidas de futsal possibilita a comissão técnica evidenciar o
padrão tático utilizado por seus adversários, bem como planejar o treinamento físico, técnico
e tático de seus atletas com o objetivo de elevar o desempenho geral da equipe. Nesse sentido,
conhecer como os gols são originados e em quais períodos da partida acontecem a maior
incidência de gols torna-se fundamental para que as comissões técnicas possam estruturar
seus treinamentos. Durante os 10 jogos analisados nesta pesquisa, verificou-se a ocorrência de um total
de 64 gols, com média de 6,4 ± 3,0, gols por partida. Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.9.n.05. mai. 2023. RESULTADOS E DISCUSSÃO Os resultados observados na tabela 1
indicam que o maior número de gols se originou de ataque posicional, com um total de 28
gols (43,8%). O ataque posicional também foi citado por Brancher et al. (2020), durante uma
competição de futsal feminino como a ação organizada com maior frequência de ocorrência
com um total de 91 gols (60,26%). Diferentemente dos achados do presente estudo, Voser et
al. (2016) constataram que os gols de ataque posicional na Liga Brasileira de Futsal em 2014
foram menores que os originados de contra-ataque, que obteve a maior Incidência na origem
dos gols de toda a competição, com 25%, totalizando 104 gols em 58 jogos, média de 1,79 por
do total dos jogos analisados. 3459 Quando analisamos os gols ocasionados pelos contra-ataques, verificamos na
presente pesquisa que nas dez rodadas do campeonato Roraimense da categoria sub-20 foram
realizados 9 gols nesse tipo de jogada, equivalente a 14% dos gols. Nesse sentido, Alves e
Bueno (2012) destacaram o contra-ataque e o ataque posicional como as principais origens
dos gols da liga futsal 2012, evidencias que também se comprovaram em nosso estudo. No futsal, um aspecto relevante e que caracteriza a modalidade é a individualidade e
a qualidade técnica dos atletas. Dessa forma, dos 64 gols analisados, 6 foram originados de
jogadas individuais, indicando um percentual de 9,5%. Outro dado a ser considerado quando
analisamos os gols oriundos de bola em jogo, ou “bola rolando” foram os que surgiram
quando uma das equipes estava com inferioridade numérica por expulsão temporária, que
ocorre eventualmente nos jogos de futsal, pois por dois minutos a equipe infratora fica com
inferioridade numérica e assim verificamos a ocorrência de 2 gols (3,1%). Analisando de forma geral os resultados dos gols que foram marcados em bolas
paradas (lateral, escanteio, falta com barreira, tiro livre dos 10 metros sem barreira e pênalti)
observamos um percentual total de 32,7% nesses tipos de gols. Esses dados, indicam que os ,
ISSN - 2675 – 3375 Revista Ibero- Americana de Humanidades, Ciências e Educação- REASE Revista Ibero- Americana de Humanidades, Ciências e Educação- REASE técnicos devem dedicar atenção especial aos treinamentos de jogadas ensaiadas de bola
parada e ao treinamento específico para cobrança de tiros livres e pênaltis. Apesar das equipes da categoria sub-20 utilizarem a estratégia do goleiro linha em
algumas das partidas analisadas, não foram verificados gols originados por essa ação tática
das equipes. RESULTADOS E DISCUSSÃO Quanto a frequência de gols e o período de ocorrência dos mesmos, de acordo com a
tabela 2 pode-se verificar que ocorreram 11 gols (17,2%) no 1o período, 13 gols (20,4%) no 2o
período, 23 gols (35,9%) no 3o período e 17 gols (26,5%) no 4o período. Esses resultados
indicam que 37,6% dos gols ocorrem no primeiro tempo e 62,4% no segundo tempo das
partidas. Ao analisarmos os períodos de ocorrência, percebemos que o 3o período foi apontado
como o de maior ocorrência dos gols, nos 10 primeiros minutos após a volta do intervalo, já
o período de menor incidência foi o 1o, provavelmente em virtude da análise inicial das
equipes quanto ao sistema de jogo e ações defensivas e ofensivas. Diferentemente dos nossos achados, alguns estudos (STAUDT e VOSER, 2011;
FUKUDA e SANTANA, 2012; VOSSER et al. 2018) observaram o 4o período como o de
maior ocorrência de gols. Esses dados se relacionam com a descrição de Gonçalves (2015),
citada por Voser et al., (2018) onde o autor descreve que: “Com relação ao tempo, percebe-se que
no segundo tempo de jogo os gols saem em maior número, principalmente nos últimos 10 minutos da
partida. É nítido também, que nesses últimos 10 minutos, o número de gols através de contra- ataque
e de goleiro linha é muito maior em relação aos outros momentos do jogo, vale ressaltar que os gols
obtidos através de erro de goleiro linha foram enquadrados em contra- ataques.” 3460 Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.9.n.05. mai. 2023. CONSIDERAÇÕES FINAIS Consideramos que os resultados do presente estudo, com base nas análises realizadas
sobre os 10 jogos de futsal da categoria sub-20, permitem-nos concluir que quanto a
ocorrência, a maioria dos gols ocorreram de ataque posicional, seguido de gols de bola parada,
especificamente de cobranças de lateral e escanteios. Já em relação a análise dos períodos de
ocorrência dos gols, verificou-se que o terceiro período de jogo foi onde ocorreu a maior
incidência de gols na competição, com atenção especial para o segundo tempo das partidas,
momento em que ocorreram com maior frequência os gols. Esses achados podem ser
relevantes para as comissões técnicas organizarem seus treinamentos e orientarem seus
atletas para a origem e ocorrência dos gols. Sugerimos que outros estudos sejam realizados
em competições de futsal de diferentes categorias e naipes, contribuindo dessa forma com ,
ISSN - 2675 – 3375 Revista Ibero- Americana de Humanidades, Ciências e Educação- REASE mais informações sobre como os gols surgem e em quais momentos dos jogos de futsal eles
ocorrem. mais informações sobre como os gols surgem e em quais momentos dos jogos de futsal eles
ocorrem. Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.9.n.05. mai. 2023.
ISSN - 2675 – 3375 REFERÊNCIAS ALVES, M.A.R.; GRAÇA, D.C.; TRAVASSOS, B. Construction and validation of an
observation tool of the imbalance pass in futsal. Rev Bras Cineantropom Desempenho
Hum, n. 24, 2022. ALVES, M.A.R.; GRAÇA, D.C.; FEITOZA, M.C.; SOARES, B.H. Produção científica
sobre análise de desempenho técnico-tático no futsal. Research, Society and Development,
v. 10, n. 12, e365101220450, 2021. ALVES, I. P.; BUENO, L. Análise dos gols na primeira fase da liga futsal 2012. Revista
Brasileira de Futsal e Futebol. São Paulo. v. 4. n. 12. p.118-123. 2012. BRANCHER, E.A.; LEITE, G.A.; SLOMP, N.N. A origem dos gols em jogos de futsal
feminino durante os jogos abertos de Santa Catarina 2019. Revista Brasileira de Futsal e
Futebol, São Paulo. v.12. n.51. p.705-712. Suplementar 1. 2020. FUKUDA, J. P. S; SANTANA, W. C. Análise dos gols em jogos da liga futsal 2011. Revista
Brasileira de Futsal e Futebol. São Paulo. Vol. 4. Núm. 11. p.62-66. 2012. GARGANTA, J. A análise da performance nos jogos desportivos. Revisão acerca da análise
do jogo. Revista Portuguesa de Ciências do Desporto. Porto. Vol. 1. p.57-64. 2001. 3461 GONÇALVES, R.B.M.; GALVÃO, R.V. Análise dos gols no futsal: uma revisão de
literatura. Revista Científica Eletrônica de Ciências Aplicadas da FAIT, Ano VIII. v 13, n 1,
maio, 2019. MORAES, J.C.; PERIN, D.; CARDOSO, M.F.S.; MONTEIRO, A.O.; VOSER, R.C. Análise das finalizações e posse de bola em relação ao resultado do jogo de futebol. R. Min. Educ. Fis. Viçosa. Edição Especial. v. 9. p.397- 403, 2013. MÜLLER, E.S.; COSTA, I.T.; GARGANTA, J. Análise tática no futsal: estudo
comparativo do desempenho de jogadores de quatro categorias de formação. Rev Bras Ciênc
Esporte, v. 40, n. 3, p. 248-256, 2018. PAULICHEN, H.M. et al. Analysis of futsal matches using a single-camera computer
vision system. Evento Online. Anais [...]. Porto Alegre: Sociedade Brasileira de
Computação, p. 134-139, 2020. PEREIRA, A.S. et al. Metodologia da pesquisa científica. [free e-book]. Santa Maria/RS:
Ed. UAB/NTE/UFSM, 2018. SANTA CRUZ, R.A.R; CAMPOS, F.A.D; GOMES, I.C.B; PELLEGRINOTTI, I.L. Percepção subjetiva do esforço em jogos oficiais de Futsal. R. bras. Ci. e Mov, v.24, n.1, p.80-
85, 2016. Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.9.n.05. mai. 2023. ISSN - 2675 – 3375 Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.9.n.05. mai. 2023. ISSN - 2675 – 3375 Revista Ibero- Americana de Humanidades, Ciências e Educação- REASE Revista Ibero- Americana de Humanidades, Ciências e Educação- REASE
STAUDT, A. REFERÊNCIAS R.; VOSER, R. C. Incidência de gols ocorridos em campeonato estudantil de
futsal masculino. Revista Digital. Lecturas: Educación Física y Desporte. Buenos Aires. Ano. 16. N. 160. 2011. VOSER
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i
d
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R
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il i
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F
b l STAUDT, A. R.; VOSER, R. C. Incidência de gols ocorridos em campeonato estudantil de
futsal masculino. Revista Digital. Lecturas: Educación Física y Desporte. Buenos Aires. Ano. 16. N. 160. 2011. STAUDT, A. R.; VOSER, R. C. Incidência de gols ocorridos em campeonato estudantil de
futsal masculino. Revista Digital. Lecturas: Educación Física y Desporte. Buenos Aires. Ano. 16. N. 160. 2011. VOSER et al. A origem dos gols da liga de futsal 2014. Revista Brasileira de Futsal e Futebol,
São Paulo. v.8. n.29. p.155-160, 2016. UEDA, L.S.C.; MENEGASSI, V.M.; AVELAR, A.; RECHENCHOSKY, L.; SILVA,
F.L.O.; BORGES, P.H. Analysis of the execution of core tactical principles and technical
efficiency of primary school futsal players. Rev Bras Cineantropom Desempenho Hum,
v.22, 2020. UEDA, L.S.C.; MENEGASSI, V.M.; AVELAR, A.; RECHENCHOSKY, L.; SILVA,
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v.22, 2020. 3462
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* Mestre em Educação. Enfermeira, doutoranda na EEUSP/EEUFMG. Professora Adjunto da Faculdade de Enfermagem da
Universidade Federal de Juiz de Fora
** Doutora em Enfermagem. Enfermeira, Professora Adjunta da Escola de Enfermagem da Universidade Federal de Minas Gerais
*** Mestre em Enfermagem Médico-Cirúrgica. Enfermeira, doutoranda na EEUSP/EEUFMG. Professora Adjunto da Escola de
Enfermagem da Universidade Federal de Minas Gerais INTRODUÇÃO norteasse as nossas pesquisas - etnográfica e
fenomenológica - deparamo-nos com diferentes
abordagens e classificações usadas pelos autores que
adotam a linha qualitativa. A partir da década de 60, nos Estados Unidos e
de 80, no Brasil, observa-se na enfermagem um crescente
interesse nas pesquisas interpretativas. Em decorrência
disso surgem diferentes nomenclaturas e referências
ontológicas e epistemológicas, dando origem a
conceituações e classificações não bem definidas ou
mesmo equivocadas. Por outro lado, identificamos a escassez de
trabalhos na área da Enfermagem que discutem os
aspectos epistemológicos e metodológicos das
abordagens interpretativas. Acreditando não ser possível desvincular nossa
visão de mundo e a intencionalidade de nossas vivências
enquanto docentes e pesquisadores, realizamos uma breve
revisão da literatura com o objetivo de delimitar o
referencial filosófico, sociológico e antropológico que
fundamentasse a elaboração dos nossos projetos de
pesquisa. Assim como afirma LOWENBERG (1993), os
pesquisadores da enfermagem que adotam as abordagens
qualitativas desenvolveram seus programas de pós-
graduação em diferentes áreas do conhecimento, como
na Sociologia, Antropologia e Psicologia, levando a uma
dificuldade de delinear significados comuns em seus
discursos. Na fase de elaboração dos projetos para
qualificação das autoras em Programas de Doutorado
deparamos com a dificuldade de identificação da corrente
filosófica que fundamentasse nossos pressupostos
teórico-metodológicos. Nossos projetos estavam
vinculados à compreensão de fenômenos situados no
cotidiano da enfermagem, buscando significados
atribuídos pelos sujeitos a suas experiências, num enfoque
sociológico e antropológico. UNITERMOS: pesquisa interpretativa, hermenêutica, fenomenologia, antropologia UNITERMOS: pesquisa interpretativa, hermenêutica, fenomenologia, antropologia Rev.latino-am.enfermagem - v. 6 - n. 2 - p. 29-35 - abril 1998 Rev.latino-am.enfermagem - v. 6 - n. 2 - p. 29-35 - abril 1998 29 O PARADIGMA HERMENÊUTICO COMO FUNDAMENTAÇÃO DAS PESQUISAS
ETNOGRÁFICAS E FENOMENOLÓGICAS Maria Cristina Pinto de Jesus*
Marisa Ribeiro Bastos Peixoto** Maria Cristina Pinto de Jesus*
Marisa Ribeiro Bastos Peixoto**
Mércia Heloísa Ferreira Cunha*** JESUS, M.C.P. de; PEIXOTO, M.R.B.; CUNHA, M.H.F. O paradigma hermenêutico como fundamentação das
pesquisas etnográficas e fenomenológicas. Rev.latino-am.enfermagem, Ribeirão Preto, v. 6, n. 2, p. 29-35, abril
1998. JESUS, M.C.P. de; PEIXOTO, M.R.B.; CUNHA, M.H.F. O paradigma hermenêutico como fundamentação das
pesquisas etnográficas e fenomenológicas. Rev.latino-am.enfermagem, Ribeirão Preto, v. 6, n. 2, p. 29-35, abril
1998. O estudo apresenta uma revisão da literatura sobre o paradigma hermenêutico como fundamentação das pesquisas
etnográficas e fenomenológicas. todo, com sua vontade, sensibilidade e
imaginação” (JAPIASSU & MARCONDES, 1995,
p.73). para o seu próprio sistema de valores e
significados”(BLEICHER,1992, p. 13). para o seu próprio sistema de valores e
significados”(BLEICHER,1992, p. 13). A hermenêutica tem sido utilizada como base
filosófica para diferentes estudos qualitativos realizados
por enfermeiros, tendo em vista que o homem e as
interações humanas são freqüentemente objeto de estudo
da enfermagem. Estudos de tal natureza remetem a
processos interpretativos do investigador, a importância
do contexto na compreensão de significados, bem como
a compreensão da linguagem oral e escrita do outro. A compreensão é enfocada por Dilthey sob o
ponto de vista interno, ou seja, a partir da psicologia
(DILTHEY, 1979). Para ele todas as formas de
conhecimento do espírito, enquanto implicar numa
relação com a história, teria como pressuposto a
capacidade do homem de se transpor para dentro da vida
psíquica do outro. Independente da natureza da pesquisa qualitativa
adotada tais abordagens têm em comum a busca da
compreensão e da interpretação do homem como ser
único e singular; com a apreensão do ser humano em sua
totalidade e do homem como melhor intérprete de si
mesmo (VIETTA, 1995, p.34-35). SIEBENEICHLER (1983) ao descrever as idéias
inspiradoras da fenomenologia para a hermenêutica
filosófica diz que Husserl, filósofo alemão, teve o mérito
de recolocar de forma crítica a ciência e a técnica no
contexto do sujeito. Husserl, em seus escritos, tomou como ponto de
partida uma ligação intencional entre espírito e mundo,
tentando explicar a situação da consciência, do sujeito
humano. Por meio do método da “epoché” e da redução
fenomenológica encontra um instrumento esclarecedor
da consciência, capaz de ultrapassar o psicologismo e o
historicismo reinantes na época. Husserl ao tentar
encaixar a problemática da consciência individual em
um contexto mais amplo verificou que era possível
“fundamentar idéias conscientes, recorrendo a uma
experiência pré-consciente, não articulada”. (SIEBENEICHLER, 1983, p.10). A arte de interpretação, hermenêutica, foi
inicialmente empregada no domínio teológico na
interpretação de textos bíblicos. A partir da idade
moderna passa a ter o sentido de “arte da compreensão”
ou seja, interpretação correta e objetiva dos textos da
Escritura (CORETH, 1973). Por outro lado, a Hermenêutica enquanto forma
de interpretação, era utilizada em textos profanos na
mitologia e na área jurídica, decorrendo numa
regionalização da hermenêutica. A HERMENÊUTICA E AS PESQUISAS
INTERPRETATIVAS A hermenêutica é definida por BLEICHER
(1992), como a teoria ou filosofia da interpretação dos
sentidos. O problema hermenêutico é fundamentado na
“... percepção de que as expressões humanas
contêm componente significativo, que tem que ser
reconhecido como tal, por um sujeito e transposto A hermenêutica é definida por BLEICHER
(1992), como a teoria ou filosofia da interpretação dos
sentidos. O problema hermenêutico é fundamentado na “... percepção de que as expressões humanas
contêm componente significativo, que tem que ser
reconhecido como tal, por um sujeito e transposto Buscando definir o referencial filosófico que O paradigma hermenêutico... Rev.latino-am.enfermagem - v. 6 - n. 2 - p. 29-35 - abril 1998 30 FREUND (1987, p.73-74) interpretando o
pensamento de Weber diz que: Toda interpretação se
propõe a vencer um afastamento, uma distância,
entre a época cultural revoluta, à qual pertence o
texto, e o próprio intérprete... Portanto, o que ele
persegue, através da compreensão do outro, é a
ampliação da própria compreensão de si mesmo. Assim, toda hermenêutica é, explícita ou
implícitamente, compreensão de si mesmo
mediante a compreensão do outro” Interessado na Sociologia weberiana, Schütz
parte para fundamentar a Sociologia da ação e
compreensão por meio da análise filosófica da
fenomenologia de Husserl. Segundo o autor a “estrutura significativa do
mundo social somente pode deduzir-se a partir das
características mais primitivas e gerais da consciência”. Visa, assim, determinar a natureza precisa do fenômeno
do significado mediante a análise da função constitutiva,
chegando a conclusão de que “a ação é uma vivência
guiada por um plano ou projeto que surge da atividade
espontânea do sujeito, sendo distinguida de todas as
outras vivências por um ato peculiar de atenção”. (SCHÜTZ, 1972, p.243) Assim, a hermenêutica busca “compreender
sentidos” ou seja o conteúdo típico humano que se
imprime a qualquer contexto histórico, no qual não
existem apenas fatos dados, acontecimentos externos,
mas, também, “significação”, “sentido” e “valores”
(DEMO, 1981, p.249). Ao se aplicar a uma disciplina a hermenêutica se
especifica ganhando feições próprias. Schütz propõe a investigação no mundo
cotidiano, onde o homem olha para esse mundo do ponto
de vista da atitude natural. Tendo nascido nesse mundo
que também é social e cultural o homem vive com seus
congêneres e dá por certa a existência destes sem
questioná-la, assim como dá por certa a existência de
objetos naturais. As pessoas interagem e compreendem todo, com sua vontade, sensibilidade e
imaginação” (JAPIASSU & MARCONDES, 1995,
p.73). A partir do século XIX há uma tentativa de
unificação dessas hermenêuticas especiais ou regionais
numa hermenêutica geral ou universal formulada por
Friedrich Schleiermacher, que introduz a questão do texto/
contexto (CORETH, 1973). A partir da herança deixada por Husserl,
Heidegger, em sua obra “Ser e Tempo”, “se afasta da fenomenologia de seu mestre Husserl
e inicia seu caminho de reflexão sobre o sentido
mais profundo da existência humana, bem como
sobre as origens da metafísica e o significado de
sua influência na formação do pensamento
ocidental” (JAPIASSU & MARCONDES, 1995,
p.116). Para Schleiermacher, a hermenêutica é a arte da
compreensão, que não visa ao saber teórico e sim ao saber
prático: “a práxis ou a técnica da boa interpretação de
um texto falado ou escrito”. O autor define a hermenêutica
como a “reconstrução histórica e divinatória objetiva e
subjetiva de um dado discurso” (CORETH, 1973, p.19). Heidegger observou que o mundo humano não
significa apenas o meio ambiente exterior e nem tão pouco
constituía-se de uma construção subjetiva e arbitrária
(SIEBENEICHLER, 1983). Wilhelm Dilthey, filósofo alemão, valorizou a
teoria da “visão do mundo” em que viver é interpretar o
mundo natural. Sua obra “Introdução ao estudo das
ciências humanas” (1883), critica a visão positivista da
“explicação” sobre o homem buscando a “compreensão”
da sua natureza social e histórica. (JAPIASSU &
MARCONDES, 1995, p.73). Em suas últimas obras, Heidegger explicita que
a compreensão se expressa na linguagem e nela se
constitui o horizonte histórico da compreensão. “A
hermenêutica torna-se, assim, a interpretação da primitiva
compreensão do homem em si e do ser” (CORETH, 1973,
p.23). Dilthey, apoiando-se em Schliermacher, formula
a dualidade de ciências da natureza e ciências do espírito
que se distinguem por um método analítico esclarecedor
e um procedimento de compreensão descritiva. Baseado em Heidegger, de quem foi seu discípulo
mais próximo e em seus estudos humanísticos, Gadamer “desenvolveu um interesse filosófico pelo diálogo
com a tradição, com as línguas e as culturas
distantes e refletiu sobre as condições históricas e
filosóficas da compreensão e da interpretação”
(HABERMAS, 1987, p.7) “Ao pretender dar um fundamento às ciências
particulares do homem, Dilthey postula a criação
de novos métodos e de conceitos psicológicos mais
sutis adaptados à vida histórica; ademais procura
evidenciar, em todas as manifestações humanas,
a totalidade da visão psíquica, a ação do homem A partir das formulações de Schleiermacher, O paradigma hermenêutico... Rev.latino-am.enfermagem - v. FREUND (1987, p.73-74) interpretando o
pensamento de Weber diz que: “são racionais a compreensão atual do sentido
de uma operação aritmética que efetua o
comportamento de um lenhador que abate uma
árvore, bem como a compreensão pelos motivos
quando vejo uma pessoa ocupada em um trabalho
a fim de ganhar sua vida. São irracionais a
compreensão atual da cólera ou a compreensão
pelos motivos quando a pessoa se serve de uma
espingarda para se vingar ou entra em cólera por
ciúme... Compreender, pode-se dizer, é captar a
evidência do sentido de uma atividade”. “são racionais a compreensão atual do sentido
de uma operação aritmética que efetua o
comportamento de um lenhador que abate uma
árvore, bem como a compreensão pelos motivos
quando vejo uma pessoa ocupada em um trabalho
a fim de ganhar sua vida. São irracionais a
compreensão atual da cólera ou a compreensão
pelos motivos quando a pessoa se serve de uma
espingarda para se vingar ou entra em cólera por
ciúme... Compreender, pode-se dizer, é captar a
evidência do sentido de uma atividade”. De seus estudos sobre Marx e Freud, Habermas
adota como preocupação central uma política
emancipatória, refletindo sobre as condições de um
“diálogo livre de dominação”, a possibilidade de uma
“comunicação isenta de coação e violência”. Para o autor, a hermenêutica, mais do que arte
de interpretar, ou tecnologia, é crítica (HABERMAS,
1987, p.6-27). Na perspectiva de RICOUER(1978, p.18), a
hermenêutica implica na compreensão dos signos e de
si. De acordo com SCHÜTZ (1972, p.35) Weber
definiu como tarefa da Sociologia não a especulação
metafísica e sim uma descrição simples e cuidadosa da
vida social. Não uma sociologia enquanto filosofia da
existência humana, mas como a ciência particular da
conduta humana e suas conseqüências que deve dar conta
da compreensão e interpretação da ação social. “Ao propor religar a linguagem simbólica à
compreensão de si, penso satisfazer o desejo mais
profundo da hermenêutica. Toda interpretação se
propõe a vencer um afastamento, uma distância,
entre a época cultural revoluta, à qual pertence o
texto, e o próprio intérprete... Portanto, o que ele
persegue, através da compreensão do outro, é a
ampliação da própria compreensão de si mesmo. Assim, toda hermenêutica é, explícita ou
implícitamente, compreensão de si mesmo
mediante a compreensão do outro” “Ao propor religar a linguagem simbólica à
compreensão de si, penso satisfazer o desejo mais
profundo da hermenêutica. todo, com sua vontade, sensibilidade e
imaginação” (JAPIASSU & MARCONDES, 1995,
p.73). 6 - n. 2 - p. 29-35 - abril 1998 31 31 Dilthey e Heidegger, Hans-Georg Gadamer elabora uma
teoria filosófica em que a Hermenêutica adquire um status
de corrente filosófica. contribuição de outros estudiosos, a exemplo do
fenomenólogo Alfred Schütz. Ao elaborar o “tipo ideal” a partir de princípios
vinculados à história, Weber pretende compreender a ação
social como a resultante de forças de relações sociais,
ação proveniente do comportamento social que tem um
significado subjetivo. Desse modo, coloca o homem como
centro das atenções já que a compreensão dos significados
se dá no sujeito. Considera “as ações motivadas por
sentimentos afetivos e as tradicionais como menos
racionais”, sendo as ações que se aproximam do tipo
racional como as mais compreensíveis (CAPALBO, 1979,
p.86). Na ótica de SIEBENEICHLER (1983, p.24)
Gadamer compreende a Hermenêutica de uma forma mais
ampla do que a arte de interpretar. Ela constitui “uma
possibilidade no horizonte da mediação entre a verdade
do nosso ser e o método da ciência”. A redescoberta fenomenológica do mundo da
vida aliada à nova compreensão da linguagem levou a
uma troca intensa de idéias entre a fenomenologia e a
Hermenêutica filosófica. Tal redescoberta coloca como possibilidade a
mediação entre a razão científica e a razão filosófica,
além de possibilitar a constituição da moderna filosofia
Hermenêutica de Gadamer e de Paul Ricoeur e da filosofia
crítica representada por Habermas. (SIEBENEICHLER,
1983, p.12). FREUND (1987, p.73-74) interpretando o
pensamento de Weber diz que: A HERMENÊUTICA E A SOCIOLOGIA
FENOMENOLÓGICA A Sociologia interpretativa teve suas raízes na
teoria de Max Weber e posteriormente recebeu a Rev.latino-am.enfermagem - v. 6 - n. 2 - p. 29-35 - abril 1998 O paradigma hermenêutico... 32 a si próprias e aos outros na realidade social. No entanto,
ressalta que somente a olhada reflexiva do observador
eleva o conteúdo da consciência do status pré-fenomênico
até o fenomênico. ação humana deve ser construído de tal maneira que um
ato humano efetuado dentro do mundo da vida, por um
ator individual, da maneira indicada pela construção
típica, seja compreensível tanto para o ator como para
seus semelhantes, em termos de interpretação de sentido
comum na vida cotidiana. Assim, fica garantida a
compatibilidade das construções do cientista social com
as construções da experiência do sentido comum na
realidade social (SCHÜTZ, 1962, p.67-68). O fenomenólogo se volta para a atitude natural e
estuda o que “já se encontra estruturado e de certo modo
interpretado, pois a realidade social já possui um sentido
para os homens que vivem em seu seio” (CAPALBO,
1979, p.36). Para Schütz a tarefa primeira da Sociologia
compreensiva consiste em descrever os processos de
estabelecimento e interpretação de significado tal como
os realizam as pessoas que vivem no mundo social. Ressalta que esta descrição pode ser empírica ou eidética. Pode tomar como tema a pessoa ou o tipo. Pode realizar-
se em situações concretas da vida cotidiana ou com alto
grau de generalidade. Além disso a Sociologia
compreensiva pode enfocar os objetos culturais e tratar
de compreender o seu significado, aplicando-lhes os
esquemas interpretativos. Para Schütz a tarefa primeira da Sociologia
compreensiva consiste em descrever os processos de
estabelecimento e interpretação de significado tal como
os realizam as pessoas que vivem no mundo social. Para SCHÜTZ (1962, p.23-24) a compreensão
de uma determinada coisa só é possível ao reduzi-la à
atividade que a criou e aos motivos que a originaram,
somente sendo possível compreender a atividade humana
compreendendo a ação correspondente. Assim, a ação
está determinada pelo projeto que inclui o “motivo para”
que é o propósito da ação e o “motivo porque” ou seja a
razão ou causa da ação. Pode tomar como tema a pessoa ou o tipo. Pode realizar-
se em situações concretas da vida cotidiana ou com alto
grau de generalidade. Além disso a Sociologia
compreensiva pode enfocar os objetos culturais e tratar
de compreender o seu significado, aplicando-lhes os
esquemas interpretativos. A HERMENÊUTICA E A SOCIOLOGIA
FENOMENOLÓGICA Para interpretar a ação sob o ponto de vista do
ator, ponto de vista subjetivo, Schütz indica um sistema
objetivo de análise cujos recursos metodológicos possam
alcançar a estrutura subjetiva de sentido, ou seja, a
construção do esquema típico-ideal. O autor elege o esquema subjetivo como o
escolhido ao estudar o mundo social tal qual é vivido
pelo homem e seus congêneres na atitude natural. O autor elege o esquema subjetivo como o
escolhido ao estudar o mundo social tal qual é vivido
pelo homem e seus congêneres na atitude natural. Por meio da construção do esquema típico-ideal
o autor chega ao “tipo ideal pessoal” que se expressa de
certa maneira e tem tais e quais vivências e ao tipo
material ou de curso de ação que é o tipo ideal do processo
expressivo mesmo (SCHÜTZ, 1972, p.216). O tipo ideal em Schütz é elaborado a partir da
vivência dos sujeitos. É o tipo vivido cujo sentido é obtido
num ato vivido. O tipo vivido concreto possibilita a compreensão
das formas significativas de vivência e a compreensão
significativa de uma subjetividade comum. Tais esquemas interpretativos diferem da
tipificação que é feita na vida cotidiana, porém devem
ser compatíveis não somente com os motivos e as
experiências subjetivas do mundo social, como também
com a experiência científica em seu conjunto. A HERMENÊUTICA E A ANTROPOLOGIA No esquema típico-ideal os contextos subjetivos
vivenciados diretamente são substituídos de forma
sucessiva por uma série de contextos objetivos de
significados, chegando-se a um modelo conceitual e
nunca a uma pessoa real. Tais modelos devem ser
compatíveis com os postulados fundamentais do método
científico e ao mesmo tempo responder às exigências do
mundo da vida. Desse modo, Schütz aponta os seguintes
postulados como requisitos para a construção desses
modelos: Postulado da Coerência Lógica, onde as
construções típicas são estabelecidas com um alto grau
de claridade e nitidez, sendo o esqueleto conceitual
totalmente compatível com os princípios da lógica formal. Este postulado garante a validade científica dos modelos
construídos. Postulado da Interpretação Subjetiva: para
explicar as ações humanas, o pesquisador deve perguntar-
se que modelo de mente individual é possível construir e
que conteúdos típicos são necessários atribuir a ele para
explicar fatos observados como resultado da atividade
da mente, numa relação compreensível. Assim, tem-se a
garantia da significação subjetiva. Segundo o Postulado
da Adequação, cada termo de um modelo científico de A Antropologia, enquanto “a reflexão do homem
sobre o homem e sua sociedade” torna-se uma ciência
com legitimidade entre outras disciplinas científicas, a
partir do século XIX. Nesta época, a antropologia,
chamada fundacional ou de gabinete, constituía-se em
narrativas e descrições das grandes viagens européias,
descobertas de outras sociedades e povos exóticos feitas
pelos observadores (viajantes ou missionários) e
analisadas pelos pesquisadores em seus gabinetes
(LAPLANTINE, 1994). No século XX a antropologia passa a ser
caracterizada pelo trabalho de campo com uma ruptura
com a antropologia dita especulativa dando início a
antropologia denominada moderna. Na antropologia moderna coexistem quatro
correntes antropológicas: a racionalista, a estrutural-
funcionalista, a culturalista e a interpretativa (CARDOSO
DE OLIVEIRA, 1988). A Antropologia Interpretativa ou
também pós-moderna, inspira-se na tradição filosófica
denominada hermenêutica, tendo em Geertz seu principal
representante. O paradigma hermenêutico... Rev.latino-am.enfermagem - v. 6 - n. 2 - p. 29-35 - abril 1998 33 (...) não é afastar-se dos dilemas existenciais da
vida em favor de algum domínio empírico de
formas não emocionalizadas; é mergulhar no meio
delas. A vocação essencial da antropologia
interpretativa não é responder às questões mais
profundas, mas colocar à nossa disposição as
respostas que os outros deram (...) e assim incluí-
las no registro de consultas sobre o que o homem
falou” (GEERTZ, 1989, p.40-41). A HERMENÊUTICA E A ANTROPOLOGIA “ao contrário, o que se verifica é uma verdadeira
dispersão de influências nessa antropologia que Embora todas as correntes dentro da antropologia
moderna se empenhem num mesmo esforço
interpretativo, Geertz se diferencia na medida em que,
na busca do significado simbólico, adota as premissas
demandadas pela hermenêutica. Enquanto no
estruturalismo, o símbolo é reduzido a um significado,
porque pressupõe uma determinada estrutura econômica,
de parentesco, entre outras, a antropologia interpretativa
de Geertz não aponta um significado e, sim, uma
complexidade deles, numa ampla relativização de
contextos. “Olhar as dimensões simbólicas da ação social A HERMENÊUTICA E A ANTROPOLOGIA A Antropologia Interpretativa, pautada no
paradigma hermenêutico, apresenta-se como uma crítica
às “antropologias tradicionais”, reconsiderando verdades
que acreditavam eternas (CARDOSO DE OLIVEIRA,
1988, CALDEIRA, 1988). Nesse novo estilo de se fazer antropologia, a
autoridade do investigador é colocada em questão, o saber
é negociado entre pesquisador e o nativo, num processo
de confrontação de horizontes. A “intersubjetividade, a
individualidade e a historicidade” passam a ser
exercitadas pelo pesquisador. Para GEERTZ (1989) uma etnografia é uma
“descrição densa” o que implica na busca da profundidade
e da contextualização dos significados. p
p
q
“... os horizontes não se excluem de um modo
absoluto, mas se interseccionam e muitas vezes se
fundem. E propiciam, por conseguinte, o exercício
pleno da intersubjetividade (...) nos domínios
privilegiados da investigação etnográfica. Investigação que realizava o pesquisador e o
pesquisado
enquanto
individualidade
explicitamente reconhecidas, uma vez que a
própria biografia deste último pode ser a do
primeiro. E ao apresentar a vida do Outro (...), o
faz em termos de historicidade, num tempo
histórico do qual ele próprio, pesquisador, não se
exclui”. (CARDOSO DE OLIVEIRA, 1988, p.100-
101). “... os horizontes não se excluem de um modo
absoluto, mas se interseccionam e muitas vezes se
fundem. E propiciam, por conseguinte, o exercício
pleno da intersubjetividade (...) nos domínios
privilegiados da investigação etnográfica. Investigação que realizava o pesquisador e o
pesquisado
enquanto
individualidade
explicitamente reconhecidas, uma vez que a
própria biografia deste último pode ser a do
primeiro. E ao apresentar a vida do Outro (...), o
faz em termos de historicidade, num tempo
histórico do qual ele próprio, pesquisador, não se
exclui”. (CARDOSO DE OLIVEIRA, 1988, p.100-
101). A descrição etnográfica na concepção de
GEERTZ (1989, p.31) é interpretativa. “... o que ela
interpreta é o fluxo social e a interpretação envolvida
consiste em tentar salvar o “dito” num tal discurso da
sua possibilidade de extinguir-se e fixá-lo em formas
pesquisáveis”. Não há uma univocidade de concepção
hermenêutica enquanto raiz filosófica da Antropologia
Interpretativa, p
“ao contrário, o que se verifica é uma verdadeira
dispersão de influências nessa antropologia que
se pretende nova. Nem a “hermenêutica
ontológica” de Heidegger e Gadamer, nem a
“hermenêutica metódica” de Betti ou de Hirsch,
nem a “hermenêutica fenomenológica” de Ricoeur
(e muito menos a “hermenêutica clássica” de
Schleiermacher e Dilthey) dominam aquilo que
prefiro chamar de “consciência hermenêutica” na
Antropologia “pós-moderna” (CARDOSO DE
OLIVEIRA, 1988, p.97). RMINOS CLAVES: investigación interpretativa, hermenéutica, fenomenología, antropología TÉRMINOS CLAVES: investigación interpretativa, hermenéutica, fenomenología, antropología TÉRMINOS CLAVES: investigación interpretativa, hermenéutica, fenomenología, antropología CONSIDERAÇÕES FINAIS HERMENEUTIC AND QUALITATIVE RESEARCH ON NURSING
The study review the literature about hermeneutics as a foundation for ethnographic and phenomenological research. KEY WORDS: interpretative research, hermeneutic, phenomenology, anthropology
LA HERMENÉUTICA Y LAS INVESTIGACIONES CUALITATIVAS EN ENFERMERÍA
El estudio presenta una revisión de la literatura sobre el paradigma hermenéutico como fundamentación de las
investigaciones etnográficas y fenomenológicas. A HERMENÉUTICA Y LAS INVESTIGACIONES CUALITATIVAS EN ENF El estudio presenta una revisión de la literatura sobre el paradigma hermenéutico como fundamentación de las
investigaciones etnográficas y fenomenológicas. HERMENEUTIC AND QUALITATIVE RESEARCH ON NURSING HERMENEUTIC AND QUALITATIVE RESEARCH ON NURSING The study review the literature about hermeneutics as a foundation for ethnographic and phenomenological research. KEY WORDS: interpretative research, hermeneutic, phenomenology, anthropology CONSIDERAÇÕES FINAIS Concordamos com
LOWENBERG (1993) quando afirma que a adoção de
abordagens interpretativas é um caminho promissor para
a enfermagem, em suas atividades de pesquisa. Da mesma
forma, enveredar por esse caminho possibilitará que a
enfermagem participe do diálogo atual entre as Ciências
Sociais e Humanas. É importante não definir os limites destas
abordagens de uma forma rígida, pois estamos numa fase
de “transição revolucionária”(LOWENBERG, 1993). Por outro lado, ao elaborar projetos de pesquisa
de natureza interpretativa sugerimos que se reserve um
espaço para mudanças, abrindo a possibilidade de
redefinir o caminho a percorrer, a partir da própria
experiência. Experiência que é única e imprevisível, visto
que se refere ao diálogo com o outro, à interação humana. HERMENEUTIC AND QUALITATIVE RESEARCH ON NURSING
The study review the literature about hermeneutics as a foundation for ethnographic and phenomenological research. KEY WORDS: interpretative research hermeneutic phenomenology anthropology Grande parte das pesquisas de enfermagem
enfoca questões do cotidiano, buscando significados
socialmente compartilhados. Concordamos com
LOWENBERG (1993) quando afirma que a adoção de
abordagens interpretativas é um caminho promissor para
a enfermagem, em suas atividades de pesquisa. Da mesma
forma, enveredar por esse caminho possibilitará que a
enfermagem participe do diálogo atual entre as Ciências
Sociais e Humanas. Acreditamos que por meio da hermenêutica
vislumbramos a possibilidade de compreender os
significados atribuídos pelos sujeitos à condição
existencial de estar no mundo e, por meio de sua
linguagem, em relatos, chegar a compreensão de sua ação. O homem revela a realidade por meio da
linguagem oral ou escrita e pela interpretação da
linguagem o pesquisador chega aos aspectos
significativos essenciais para a compreensão do ser. Portanto, a linguagem pode ser um elemento mediador
que permite essa compreensão/interpretação, tanto dos
processos cognitivos individuais, quanto dos aspectos
sociais e culturais. É importante não definir os limites destas
abordagens de uma forma rígida, pois estamos numa fase
de “transição revolucionária”(LOWENBERG, 1993). Por outro lado, ao elaborar projetos de pesquisa
de natureza interpretativa sugerimos que se reserve um
espaço para mudanças, abrindo a possibilidade de
redefinir o caminho a percorrer, a partir da própria
experiência. Experiência que é única e imprevisível, visto
que se refere ao diálogo com o outro, à interação humana. Adotar na enfermagem pesquisas de natureza
interpretativa representa, cada vez mais, a possibilidade
para que os enfermeiros analisem fenômenos no cotidiano
da saúde, a partir do ponto de vista de quem o vivencia. CONSIDERAÇÕES FINAIS Uma das tradições na Sociologia e Antropologia
se inspira nos fundamentos da Filosofia da compreensão
alemã, representada por Weber na Sociologia e em
diferentes concepções hermenêuticas, na Antropologia. O conceito de cultura de Geertz é essencialmente
semiótico, como explica: “Acreditando, como Max Weber, que o homem é
um animal amarrado a teias de significados que
ele mesmo teceu, assumo a cultura sendo essas
teias e a sua análise; portanto, não como uma
ciência experimental em busca de leis, mas como
uma ciência interpretativa, à procura do
significado”. (GEERTZ, 1989, p.15). A hermenêutica enquanto paradigma filosófico
tem fundamentado as pesquisas qualitativas da
enfermagem. Ao considerar as situações cotidianas do
enfermeiro no contexto da saúde, buscando o ponto de
vista dos sujeitos, sobre suas condições no processo
saúde-doença, esse profissional tem aproximado mais as
pesquisas da assistência de enfermagem, voltando-se à
compreensão/interpretação dos fenômenos pesquisados. Assim, a Antropologia chamada pós-moderna se
preocupa muito mais com os fundamentos do que com
as técnicas. Na ótica de Geertz não há uma receita para o
diálogo na busca da compreensão de significados,
comportamentos e ações do outro, o que para ele
pressupõe um controle, rigor ou preocupação com a
objetividade. A compreensão e interpretação do fenômeno que
interessa ao enfermeiro se dá “em sua natureza material
e significativa e não em sua natureza formal e estrutural
que
caberia
à
ciência
e
aos
cuidados
médicos”(CAPALBO, 1994, p.196). “Olhar as dimensões simbólicas da ação social “Olhar as dimensões simbólicas da ação social O paradigma hermenêutico... Rev.latino-am.enfermagem - v. 6 - n. 2 - p. 29-35 - abril 1998 34 Acreditamos que por meio da hermenêutica
vislumbramos a possibilidade de compreender os
significados atribuídos pelos sujeitos à condição
existencial de estar no mundo e, por meio de sua
linguagem, em relatos, chegar a compreensão de sua ação. O homem revela a realidade por meio da
linguagem oral ou escrita e pela interpretação da
linguagem o pesquisador chega aos aspectos
significativos essenciais para a compreensão do ser. Portanto, a linguagem pode ser um elemento mediador
que permite essa compreensão/interpretação, tanto dos
processos cognitivos individuais, quanto dos aspectos
sociais e culturais. Adotar na enfermagem pesquisas de natureza
interpretativa representa, cada vez mais, a possibilidade
para que os enfermeiros analisem fenômenos no cotidiano
da saúde, a partir do ponto de vista de quem o vivencia. Grande parte das pesquisas de enfermagem
enfoca questões do cotidiano, buscando significados
socialmente compartilhados. 17. SCHÜTZ, A. Fenomenologia del mundo social:
introdución a la sociologia compreensiva. Buenos
Aires: Paidós, 1972. 19. VIETTA, E.P. Configuração triádica, humanista-
existencial-personalista: uma abordagem
teórico-metodológica de aplicação nas
pesquisas de enfermagem psiquiátrica e saúde
mental. Rev. latino am. enfermagem, v. 3, n.1,
p.31-43, 1995. 17. SCHÜTZ, A. Fenomenologia del mundo social:
introdución a la sociologia compreensiva. Buenos
Aires: Paidós, 1972.
18. SIEBENEICHLER, F.B. Fenomenologia e
hermenêutica. In: CAPALBO, C. Fenomenologia
e hermenêutica. Rio de Janeiro: Âmbito
Cultural, 1983. Cap. 1, p.9-34. REFERÊNCIAS BIBLIOGRÁFICAS 08. DILTHEY, W. Essência da filosofia. Lisboa:
Presença, 1979. 01. BLEICHER, J. Hermenêutica contemporânea. Rio
de Janeiro: Edições 70, 1992. 09. FREUND, J. Sociologia de Max Weber. 4. ed. Rio
de Janeiro: Forense Universitária, 1987. 10. GEERTZ, C. A interpretação das culturas. Rio de
Janeiro: Guanabara, 1989. 02. CALDEIRA, T.P. do R. A presença do autor e a pós-
modernidade. Novos Estudos, n.21, p.133-57,
julho 1988. 11. JAPIASSU, H.; MARCONDES, D. Dicionário
básico de filosofia. 2. ed. Rio de Janeiro: Zahar,
1995. 03. CAPALBO, C. Considerações sobre o método
fenomenológico e a enfermagem. Rev. Enferm. UERJ, v.2, n.2, p.192-197, 1994. 12. HABERMAS, J. Dialética e hermenêutica. Porto
Alegre: L&PM, 1987. 04. CAPALBO, C. Metodologia das ciências sociais: a
fenomenologia de Alfred Schütz. Rio de Janeiro:
Antares, 1979. 13. LAPLANTINE, F. Aprender antropologia. 8. ed. São Paulo: Brasiliense,1994. 14. LOWENBERG, J. Interpretive research methodology:
broadening the dialogue. Adv. Nurs. Sci., v.16,
n.2, p.57-69, 1993. 05. CARDOSO DE OLIVEIRA, R. Sobre o pensamento
antropológico. Rio de Janeiro: Tempo Brasileiro,
1988. 15. RICOUER, P. O conflito das interpretações: ensaios
de hermenêutica. Rio de Janeiro: Imago, 1978. 06. CORETH, E. Questões fundamentais de
hermenêutica. São Paulo: EPU/EDUSP, 1973. 16. SCHÜTZ, A. El problema de la realidad social. Buenos Aires: Amorrortu, 1962. 07. DEMO, P. Metodologia científica em ciências
sociais. 3. ed. São Paulo: Artes, 1981. O paradigma hermenêutico... 35
Rev.latino-am.enfermagem - v. 6 - n. 2 - p. 29-35 - abril 1998 35 17. SCHÜTZ, A. Fenomenologia del mundo social:
introdución a la sociologia compreensiva. Buenos
Aires: Paidós, 1972. 19. VIETTA, E.P. Configuração triádica, humanista-
existencial-personalista: uma abordagem
teórico-metodológica de aplicação nas
pesquisas de enfermagem psiquiátrica e saúde
mental. Rev. latino am. enfermagem, v. 3, n.1,
p.31-43, 1995. 18. SIEBENEICHLER, F.B. Fenomenologia e
hermenêutica. In: CAPALBO, C. Fenomenologia
e hermenêutica. Rio de Janeiro: Âmbito
Cultural, 1983. Cap. 1, p.9-34.
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Introdução à Mecânica Quântica: uma proposta de minicurso para o ensino de conceitos e postulados fundamentais
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Revista brasileira de ensino de ciência e tecnologia
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Resumo Analisa-se a viabilidade de uma proposta pedagógica de curta duração
para o ensino de Mecânica Quântica (MQ) em nível introdutório. Foi elaborado
um minicurso visando criar situações propícias para o ensino dos conceitos
fundamentos contidos nos primeiros princípios da MQ. O minicurso teve três
apresentações, a primeira ocorrendo no II Encontro Estadual de Ensino de Física,
realizado na Universidade Federal do Rio Grande do Sul, em Porto Alegre, RS, e as
outras duas na VII Semana da Física, na Universidade do Estado de Santa Catarina,
em Joinville, SC, Brasil. Palavras-chave: Mecânica Quântica, proposta pedagógica, conceitos
fundamentais. Introdução à Mecânica Quântica: uma
proposta de minicurso para
o ensino de conceitos e postulados
fundamentais
Introduction to Quantum Mechanics: a
proposal for a short course for
the teaching of fundamental concepts and
postulates Carlos Raphael Rocha Victoria Elniecave Herscovitz Marco Antonio Moreira Abstract We analyze the viability of a short-term pedagogical proposal about
Quantum Mechanics teaching at an introductory level. The course was intended to
create situations to enhance the acquisition and retention of the fundamental R. B. E. C. T., vol 3, núm 1, jan./abr. 2010
ISSN R. B. E. C. T., vol 3, núm 1, jan./abr. 2010
ISSN - 1982-873X
1 ISSN - 1982-873X R. B. E. C. T., vol 3, núm 1, jan./abr. 2010 1 concepts present in the first postulates of Quantum Mechanics. The short-term
course had three presentations, the first one in the II State Meeting on Physics
Teaching, at the Federal University of Rio Grande do Sul, at UFRGS, Porto Alegre,
RS and the other two during a physics meeting at the State University of Santa
Catarina, UDESC, Joinville, SC, Brazil. Keywords: Quantum Mechanics, pedagogical proposal, fundamental
concepts. Introdução O desenvolvimento da Mecânica Quântica (MQ) no século passado ocasionou uma
importante alteração no modo científico de pensar. Esta alteração produziu grandes implicações
tecnológicas e educacionais, tanto que não se pode hoje deixar de incluir o ensino de MQ em
cursos de graduação relacionados à Física. Observa-se, porém, que em vários cursos o ensino
desta disciplina trabalha muito com a resolução de problemas envolvendo equações diferenciais e
pouco com aspectos relacionados aos conceitos fundamentais e postulados da MQ. Adicionalmente, há uma necessidade inerente à reforma curricular do Ensino Médio, de
apresentar-se, nas disciplinas de Física entre outras, teorias científicas desenvolvidas mais
recentemente. Pode-se dizer que, na maioria dos cursos de nível médio, a Física que se ensina nas
escolas está defasada de cerca de dois séculos. Para que se saliente o desenvolvimento contínuo
da ciência, faz-se necessário atualizar constantemente o currículo educacional e para que esta
atualização chegue às salas de aula do Ensino Médio é primordial que haja um adequado preparo
dos professores, propiciando que estes estejam seguros de seu conhecimento. Cursos complementares para professores e alunos de licenciatura ou de outras áreas, de
duração menor do que a duração usual curricular, em que sejam abordados aspectos de MQ
numa linguagem menos matemática, mas fiel à Teoria são, então, alternativas recomendáveis. Esta linha tem sido adotada em Projeto em execução por alguns professores e estudantes de Pós-
Graduação em Ensino de Física do IFUFRGS. Uma destas propostas, desenvolvida por Greca et al. (2001), apresenta um módulo instrucional com duração aproximada de 24 horas-aula (12 sessões)
que foi oferecido a estudantes de Física Geral de cursos de Engenharia, como forma de levá-los a
compreender as bases de várias implicações tecnológicas recentes e de impacto, da teoria. Isto
porque a MQ interessa não somente aos físicos, mas está, também, na base de desenvolvimentos
de várias áreas, como as de engenharia, informação, comunicação e saúde, entre muitas outras
que utilizam tecnologia em seu cerne. Tal abordagem muito pouco recorreu a desenvolvimentos
matemáticos, preocupando-se mais com aspectos de compreensão de determinados fenômenos R. B. E. C. T., vol 3, núm 1, jan./abr. 2010 2 não explicados pela Física Clássica, mostrando-se positiva tanto nos aspectos motivadores, como
nos de compreensão dos fenômenos enfocados. Introdução Outra proposição, a atual, é a de que uma abordagem da MQ que enfoque os primeiros
conceitos e postulados da Teoria pode ser uma via de acesso a esta área da Física Moderna, para
o Ensino Médio. Assim, o que se busca neste trabalho é analisar a viabilidade de um projeto
pedagógico de curta duração para o ensino de MQ, focado nos primeiríssimos princípios desta
teoria e oferecido a professores de Física em exercício no Ensino Médio ou em formação. Outrossim, deseja-se verificar quais dos tópicos apresentados são de interesse do público-alvo e
se tais tópicos podem ser adequadamente trabalhados junto a alunos de Ensino Médio. Os
princípios fundamentais da MQ abordados foram selecionados visando preencher uma lacuna
que perdura, às vezes, mesmo após a conclusão pelos licenciados, do curso de graduação. Isto
porque, como já citado, em vários cursos de graduação são mais salientados certos aspectos
matemáticos desta teoria (em que os alunos encontram dificuldades), faltando tempo para uma
discussão mais aprofundada da parte conceitual, importante para a compreensão de qualquer
princípio em toda teoria e especialmente nesta, pelo ineditismo não intuitivo. Procurou-se, além
disto, levantar informação sobre outros tópicos que estimulassem o interesse pelo conhecimento
e ensino de MQ. Em suma, o principal intuito do minicurso foi o de promover uma complementação ao
estudo da MQ, sob enfoque pouco explorado em cursos de licenciatura e, ao mesmo tempo,
analisar a viabilidade e o interesse de uma proposta pedagógica menos tradicional para o ensino
dos princípios básicos da MQ, sem a necessidade de conhecimentos prévios sobre o assunto, e
que pudesse ser um veículo para a promoção da atualização curricular. Para analisar tais pontos o mini-curso foi apresentado três vezes, em duas situações
distintas, conforme exposto adiante. R. B. E. C. T., vol 3, núm 1, jan./abr. 2010
ISSN - 1982-873X Fundamentação teórica As manifestações dos participantes no decorrer dos mini-cursos foram analisadas
utilizando-se elementos da Teoria da Aprendizagem Significativa de David Ausubel e da Teoria dos
Campos Conceituais de Gerárd Vergnaud. Segundo Ausubel (1968), se fosse possível reduzir a psicologia educacional a um princípio,
este seria: “o fator mais importante a contribuir para o aprendizado é aquilo que o aprendiz já
sabe”. Ao educador cabe, então, averiguar o que o aprendiz sabe e ensinar de acordo. Esse
conhecimento prévio normalmente funciona como ancoradouro cognitivo para a aquisição
significativa de novos conhecimentos, mas pode também atuar como obstáculo epistemológico. R. B. E. C. T., vol 3, núm 1, jan./abr. 2010
ISSN - 1982-873X R. B. E. C. T., vol 3, núm 1, jan./abr. 2010
ISSN - 1982-873X
3 R. B. E. C. T., vol 3, núm 1, jan./abr. 2010 ISSN - 1982-873X 3 Ao iniciar o estudo da MQ, o aprendiz já possui um conhecimento intuitivo oriundo da Mecânica
Clássica (seja pelo aprendizado formal ou pela vivência no cotidiano). Como a MQ, contudo, não é
um caso particular da Mecânica Clássica, deve-se buscar abordá-la de forma que não se apóie em
fundamentos clássicos. No entanto, a utilização de analogias entre a MQ e a Mecânica Clássica
pode ser útil para a compreensão da estrutura da MQ sem que com isto se pretenda explicar os
fenômenos quânticos com base em princípios clássicos. Como todo processo de aprendizagem, a aprendizagem significativa também é uma
negociação (e aquisição) de significados, existindo algumas condições iniciais para que possa
ocorrer, a saber (op. cit., p.1): •
a estrutura cognitiva do aprendiz deve conter idéias relevantes com as quais o
novo material deve se relacionar; •
a relação com esse conhecimento prévio deve ser não-arbitrária e não-literal. Outra condição para a ocorrência da aprendizagem significativa é que o aprendiz deve
apresentar uma pré-disposição para aprender. Porém, não se deve confundir pré-disposição com
motivação, no sentido de gostar, de entusiasmar-se. O aprendiz pode não estar motivado para
aprender, mas predisposto. Vale salientar que de nada adianta o material ser potencialmente
significativo se o aluno não estiver disposto a relacioná-lo interativamente com seus subsunçores,
ou seja, com conhecimentos prévios que servem de “ancoradouro” cognitivo para o novo
conhecimento. É importante observar que, como o processo é interativo, além de dar significado
ao novo conhecimento, o subsunçor também se modifica, adquirindo novos significados, ficando
mais estável, mais elaborado, diferenciado. Fundamentação teórica Outro conceito muito importante introduzido por Ausubel é o de organizador prévio
(2000, p. 11). O organizador prévio é um recurso pedagógico que pretende suprir eventuais
deficiências de subsunçores necessários para que ocorra aprendizagem significativa. Ele serve de
ponte entre aquilo que o aluno já sabe e aquilo que deveria saber para que pudesse aprender de
forma significativa e deve ser apresentado em um nível de abstração, generalidade e inclusividade
mais alto que o do novo material a ser aprendido (ibid.).No caso em foco, o aprendiz vem à aula
com conhecimento criado a partir de vivências explicadas pela Mecânica Clássica, as quais podem
servir para construir organizadores prévios, mas que, na nossa perspectiva, devem servir como
subsunçores provisórios que visem facilitar o aprendizado da MQ sem a necessidade de se apoiar
em “pilares clássicos”. Apesar de ter sido aluno de Piaget, Vergnaud foca seu estudo no sujeito em situação. São
as situações que darão sentido aos conceitos (Moreira, 2002, p. 10). Um conceito, porém, não é
uma mera definição. Ele se refere a um conjunto de situações, envolve um conjunto de diferentes
invariantes operatórios e suas propriedades podem ser expressas por diferentes representações 4 R. B. E. C. T., vol 3, núm 1, jan./abr. 2010 simbólicas e lingüísticas (Vergnaud, 1999, p. 177). Além das situações, os conceitos também são
constituídos por invariantes operatórios e representações. Os invariantes operatórios definem a
operacionalidade e o significado do conceito (Moreira, 2002, p. 10). São conhecimentos-em-ação:
teoremas-em-ação (regras que o sujeito crê que são válidas sobre a realidade) e conceitos-em-
ação (propriedades, atributos, pertinentes à situação). As representações são utilizadas para
indicar simbolicamente os invariantes e, assim, representar as situações e os procedimentos para
lidar com elas (ibid.). Outro aspecto da teoria de Vergnaud é concernente ao fato de que o aprendiz leva, em
geral, algum tempo para dominar determinado campo conceitual. Com isto, considerando a MQ
como um campo conceitual, pode-se concluir que em um primeiro contacto com o assunto o
estudante dará seus primeiros passos nesse campo, ou seja, começará a dar significados aos
principais conceitos e postulados da MQ. Esses primeiros passos são extremamente importantes
para uma aprendizagem significativa. Fundamentação teórica Ademais, mesmo que o aprendiz já tenha tido a
oportunidade de estudar alguns aspectos da MQ, um curso de curta duração sobre os princípios
básicos da MQ propiciará vincular seus conhecimentos anteriores aos postulados, permitindo
elevar seu nível de compreensão sobre esta teoria. Em paralelo, um curso de curta duração com
tal enfoque conduzirá a uma visão mais correta do que é a MQ, quando seguido de cursos mais
longos. A proposta A proposta inicial do projeto foi a de preparar um minicurso destinado a licenciados e
licenciandos de Física, para avaliar a viabilidade de uma modalidade de ensino introdutório ou
complementar de MQ, com foco em conceitos fundamentais e primeiros postulados da Teoria,
acompanhados de algumas de suas marcantes implicações. A expectativa é de que isto possa
servir de estímulo e apoio para que os professores passem a incluir tópicos desta natureza em
suas aulas no Ensino Médio. O mini-curso foi apresentado três vezes, uma delas na UFRGS e duas outras na UDESC,
conforme detalhado mais adiante. A ementa é apresentada a seguir. Fundamentos da Mecânica Quântica
o Introdução
o Experimentos de dupla fenda
o Superposição de ondas Fundamentos da Mecânica Quântica
o Introdução
o Experimentos de dupla fenda
o Superposição de ondas o Superposição de ondas 5 o Superposição de vetores do plano
o Estados quânticos e observáveis
o Espaços lineares
Primeiros Postulados da Mecânica Quântica
o Superposição de estados
o Medições e observáveis
o Estados de um sistema quântico e observáveis
o Evolução temporal de um sistema quântico
Implicações dos Primeiros Postulados
o Emaranhamento quântico
o Relógios atômicos
o Criptografia quântica o Superposição de vetores do plano
o Estados quânticos e observáveis
o Espaços lineares
Primeiros Postulados da Mecânica Quântica
o Superposição de estados
o Medições e observáveis
o Estados de um sistema quântico e observáveis
o Evolução temporal de um sistema quântico
Implicações dos Primeiros Postulados
o Emaranhamento quântico
o Relógios atômicos
o Criptografia quântica Por se tratar de um curso de curta duração, optou-se por trabalhar com apenas alguns
dos conceitos da MQ e com implicações referentes somente aos primeiros postulados. Não houve
pretensão de avaliar formalmente a aprendizagem dos participantes no transcorrer do curso, mas
seu interesse pelo conteúdo e seu envolvimento com o mesmo foram observados. A rigor, o
objeto de avaliação foi o curso em si, no entanto, em todo o decorrer das atividades, os alunos
foram instados a apresentar questionamentos e a responder perguntas, para promover a
participação dos mesmos no assunto, tornando-os agentes ativos no processo de aprendizagem. Já na Introdução, buscou-se apresentar alguma situação que pudesse motivar os alunos
para o aprendizado da MQ. Para tanto, apresentou-se um exemplo do fenômeno de
emaranhamento quântico, através do trabalho de Ursin et al. A proposta (2007) em que os autores
conseguiram obter um par de fótons emaranhados (em termos de auto-estados de polarização)
distanciados de 144 km. No curso ministrado na UFRGS foram trabalhados na primeira aula os tópicos
relacionados aos Fundamentos da MQ ( Introdução, Experimentos de Dupla Fenda, Superposição
de Ondas e de Vetores do Plano, Estados Quânticos e Observáveis e Espaços Lineares). Neste
encontro buscou-se formar alguns conceitos que pudessem servir de âncora para a discussão e
aprendizado subseqüente de alguns dos postulados da MQ. Foram apresentados vários conceitos
característicos de espaços lineares, a saber, os de vetor, superposição linear de vetores, operador,
auto-vetores, autovalores e autofunções, para servirem como organizadores prévios. Outros 6 R. B. E. C. T., vol 3, núm 1, jan./abr. 2010 itens, como o do Experimento de Dupla Fenda, funcionariam já como subsunçores para a
aceitação dos primeiros postulados. Na segunda aula foram apresentados alguns dos primeiros
postulados da MQ, na descrição de Schrödinger, a saber, o referente ao estado quântico como
depositário das informações sobre as grandezas físicas de um sistema quântico em um dado
instante de tempo, o que denota a correspondência entre grandezas físicas e operadores lineares
auto-adjuntos, o relativo ao colapso do vetor de estado no ato de medida de um observável físico
e o da evolução temporal do estado de um sistema quântico, regida pela equação de Schrödinger. Tais postulados foram trabalhados, em sua maior parte, de forma conceitual e com exemplos,
visando preencher lacunas existentes ou remanescentes, mesmo após a conclusão do ensino
formal pelos participantes. Na última aula, foram apresentadas algumas aplicações importantes e
atuais da MQ, implicações formalmente simples, embora surpreendentes, dos primeiros
postulados trabalhados, como o emaranhamento quântico e a criptografia quântica, para tentar
promover a contextualização, através da utilização dos conceitos abordados. Nos cursos apresentados na UDESC, os dois primeiros encontros supracitados foram
condensados em uma única apresentação e a segunda aula correspondeu à terceira anterior. Além disto, foram propostos exercícios aos alunos, para fixação do conteúdo abordado,
exercícios estes resolvidos e discutidos na própria aula. Procurou-se também instigar os alunos a
participar criticamente e a se interessar pelo assunto com questões do tipo “Pense !”, fazendo
com que conseguissem perceber algumas “estranhezas” do mundo quântico. A implementação do minicurso Após a elaboração da proposta do curso e do material didático a ser entregue aos
participantes, o minicurso foi ministrado três vezes, em duas ocasiões distintas, em setembro de
2007. O primeiro deles foi apresentado no II Encontro Estadual de Ensino de Física (II EEEFis), no
Instituto de Física da UFRGS, em Porto Alegre, RS. Tal mini-curso teve seis horas-aula divididas em
três encontros de igual duração, nos dias 13, 14 e 15 de setembro. Neste curso, o público-alvo
esteve constituído de professores de Física (profissionais em exercício, a maioria no Ensino
Médio) e de licenciandos de Física, quase todos do Rio Grande do Sul. Estavam inscritos no mini-curso, antecipadamente, 15 participantes, mas foram aceitas no
dia do início do Encontro outras sete inscrições, totalizando a presença de 22 alunos. A estrutura
disponibilizada para ministrar o curso constou de um laboratório de informática equipado com 20
computadores (capazes de rodar as simulações utilizadas nas aulas) e um projetor multimídia,
acoplado a um dos computadores. R. B. E. C. T., vol 3, núm 1, jan./abr. 2010
ISSN - 1982-873X R. B. E. C. T., vol 3, núm 1, jan./abr. 2010 ISSN - 1982-873X 7 Os outros dois mini-cursos foram apresentados na VII Semana da Física, na Universidade
do Estado de Santa Catarina, em Joinville, SC. Estes cursos tiveram quatro horas-aula de duração
cada, divididas em dois encontros, e ocorreram nos dias 19 e 20 de setembro. A comissão
organizadora do evento ofertou 20 vagas para cada turma, mas a procura pelo curso (sobretudo
para a primeira turma) foi tamanha, que resultou em uma fila de espera com mais de 40 pessoas. Para a primeira turma, foram selecionados 23 alunos e para a segunda, 16. Por ser um evento
acadêmico (promovido pela Universidade), o público-alvo era formado por estudantes do curso
de Licenciatura em Física da UDESC, público este que se revelou heterogêneo, incluindo tanto
alunos dos primeiros semestres do curso, como dos últimos. Esta diversidade possibilita analisar-
se a importância do conhecimento de conteúdos prévios, para a proposta. Estes dois cursos foram, ambos, ministrados em dois ambientes distintos. No primeiro dia,
utilizou-se um laboratório de informática com 15 computadores, enquanto no segundo dia do
curso o encontro ocorreu em uma sala de seminários equipada com um computador portátil e um
projetor multimídia. Em nenhum dos mini-cursos, houve solicitação de pré-requisitos (além de serem
oferecidos no âmbito universitário). A implementação do minicurso Considerou-se que, em princípio, se é possível ensinar MQ a
alunos sem que eles tenham tido um contato inicial com a mesma, é viável também, ter alunos
universitários com níveis de formação distintos, compartilhando o mesmo curso. Como dito anteriormente, não houve avaliação formal da aprendizagem dos alunos
participantes, mas, com o fito de analisar a adequação e a viabilidade do curso, foi programado e
realizado um registro das ocorrências em sala de aula, no decorrer das apresentações (diário de
bordo) com o intuito de colher dados para uma análise posterior. Resultados Não havendo a intenção de avaliar a aprendizagem, o relato sobre as atividades
desenvolvidas e os questionamentos formulados pelos alunos no decorrer dos três mini-cursos
são apresentados de forma conjunta, considerando ser a proposta, única, e não especialmente
adequada para análises isoladas. Material didático produzido especialmente para as aulas foi distribuído aos alunos no
início de cada encontro e um projetor multimídia foi utilizado durante toda a duração do curso,
para apresentação do material, de modo a tornar mais atraente a atividade desenvolvida. Visando motivar os alunos e criar uma condição de pré-disposição para o aprendizado, as
aulas dos minicursos foram iniciadas com informação sobre a verificação experimental de estados
emaranhados (fortemente correlacionados) separados espacialmente por 144 km (Ursin et al., R. B. E. C. T., vol 3, núm 1, jan./abr. 2010 8 2007). Apesar de se tratar de evento recente e fortemente não-intuitivo, a menção não causou
impacto sobre os alunos e nem os motivou a ponto de demonstrarem interesse no tópico. Como
se considera que o emaranhamento quântico é uma das principais e mais marcantes aplicações
atuais dos primeiros princípios da MQ, concluiu-se pela modificação e aprimoramento da
apresentação, em intervenções posteriores, para que se consiga o efeito esperado. Em princípio,
a apresentação do emaranhamento quântico no estudo de conceitos fundamentais de MQ, dadas
as suas surpreendentes e não clássicas conseqüências, é um ótimo exemplo, pois decorre de
modo simples de conceitos fundamentais, como o de estado de sistema quântico e de
superposição linear de estados de sistemas quânticos. Quanto aos experimentos de dupla fenda com ondas, com partículas clássicas e
com partículas quânticas, além da descrição e explicação dos mesmos, foi usada uma
simulação em computador1 para enriquecer o aprendizado. Percebeu-se que, na
discussão sobre o caso quântico, os participantes dos cursos não utilizavam o conceito de
estado e a propriedade de superposição linear para a explicação dos eventos
apresentados, mas sim, em geral, a dualidade onda-partícula (não enfatizada nos
minicursos). Foi possível notar, também, que os conceitos quânticos mais básicos não
pareciam bem arraigados nos participantes (embora muitos destes fossem professores ou
alunos de final de curso de Licenciatura), pois na discussão dos experimentos, os alunos
cometeram vários erros, demonstrando insegurança e falta de conhecimento (e
preparo). Isto mostra que os subsunçores presentes na estrutura cognitiva dos alunos não
eram adequados para ancorar uma explicação correta dos fenômenos quânticos. 1 Simulação disponível em
http://phet.colorado.edu/simulations/sims.php?sim=Quantum_Wave_Interference. http://phet.colorado.edu/simulations/sims.php?sim=Quantum_Wave_Interference. Resultados Depois
de analisar o experimento com partículas quânticas em termos de “possibilidades”, foi
alentador verificar que os alunos conseguiam explicar melhor a existência de um padrão
de interferência, mesmo quando o experimento era realizado com um único elétron
sendo detectado a cada vez. Para a superposição de ondas, os alunos receberam a tarefa de compor combinações
que possibilitavam verificar esta propriedade. Foram apresentados, também, exemplos de
superposições de ondas comuns no campo da Eletrônica, como as ondas triangulares. Verificou-se
que os participantes apresentavam dificuldades na construção de gráficos em que era necessário
somar duas (ou mais) ondas. Apesar disto, os alunos aceitaram facilmente que a superposição de
duas ou mais ondas gera uma nova onda e que se multiplicarmos a função que representa uma
onda por um escalar também será gerada uma onda. Tal compreensão é bastante útil para se R. B. E. C. T., vol 3, núm 1, jan./abr. 2010
ISSN - 1982-873X ISSN - 1982-873X R. B. E. C. T., vol 3, núm 1, jan./abr. 2010 9 possa fazer a transposição para a superposição linear de estados quânticos (organizador prévio). Nesta instância dos mini-cursos, os alunos utilizaram uma simulação em computador que
possibilita alterar o comprimento de onda e a amplitude de duas ondas e observar a onda
resultante na soma³1. Também uma questão do tipo ”Pense!” foi proposta aos alunos, a saber,
“Qual a ligação entre a superposição de ondas e o resultado do experimento de dupla fenda com
partículas quânticas?”, com respostas bastante adequadas. A superposição de vetores geométricos não gerou dúvidas. Possivelmente devido à
familiaridade com a utilização freqüente de vetores em Matemática e a aplicação de vetores em
Mecânica Clássica, os alunos acolheram prontamente a idéia de que a soma (usual) de dois
vetores e a multiplicação de vetor por escalar resultam em outros vetores do mesmo espaço. Mostrou-se, então, bastante simples, promover a generalização e a abstração das
propriedades de ondas (funções) e de vetores geométricos, para espaços lineares. Revelando-se
devidamente arraigado o conceito de superposição linear (de ondas e de vetores geométricos),
buscou-se fazer a ligação com o conceito de superposição linear de estados de um sistema
quântico. 1 Simulação disponível em http://www.colorado.edu/physics/2000/schroedinger/index.html. Resultados Após utilizar a superposição de ondas e a de vetores geométricos como organizadores
prévios do conhecimento sobre espaços lineares e de proceder-se à análise daquelas
superposições sob enfoque mais amplo de vetores de um espaço linear, foram introduzidos os
conceitos de estado quântico e de superposição linear de estados quânticos, salientando-se que,
tal como ondas e vetores geométricos, os estados quânticos também podem ser superpostos,
gerando outros estados quânticos, para o que foi importante o resultado da experiência de dupla
fenda para elétrons. Fez-se, então, detida menção sobre o fato de que a caracterização do estado
de um sistema quântico como vetor de um espaço linear conduz a conseqüências não explicáveis
pela Mecânica Newtoniana (e explicáveis pela nova Teoria). Salientou-se que a propriedade de
superposição linear dá conotação não clássica aos estados dos sistemas quânticos. Questionamentos sobre a caracterização do estado de um sistema clássico e do de um
sistema quântico, revelaram que a grande dificuldade dos alunos residia em não saber como
obter informações a respeito. Contudo, ao induzir os alunos a pensar em medidas de
propriedades físicas como modo de obter informação sobre o estado de objetos (sistemas)
clássicos, foram transpostas várias das dificuldades reveladas pouco antes, inclusive para a
caracterização de estados de sistemas quânticos. O uso deste específico conhecimento sobre um
sistema clássico serviu como subsunçor para a MQ. Não foi, contudo, proposto (ou incentivado) 1 Simulação disponível em http://www.colorado.edu/physics/2000/schroedinger/index.html. R. B. E. C. T., vol 3, núm 1, jan./abr. 2010 10 em qualquer momento que os alunos interpretassem os fenômenos quânticos com conceitos
clássicos. Vencido este ponto, foi introduzida, apenas como um simbolismo, sem maior rigor, a
notação de Dirac para (vetores de) estados de sistemas quânticos. Foi ponto comum em todos os
mini-cursos o grande impacto causado por esta notação. Como muitos dos alunos nunca haviam
tido contato com ela, afirmavam inicialmente que era difícil entender e utilizar a simbologia e que
não conseguiam compreender o seu significado. Porém, no transcorrer do curso, com a
introdução e aplicação dos conceitos de estado e de observável, os alunos passaram a se habituar
com a notação de Dirac e a considerá-la útil e, ao final, até já a utilizavam em suas perguntas. Percebeu-se que tal notação auxiliou na generalização de muitas das idéias que envolvem vetores
de espaços lineares e estados de objetos quânticos. Recorrendo novamente a uma simulação em computador do experimento de dupla fenda
para elétrons, os alunos perceberam que a medida (da posição da partícula) é uma operação que
pode alterar o padrão de interferência e, assim sendo, modificar o estado do objeto quântico em
análise. Ficou perceptível, aqui, que a utilização das simulações em computador facilitou a
compreensão dos conceitos quânticos trabalhados. A seguir foi analisado o postulado relativo ao “colapso do vetor de estado no ato da
medida”, importante quesito dentre os primeiros princípios da MQ. Na discussão deste postulado,
face aos questionamentos apresentados, foi possível perceber que os alunos compreenderam que
esta premissa permite explicar conseqüências da medição observadas (experimentalmente), um
dos principais objetivos do curso. Este foi um dos tópicos em que os participantes mais
demonstraram interesse, possivelmente por estar constantemente presente em artigos, livros e
filmes de divulgação científica ou fantasiosos, recentemente produzidos. Com o tempo da segunda aula do minicurso apresentado na UFRGS praticamente
esgotado, foi dada apenas uma rápida explanação sobre o postulado da evolução temporal dos
estados de sistemas quânticos (Equação de Schrödinger), não tendo sido possível elaborar uma
discussão a respeito, para bem compreendê-lo. Muitos alunos, mesmo considerando a existência
de uma “lei” para a evolução temporal dos sistemas, afirmavam não ser possível determinar o
estado de um sistema quântico em um instante posterior a um dado, face ao Princípio de
Incerteza. 1 Simulação disponível em http://www.colorado.edu/physics/2000/schroedinger/index.html. Apesar de sua importância na MQ, o Princípio da Incerteza (que pode ser deduzido a
partir dos primeiros postulados e, portanto, não ser considerado, de fato, um princípio), talvez
devido a freqüentes citações e “usos” por leigos pode, em certos casos, tornar-se um obstáculo
pedagógico para o aprendizado dos primeiros princípios da MQ, por ser mal compreendido. Na segunda aula do mini-curso apresentado no II EEEFis, um aluno comentou que achava
muito interessante a abordagem utilizada. Segundo ele, praticamente todos os cursos de R. B. E. C. T., vol 3, núm 1, jan./abr. 2010 ISSN - 1982-873X 11 Mecânica Quântica que havia feito se caracterizaram por excessiva ênfase na resolução de
problemas de autovalores para a equação de Schrödinger independente do tempo. Esta, aliás, é
uma das reclamações que os alunos de disciplinas de MQ de cursos de graduação, costumam
apresentar. Em geral, a abordagem dos mini-cursos, enfocando conceitos iniciais da MQ, foi bem
recebida por outros alunos também 41. Quanto ao fenômeno do emaranhamento quântico, abordado após os conceitos
principais, houve grande interesse pelo assunto e, sobretudo, em saber como eram produzidos os
pares emaranhados. Também consideraram os alunos que as críticas ao emaranhamento
quântico, feitas por Einstein (e Podolski e Rosen), não tinham sido totalmente rebatidas, ou seja,
os alunos não “aceitaram” as soluções apresentadas ao paradoxo EPR, evidenciando certa
resistência à aceitação da idéia de não-localidade na MQ e, conseqüentemente, da plenitude da
validade do princípio da superposição linear de estados. Contudo, como regra geral, os alunos
manifestaram ser extremamente interessante estudar propriedades e fenômenos que não estão
explicitados nos livros com os quais estão acostumados a trabalhar na graduação. O tema relativo a relógios atômicos, em que se utilizou o emaranhamento quântico para
melhorar a precisão do relógio, não instigou os alunos para a compreensão do fenômeno. Aliás,
como não surtiu o efeito desejado na motivação dos alunos, esta aplicação mais atrapalhou, pelo
tempo que ocupou, do que auxiliou no processo de aprendizagem. Como é recomendável
apresentar o maior número possível de situações-problema para promover a conceitualização,
este exemplo será eliminado, mas outros serão inseridos no material de apoio dos cursos. Além
disto, algumas das situações ali constantes serão mais salientadas, para que se consiga melhor
promover a aprendizagem significativa dos conceitos fundamentais da MQ. Quanto à criptografia quântica, a opção adotada de detalhar seus procedimentos
conduziu a algumas dificuldades. 1 Comentários elogiosos sobre a proposta do curso foram apresentados por alguns dos
participantes, ao final dos dois primeiros encontros do minicurso oferecido na UFRGS 1 Simulação disponível em http://www.colorado.edu/physics/2000/schroedinger/index.html. O primeiro problema apresentado pelos alunos na compreensão
do tópico foi a dificuldade de entender o conceito de monitoramento passivo. Apesar de ser um
conceito simples e tido como intuitivo, sempre que o conceito vinha à tona era necessária uma
nova, embora breve, explicação. Escolheu-se como protocolo de transmissão de chaves criptográficas a ser apresentado
aos alunos o protocolo BB84, que utiliza fótons polarizados para a criação da chave. A escolha 1 Comentários elogiosos sobre a proposta do curso foram apresentados por alguns dos
participantes, ao final dos dois primeiros encontros do minicurso oferecido na UFRGS R. B. E. C. T., vol 3, núm 1, jan./abr. 2010 12 priorizou a simplicidade do processo, embasado principalmente na superposição linear de estados
de polarização da luz (horizontal e vertical). No início os alunos aparentaram estar assustados com os passos utilizados no decorrer do
processo de transmissão da chave criptográfica e, principalmente, com a quantidade de
informações tornadas públicas sem que isso afetasse a segurança do processo. Este exemplo,
entretanto, gerou um interesse muito grande, compensando assim as dificuldades apresentadas. De fato, no mini-curso oferecido durante o II EEEFis, a última aula ocorreu em um sábado
à tarde, dia e horário em que, muitas vezes, por ser a última atividade oferecida, alguns inscritos
faltam, inclusive para retornar às cidades em que residem. Contudo, o interesse pelas atividades
do mini-curso foi tão grande que apenas um dos inscritos, que desde o início informou ter um
compromisso inadiável neste dia, não compareceu. Além disto, ao final do curso, muitos alunos
continuaram na sala de aula fazendo perguntas a respeito dos diversos tópicos apresentados. Também ao final dos minicursos ministrados na VII SEFIS, alguns alunos passaram a repetir o
processo de criptografia no quadro, enquanto outros discutiam o paradoxo EPR. Isto mostra que
tais tópicos efetivamente motivaram os participantes para o aprendizado de MQ. Recomenda-se,
então, que tais exemplos, constituindo-se em situações-problema muito atrativas, sejam
utilizados para auxiliar na aprendizagem significativa dos conceitos neles presentes. No encerramento dos minicursos, muitos alunos se mostraram agradecidos pela nova
abordagem e comentaram julgar possível começar a trabalhar a MQ no Ensino Médio desta
forma. Conclusão A receptividade dos tópicos abordados nos mini-cursos sugere que a ausência de
discussões relativas aos conceitos fundamentais da MQ em cursos de graduação é sentida pelos
alunos, dado o entusiasmo com que alguns pontos foram trabalhados. Em particular, os conceitos
de estado e de superposição linear de estados de um sistema quântico não foram tidos como
difíceis pelos alunos, face às situações que foram apresentadas. Isto nos estimula a buscar mais
formas de trabalhar tais conceitos (e os demais princípios básicos) visando a conceitualização e,
assim, fazer com que os professores tenham segurança ao abordar tópicos de Física Moderna com
seus alunos. Também se pode afirmar que os cursos apresentados trouxeram à tona exemplos de
situações-problema úteis para a compreensão dos fenômenos quânticos. Mesmo que algumas
das situações-problema utilizadas não tenham sido totalmente proveitosas, talvez por exigüidade
de tempo, não se faz necessário descartar qualquer delas (exceto o enfoque do tema relativo aos R. B. E. C. T., vol 3, núm 1, jan./abr. 2010 ISSN - 1982-873X 13 relógios atômicos que, pelo motivo antes mencionado, será substituído por outro). Na verdade, o
ideal seria a inclusão de ainda mais situações-problema, o que pode ser feito em cursos de não
tão curta duração. Recordemos que, segundo Vergnaud, o grande número e diversidade de
situações-problema em que os conceitos são aplicados favorece a conceitualização. Situações como o emaranhamento quântico e os experimentos de dupla fenda se
apresentaram como bons meios para a compreensão de alguns dos mais importantes conceitos
da MQ, tais como o de estado quântico, o de superposição linear de estados e o de colapso do
vetor de estado, entre outros. Crê-se, então, que as situações-problema utilizadas, agregadas a outras de igual
relevância na MQ, tais como o experimento de Stern-Gerlach e a polarização da luz, podem se
apresentar como de suma importância para a compreensão dos principais conceitos desta teoria. A utilização de simulações em computador se mostrou eficiente tanto para o interesse
quanto para a compreensão dos fenômenos observados conduzindo, então, a agregar-se
aplicativos também das novas situações-problema a serem incorporadas (minimizando o fato de
que aparatos científicos de grande número de experimentos de MQ não é acessível aos centros
universitários e escolas de Ensino Médio do país). Mostrou-se ponto importante dos minicursos a não utilização de conceitos relativos à
Mecânica Clássica para explicar os fenômenos quânticos. Conclusão Desde o início, buscou-se trabalhar as
situações-problema com conceitos oriundos diretamente da MQ, evitando que as leis da Física
Clássica fossem equivocadamente arroladas como subsunçor para o aprendizado da MQ. Isto
acabou por obviar alguns possíveis obstáculos epistemológicos para um prosseguimento no
estudo da MQ. A proposta aqui analisada permite, então, considerar que o ensino introdutório de MQ
pode ser feito sem o uso de conceitos da Mecânica Clássica e com o auxílio de inúmeras
situações-problema em que os conceitos fundamentais da MQ são aplicados. Contudo, eventuais
analogias com estruturas clássicas, tais como as de superposição de vetores no plano e de ondas,
podem ser úteis para a construção de subsunçores para a MQ. Referências AUSUBEL, D. P. Acquisition and retention of knowledge: a cognitive view. Dordrecht: Kluwer
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ctoria@if.ufrgs.br Marco Antonio Moreira. Universidade Federal do Rio Grande do Sul. Instituto de Física. Professor
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15 R. B. E. C. T., vol 3, núm 1, jan./abr. 2010
ISSN - 1982-8 R. B. E. C. T., vol 3, núm 1, jan./abr. 2010 ISSN - 1982-873X 15
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A scoping review of competencies for scientific editors of biomedical journals
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© 2016 Galipeau et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. RESEARCH ARTICLE
Open Access
A scoping review of competencies for
scientific editors of biomedical journals
James Galipeau1*
, Virginia Barbour2, Patricia Baskin3,13, Sally Bell-Syer4,14, Kelly Cobey1,15, Miranda Cumpston5,16,
Jon Deeks6, Paul Garner7, Harriet MacLehose8, Larissa Shamseer1,10, Sharon Straus9, Peter Tugwell1,10,17,
Elizabeth Wager11, Margaret Winker12 and David Moher1,10 * Correspondence: jgalipeau@ohri.ca
1Clinical Epidemiology Program, Ottawa Hospital Research Institute, Ottawa,
Canada
Full list of author information is available at the end of the article A scoping review of competencies for
scientific editors of biomedical journals James Galipeau1*
, Virginia Barbour2, Patricia Baskin3,13, Sally Bell-Syer4,14, Kelly Cobey1,15, Miranda Cumpston5,16,
Jon Deeks6, Paul Garner7, Harriet MacLehose8, Larissa Shamseer1,10, Sharon Straus9, Peter Tugwell1,10,17,
Elizabeth Wager11, Margaret Winker12 and David Moher1,10 James Galipeau1*
, Virginia Barbour2, Patricia Baskin3,13, Sally Bell-Syer4,14, Kelly Cobey1,15, Miranda Cumpston5,16,
Jon Deeks6, Paul Garner7, Harriet MacLehose8, Larissa Shamseer1,10, Sharon Straus9, Peter Tugwell1,10,17,
Elizabeth Wager11, Margaret Winker12 and David Moher1,10 Abstract Background: Biomedical journals are the main route for disseminating the results of health-related research. Despite this, their editors operate largely without formal training or certification. To our knowledge, no body of
literature systematically identifying core competencies for scientific editors of biomedical journals exists. Therefore,
we aimed to conduct a scoping review to determine what is known on the competency requirements for scientific
editors of biomedical journals. Methods: We searched the MEDLINE®, Cochrane Library, Embase®, CINAHL, PsycINFO, and ERIC databases (from
inception to November 2014) and conducted a grey literature search for research and non-research articles with
competency-related statements (i.e. competencies, knowledge, skills, behaviors, and tasks) pertaining to the role of
scientific editors of peer-reviewed health-related journals. We also conducted an environmental scan, searched the
results of a previous environmental scan, and searched the websites of existing networks, major biomedical journal
publishers, and organizations that offer resources for editors. Results: A total of 225 full-text publications were included, 25 of which were research articles. We extracted a total
of 1,566 statements possibly related to core competencies for scientific editors of biomedical journals from these
publications. We then collated overlapping or duplicate statements which produced a list of 203 unique statements. Finally, we grouped these statements into seven emergent themes: (1) dealing with authors, (2) dealing with peer
reviewers, (3) journal publishing, (4) journal promotion, (5) editing, (6) ethics and integrity, and (7) qualities and
characteristics of editors. Discussion: To our knowledge, this scoping review is the first attempt to systematically identify possible competencies
of editors. Limitations are that (1) we may not have captured all aspects of a biomedical editor’s work in our searches,
(2) removing redundant and overlapping items may have led to the elimination of some nuances between
items, (3) restricting to certain databases, and only French and English publications, may have excluded
relevant publications, and (4) some statements may not necessarily be competencies. Conclusion: This scoping review is the first step of a program to develop a minimum set of core
competencies for scientific editors of biomedical journals which will be followed by a training needs
assessment, a Delphi exercise, and a consensus meeting. Keywords: Biomedical, Competencies, Journal, Scientific editor, Scoping review Full list of author information is available at the end of the article © 2016 Galipeau et al. Galipeau et al. BMC Medicine (2016) 14:16
DOI 10.1186/s12916-016-0561-2 Galipeau et al. BMC Medicine (2016) 14:16
DOI 10.1186/s12916-016-0561-2 Background There are also individual
websites and blogs, such as “Journalology” [8], that pro-
vide thoughtful commentary on current issues related to
publication science. Several commercial groups offer short
training courses for editors [9, 10]. However, for any com-
prehensive editor training program to work effectively and
be assimilated into practice, it must be based on what the
broader biomedical editor community considers to be
core competencies. Unlike many other professional groups, such as clini-
cians and healthcare professionals, many scientific edi-
tors of biomedical journals operate largely without
formal training and universal certification is not yet a
high priority [5]. Instead, editors generally are invited to
serve in their role by publishers, based on their expertise
and stature in the field, since such expertise is essential
for evaluating research and stature is important for es-
tablishing the reputation of the journal and attracting
submissions. However, such expertise does not guaran-
tee that editors have the background or training neces-
sary to carry out their roles and responsibilities. Editors
may or may not be paid for their role and financial sup-
port for the editorial role often does not include travel
or training funds. Most editors work part-time as they
continue their academic responsibilities in research
and/or clinical work, with time for completing editorial
responsibilities – much less training – being at a pre-
mium. Researchers and peer reviewers similarly have no
international standardized formal training or certifica-
tion as to research conduct, reporting, and evaluation,
making the editor’s job even more demanding. This
situation is highly problematic given that the conse-
quences of deciding what gets published and the degree
of quality that is acceptable impacts future research, de-
cisions, and healthcare directly. Our view is that the We are unaware of any body of literature systematic-
ally identifying core competencies for biomedical editors,
nor any agreement on or attempt at a consensus process
to determine what they should be. For the purposes of
this
research,
we
borrow
from
the
literature
on
competency-based continuing professional development
to define competence as “the array of abilities across
multiple domains or aspects of [practitioner] perform-
ance in a certain context” [11]. We thus define core
competencies as the essential knowledge, skills, and be-
haviors necessary for the practice of scientific editing of
biomedical journals. Background lack of consistent training of editors reduces the value
of the published literature, including its quality, trans-
parency, and reproducibility, thereby reducing value for
money to funders and the usability of research findings,
ultimately degrading public trust in the research record
[3]. However, we are unaware of any research that dir-
ectly addresses this topic. Additionally, while the train-
ing of biomedical editors is an important mechanism to
ensure the quality of the published literature, other im-
portant changes in tandem with this, including re-
examining the training offered to peer-reviewers and
training graduate students in study conduct, analysis,
interpretation, and reporting, could also have a benefi-
cial effect. “…journals, some of which have reported research for
many decades, are still not producing articles that are
clear enough to really judge a study’s conduct, quality,
and importance—let alone to allow other researchers to
reproduce it or build on it” [1]. Biomedical journals are the main route for disseminat-
ing the results of health-related research [2]. However,
when examined more closely, the articles that journals
publish are problematic; critical details are often missing
or poorly reported, consequently reducing their quality,
transparency, reproducibility, and usefulness for decision
makers [3] – this is wasteful, diminishes scientific and
fiscal value, and is unethical [4]. Authors and scientific
journals share the majority of the responsibility for these
shortcomings, as the former are accountable for the in-
tegrity of a study’s conduct and the accuracy of reporting
of the content within the manuscript, while the latter
are accountable for decisions regarding its publication. On the side of journals, it is scientific editors (by which
we mean editors, and ultimately the Editor-in-Chief,
who are tasked with making decisions about the content
and policies of journals) who are accountable for all ma-
terial published in their journals. Readers have a right to
expect these editors to implement all reasonable actions
that could lead to best practices within their journals, as
well as journals having processes in place to ensure the
quality of the papers they publish. Some organizations, for example, the World Association
of Medical Editors (WAME) [6] and the Committee on
Publication
Ethics
(COPE)
[7],
provide
rigorously-
developed resources for biomedical journal editors free of
charge, including guidance on the role of the medical edi-
tor, editorial policies, and listservs on which editors’ ques-
tions and issues are discussed. Background We believe it is important to de-
velop a set of core competencies so that training pro-
grams can then be developed and tailored with the
intent that all editors meet some basic globally agreed-
upon standards. Other stakeholders, such as publishers
(including medical associations who have their own jour-
nals), peer reviewers, and authors (researchers), also
need to contribute to this effort. Herein, as a starting
point, we report a scoping review of possible core com-
petencies of scientific editors of biomedical journals. Abstract 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 17 Page 2 of 17 Galipeau et al. BMC Medicine (2016) 14:16 Objectives The objective of this scoping review was to conduct a
systematic search of the literature on the competencies Galipeau et al. BMC Medicine (2016) 14:16 Page 3 of 17 Page 3 of 17 journals, and an associated environmental scan that
found only a few training opportunities for these editors
[18]. Additionally, our anecdotal experience was that
most descriptions of editor competencies are found in
editorial-type publications (e.g. commentaries), job post-
ings, and guidance documents (e.g. COPE Guidelines for
Editors) – all of which would not generally be captured
in a traditional scoping review of published research. required for scientific editors of biomedical journals to
effectively and efficiently produce transparently reported
and correctly analyzed and interpreted publications. Our
specific aim was to answer the research question: “What
is known from the literature on the competency require-
ments of scientific editors of peer reviewed biomedical
journals?” with the goal of summarizing the existing lit-
erature. The purpose of this scoping review is to inform
the future development of a set of core competencies for
scientific editors of
biomedical journals, which we
hypothesize will ultimately lead to improvements in the
quality of the published literature. The expanded scoping exercise included (1) searching
the databases from the search strategy and grey litera-
ture for non-research-based publications, (2) search-
ing the results of an environmental scan from a
previous related project [17, 18], (3) conducting a
new environmental scan with additional search terms,
and (4) searching the websites of existing networks
(i.e. EQUATOR Network), major biomedical journal
publishers (i.e. Wiley-Blackwell, Elsevier, BioMed Central,
BMJ Publishing Group, Springer), and organizations that
offer resources for editors (i.e. COPE, WAME, Council of
Science Editors, European Association of Science Editors,
and International Committee of Medical Journal Editors). For the database searches, the full-text of all potentially
relevant documents were retrieved and independently
reviewed for eligibility, in duplicate, by members of the
team using a priori eligibility criteria. Disagreements
between reviewers were resolved by consensus or by a
third member of the research team. Both environmen-
tal scans employed the same methodology, which involved
the use of the Google search engine to run a series of two-
and three-word keyword searches (Additional file 1). For
each set of search outcomes, the first 50 Google results
were screened for relevant information. If any of the last
10 results contained useful information, another 10 results
were screened. Search strategy We searched the MEDLINE®, Cochrane Library, Embase®,
CINAHL, PsycINFO, and ERIC databases, all from incep-
tion to November 10th, 2014 (Additional file 1). The spe-
cific search strategies were created by a Health Sciences
Librarian with expertise in scoping review searching. The
MEDLINE strategy was developed with input from the
project team, then peer reviewed by a second librarian not
otherwise associated with the project using the PRESS
standard [15]. We also conducted a grey literature search,
which included hand searching the reference lists of in-
cluded articles, as well as searching key journals, in par-
ticular JAMA from 1989, and BMJ, PLoS Medicine,
European Science Editing, Annals of Internal Medicine,
and CMAJ from 2004 onward to identify publications re-
lated to the Peer Review Congresses [16]. Expanded scoping exercise Given that this scoping review is part of a larger pro-
gram to develop core competencies for scientific editors
of biomedical journals, in addition to seeking research
literature, we also incorporated an expanded scoping
exercise that included non-research-based (published
and unpublished) materials to fill an expected gap in re-
search on competencies of scientific editors of biomed-
ical journals. This expectation was based on a previous
systematic review [17], which found no comparative
studies on training for scientific editors of biomedical Methods The protocol for this project has previously been depos-
ited in the University of Ottawa’s Digital Repository
(uOttawa Research) prior to beginning the screening
phase [12]. Our methodological approach was guided by
the Arksey and O’Malley Framework [13], as well as the
additional suggestions to this framework made by Levac
[14]. Specifically, we undertook the six-step process of:
(1) identifying the research question; (2) identifying rele-
vant studies; (3) study selection; (4) charting the data;
(5) collating, summarizing, and reporting results; and
(6) consultation. Objectives This process was continued until a set of
10 results with no relevant information was found [18]. Study selection: inclusion criteria
Population Articles
with
statements
mentioning
competencies,
knowledge, skills, behaviors, and tasks (henceforth re-
ferred to as ‘competency-related statements’) pertaining
to the role of scientific editors of peer-reviewed health-
related journals (including Editors-in-Chief and associate/
academic editors, and full-time professional journal edi-
tors) were included. Articles related to all other types of
editor not directly involved in evaluation, peer review, re-
vision, and selection of content (e.g. managing editors,
technical editors, copy editors) were excluded. Disciplines We adopted MEDLINE’s journal selection criteria for
our definition of health [19]. This definition includes
journals that are “predominantly devoted to reporting
original investigations in the biomedical and health Page 4 of 17 Page 4 of 17 Galipeau et al. BMC Medicine (2016) 14:16 sciences, including research in the basic sciences; clinical
trials of therapeutic agents; effectiveness of diagnostic or
therapeutic techniques; or studies relating to the be-
havioral, epidemiological, or educational aspects of
medicine” [19]. This definition encompasses biomedical
journals as well as those in the disciplines of psychology
and education. For feasibility purposes, we did not in-
clude journals from the physical or natural sciences. sciences, including research in the basic sciences; clinical
trials of therapeutic agents; effectiveness of diagnostic or
therapeutic techniques; or studies relating to the be-
havioral, epidemiological, or educational aspects of
medicine” [19]. This definition encompasses biomedical
journals as well as those in the disciplines of psychology
and education. For feasibility purposes, we did not in-
clude journals from the physical or natural sciences. scans we extracted the URL, title of the document, lan-
guage of publication, and who produced the document
(affiliation). For all documents, we collected descriptions
of any statements potentially relating to the competen-
cies of scientific editors of biomedical journals, such as
descriptions of particular skills, knowledge, attitudes,
behaviors, tasks, and training. Collating, summarizing, and reporting the results
In an effort to create a useful summary of the data for
the next steps of our program to develop core compe-
tencies for scientific editors of biomedical journals, we
combined the competency-related statements retrieved
from all sources. First, two people (JG, KDC) classified
all statements pertaining only to Editors-in-Chief into a
single category, since these would be considered to be
beyond the core competencies of scientific editors more
generally. They then collated overlapping or duplicate
statements to produce a list of unique statements. Fi-
nally, they grouped statements into emergent themes to
make them more manageable for future use (e.g. in an
upcoming Delphi exercise), and so that they would be
understandable to readers. While some of the wording
of particular statements was modified to assimilate over-
lapping statements, where statements were expressed as
knowledge, skills, behaviors, or tasks that implied com-
petencies, but not as competencies themselves, we did
not edit or translate them to express competencies, in
order to preserve the original intent. Charting the data A data extraction form was developed a priori to capture
information on each document included in the review. It
was piloted and refined based on feedback from the ex-
ercise. Three people (JG, KDC, LS) carried out data ex-
traction in the following manner: data were extracted by
one reviewer and a second reviewer conducted verifica-
tion of the data for all records. Disagreements between
reviewers were resolved by consensus. General study
characteristics extracted for the database search were:
first author name and contact information (of corre-
sponding author), year of publication, institutional affili-
ation of first author, country, language of publication,
study design, and funding source. For the environmental Database search We screened 5,837 titles and abstracts, of which 360
were screened in full-text (Fig. 1). Of these, 206 were ex-
cluded, leaving 154 publications meeting the inclusion
criteria. Twenty-five of these publications were research
based (Table 1) and the remaining 136 were editorial in
nature (Additional file 2). Disciplines The relationships
between behaviors, tasks, and competencies will be the
subject of discussion and translation undertaken as part
of the consensus meeting phase of this project. Screening Following the execution of the search strategy, the iden-
tified
records
(titles
and
available
abstracts)
were
collated in a Reference Manager [20] database for de-
duplication. The final unique record set of potentially
eligible studies was exported to Internet-based soft-
ware, DistillerSR (Evidence Partners, Ottawa, Canada),
through which screening of records and data extrac-
tion were carried out. Each title and abstract was
screened by two of four reviewers (LS, JG, JT (see
Acknowledgements), and MG (see Acknowledgements))
using a ‘liberal accelerated’ method [21] (i.e. one reviewer
screened each record and a second reviewer screened only
excluded records, independently). The full-text of all
remaining potentially eligible papers was then retrieved,
uploaded into DistillerSR, and reviewed for eligibility, in-
dependently, by two members of the team (LS and JG)
using a priori eligibility criteria. Disagreements between
reviewers at this stage were resolved by consensus or by a
third member of the research team. Study designs
Th The review included all study designs as well as edito-
rials
and
commentaries. Economic
evaluations
and
letters were excluded, as neither was expected to con-
tribute useful data for the purposes of this scoping
review. For feasibility purposes, we included articles
written in English and French only. We did not include
English or French abstracts of papers written in another
language. Charting the data
General characteristics Research-based publications A total of 18 publications
from the database search presenting the results of re-
search (subsequently termed ‘research-based publica-
tions’) were considered relevant to core competencies
for scientific editors of biomedical journals, along with
another seven articles found in the grey literature search
(including six conference abstracts for which there ap-
pears to be no full text publication) (Table 1). None of
these 25 articles had an outline or description of core
competencies of scientific editors as an objective of the Galipeau et al. BMC Medicine (2016) 14:16 Page 5 of 17 Fig. 1 Study flow diagram scientific editors. Overall, 133 publications were editorial
in nature, while the remaining three included a lecture, a
job description, and an interview. Seventeen journals
had multiple included publications, with three of them
(Australian and New Zealand Journal of Obstetrics and
Gynaecology, Croatian Medical Journal, and Indian
Journal of Medical Sciences) producing three publica-
tions each while the other 14 journals had two publica-
tions each. JAMA also had four publications across three
journals
in
its
collection
(JAMA,
JAMA
Internal
Medicine, JAMA Ophthalmology). The rest of the sample
consisted of a single publication per journal. The date of
publication ranged from 1954 to 2015; 2011 (n = 20)
and 2012 (n = 17) were the 2 years with the most
studies. The sample included 66 studies with first au-
thors originating from the USA and 19 from the
United Kingdom, with representation from another 18
countries as well among first authors. The individual
publications yielded between zero and 15 competency-
related statements, with a median of two statements per
publication. research. Fifteen studies were survey-based research,
three were descriptive studies, three were case studies,
and two were final project reports (one from a task force
and the other from a study funded by the US National
Institute of Education). The remaining two studies were
a systematic review and a mixed-methods study. Five
studies reported receiving funding. Nineteen studies
were published in different journals and the remaining
six were part of the Peer Review Congress Proceedings. Publication dates ranged from 1975 to 2014; 2009 was
the year with the most publications (n = 3), followed by
2011 (n = 2) and 2013 (n = 2). Twelve of the studies’ first
authors were from the USA, 10 from the United Kingdom,
and one each from Iran, India, and Canada. Charting the data
General characteristics The pub-
lications produced a total of 40 competency-related
statements (i.e. possible competencies), with individual
publications yielding between zero and 14 statements
and a median of one statement per publication. Expanded scoping exercise Non research-based publications A total of 136 non-
research-based publications were considered relevant to
core competencies for scientific editors of biomedical
journals, yielding a total of 537 competency-related
statements (Additional file 2). Similar to the research-
based literature, none of these publications had the ex-
plicit goal of outlining a set of core competencies for Environmental scan of training in Journalology We
reviewed all 258 documents listed in the Repository of
Ongoing Training Opportunities in Journalology [20],
which houses all of the data from an environmental scan
of training in Journalology carried out by members of Galipeau et al. BMC Medicine (2016) 14:16 Page 6 of 17 Table 1 Included research-based publications
First author
Affiliation
Country Journal
Year
Design
# a Item(s) b
Albert, T
Tim Albert Training
UK
Learned Publishing
2002 Survey
0
N/A
Barnes, M
University of Nebraska-Lincoln
USA
The Review of Higher Education
1986 Survey
5
5, 72, 170, 171, 185
Carroll-Johnson, R Oncology Nursing Society
USA
Nurse Author & Editor
1996 Survey
1
137
Davis, RM
Henry Ford Health System
USA
Science & Engineering Ethics
2002 Survey
1
137
de Jesus Mari, J
King’s College, University
of London
UK
African Journal of Psychiatry
2009 Task Force Report
0
N/A
Etemadi, A
Shaheed Beheshti University
of Medical Sciences
Iran
Saudi Medical Journal
2004 Survey
1
141
Freda, M
Journal of Nursing Scholarship
USA
Journal of Nursing Scholarship
2005 Survey
0
N/A
Froehle, T
Indiana University, Bloomington
USA
Counselor Education & Supervision 1990 Descriptive Study
0
N/A
Galipeau, J
Ottawa Hospital Research
Institute
Canada
Systematic Reviews
2013 Systematic Review
0
N/A
Garrow, J
LOCKNET Peer Review Research
Group: European Journal of
Clinical Nutrition
UK
Journal of the American Medical
Association
1998 Survey
0
N/A
Grindlay, D
Centre for Evidence-based
Veterinary Medicine, School
of Veterinary Medicine and
Science, The University of
Nottingham
UK
BMC Veterinary Research
2014 Survey
1
148
Hing, C
Department of Trauma &
Orthopaedics, St George’s
Hospital, Tooting, UK
UK
Journal of Orthopaedic Surgery &
Research
2011 Survey
0
N/A
Kearney, M
University of Rochester School
of Nursing
USA
Nursing Outlook
2006 Descriptive Study
13
5, 26, 30, 35, 69, 72, 83,
84. Expanded scoping exercise 85, 91, 102, 178, 203
Kleinert, S
The Lancet
UK
Peer Review Congress (Abstract)
2005 Observational Study 0
N/A
Lebeau, DL
Tulane University Medical Center
USA
Peer Review Congress (Abstract)
1997 Survey
1
72
Logothetti, H
Obstetrics & Gynecology
USA
Peer Review Congress (Abstract)
2009 Case-Control
1
101
Patrone, D
Philosophy Department, State
University of New York at
Oneonta/Broome Community
College, Binghamton, New York
USA
Biosecurity and Bioterrorism:
Biodefense Strategy, Practice,
and Science
2012 Survey
2
147, 203
Radford, D
Division of Prosthetic Dentistry,
King’s and St Thomas’ Dental
Institute, London
UK
British Dental Journal
1999 Survey
0
N/A
Reynolds, T
Highland Hospital
USA
Peer Review Congress (Abstract)
2009 Survey
0
N/A
Silverman, R
Ohio State University
USA
None (Final Report)
1975 Final Report
6
79, 91, 101, 166, 194, 203
Srinivasan, S
Indian J Medical Ethics
India
Peer Review Congress (Abstract)
2013 Survey
0
N/A
Wager, E
Sideview, Princes Risborough
UK
Peer Review Congress (Abstract)
2009 Case Analysis
0
N/A
Wager, E
Sideview, Princes Risborough
UK
The British Medical Journal
2013 Quantitative +
Interviews
1
136
Williams, P
University College London
UK
Science and Engineering Ethics
2011 Case Studies
2
58, 203
Wong, V
Department of Neurology,
University of Michigan,
Ann Arbor
USA
Journal of Clinical Epidemiology
2011 Survey
5
2, 118, 123, 203(2)
TOTAL
40
a Number of competency statements extracted from the document
b Corresponds to the item number from the list of competency-related statements (Table 3) a Number of competency statements extracted from the document
b Corresponds to the item number from the list of competency-related statements (Table 3) a Number of competency statements extracted from the document
b Corresponds to the item number from the list of competency-related statements (Table 3) relating to scientific editors of
biomedical journals
(Table 2). Seven of these documents were from organiza-
tions that provide guidance to editors (e.g. WAME, our team in 2013. From this repository, we extracted 11
relevant non-research-based articles from which we were
able
to
retrieve 556 competency-related statements Page 7 of 17 Galipeau et al. Expanded scoping exercise BMC Medicine (2016) 14:16 Table 2 Included documents from expanded scoping exercise
GUIDANCE FROM EDITORIAL GROUPS
Title
Editorial group/organization
Scanning source
# a
Item(s) b
International Standards for Authors
Committee on Publication
Ethics (COPE)
Training
58
6(2), 9, 10, 11, 12(2), 20(6), 22(3), 25, 26,
34, 35(3), 39, 46, 57(2), 64, 72(5), 79(2),
108(2), 137(2), 138(4), 140, 141(4), 143,
144, 145, 146(3), 150, 159, 198, 199, 203
Guidelines for Editors
COPE
Training
69
2, 6, 16, 19, 20, 21,22(2), 24, 25, 26(2),
27, 28, 30(2), 32, 34, 35(4), 39, 41, 42,
46, 54, 61, 67, 72(6), 79(2), 80, 83, 87(2),
88, 104, 111, 137(2), 138, 139, 141(2),
143, 146, 150, 153(2), 159, 189, 194,
197, 198, 199(2), 203(7)
A Short Guide to Ethical Editing for
New Editors
COPE
Training
34
6(2), 10, 20(3), 22, 26(2), 30, 32, 35(2),
36, 48(2), 62, 68, 72, 104, 116, 136,
137(2), 138, 144(2), 189, 197, 203(5)
A Science Editing Course for
Graduate Students
Council of Science Editors (CSE)
Core competencies
6
5, 95, 98, 135, 138, 178
Can Non-Native-English-Speaking
Editors be Effective Editors of
English-Language Writing? Alliance of Crop, Soil, and
Environmental Sciences Society Expanded scoping exercise CSE
Core competencies
13
25, 78, 95, 100(2), 126(2), 127, 132,
138, 141, 178, 199
Guidelines for Reporting Health
Research: How to Promote their
Use in Your Journal
EQUATOR Network
Networks search
0
N/A
Research Ethics, Publication Ethics
and Good Practice Guidelines
EQUATOR Network
Networks search
0
N/A
European Association of Science Editors
(EASE) Toolkit for Journal Editors
EASE
Core competencies
0
N/A
Editor’s Handbook (2nd Edition)
EASE
Core competencies
166
2(2), 7, 8, 9, 10, 15(2), 16, 18, 20(2),
22(4), 24(2), 25, 26(2), 27(2), 28, 30, 31,
32 33 35(3) 36 39(2) 48 50(3) 52(2) 201(3), 202(2), 203(8)
Core competencies
10
6, 15, 35, 72(2), 85, 89, 111, 138, 149
Core competencies
2
35, 140
Training
9
1, 13, 38, 46, 47(2), 140(3)
Training
56
6, 10, 14, 18, 20(4), 26(3), 29, 35, 43,
47, 48(2), 52, 57(6), 61(2), 62, 72(2), 79,
83, 84, 91, 92, 106, 113, 119, 137, 139,
141(2), 146(2), 159, 189, 197(2), 203(9)
Training
84
17, 20(2), 22, 26(5), 33, 37, 48, 50, 51,
52, 56(2), 59, 61, 64(3), 65(3), 66, 68(5),
70, 72, 74, 75, 76, 80, 81, 84, 90, 91(2),
106, 109, 113(4), 124(3), 127, 128, 132,
133(2), 135(2), 136, 137, 140, 141(5),
142, 144, 145, 146, 152, 154, 155(3),
159, 162(2), 166, 198, 203(4)
Core competencies
0
N/A Core competencies
10
6, 15, 35, 72(2), 85, 89, 111, 138, 149
Core competencies
2
35, 140 ASSOCIATIONS, JOURNALS, PUBLISHERS
Editor Handbook
Alliance of Crop, Soil, and
Environmental Sciences Society ASSOCIATIONS, JOURNALS, PUBLISHERS
Editor Handbook
Alliance of Crop, Soil, and
Environmental Sciences Society Core competencies
0
N/A Alliance of Crop, Soil, and
Environmental Sciences Society Galipeau et al. Expanded scoping exercise BMC Medicine (2016) 14:16 Page 8 of 17 Table 2 Included documents from expanded scoping exercise (Continued) Table 2 Included documents from expanded scoping exercise (Continued)
Editors and Reviewers
Alliance of Crop, Soil, and
Environmental Sciences Society
Core competencies
11
6(3), 35(2), 47, 61, 112(2), 140, 141
Editor Handbook
American Association of
Pharmaceutical Scientists Journal
Core competencies
9
35, 91, 116(2), 203(5)
Editor-In-Chief: Position Description
American Geophysical Union
Core competencies
25
69, 72, 84, 95, 140, 159, 160, 161,
162(2), 166, 167, 170(2), 171(2), 178,
181(2), 182, 189, 191(2), 199, 203
Position Description for the AJNR Editor
and Basic Qualifications
American Journal of Neuroradiology
Core competencies
5
5, 70, 91, 117, 168
Responsibilities of an Editor
Annals, Academy of Medicine,
Singapore
Training
30
6(2), 17, 19, 35(2), 61, 73, 77, 79, 80,
81, 83, 90(4), 91, 92, 137(4), 138, 151,
172, 192, 203(3)
What does an Associate Editor
Actually do? Association for Computing Machinery
Core competencies
3
34, 40, 72
The Role of the Scientific Editor
British Dental Journal
Core competencies
3
8, 14, 20
Recruiting a Journal Editor: An HSS
Challenge
Cambridge Journals Blog
Core competencies
0
N/A
Editorial: on Editing and Being an Editor
Cultural Studies of Science Education
Core competencies
3
6, 33, 34
Editor’s Pack
Elsevier
Publishers search
0
N/A
How do Publishers Choose Editors, and
How do they Work Together? Expanded scoping exercise ResearchGate
Core competencies
5
44, 72, 79, 159, 203
Job Description of an Editor-in-Chief
Study.com
Core competencies
0
N/A
So, you want to be a Science Writer
when you grow up…
The Black Hole
Core competencies
0
N/A
Ideas for a Topical Outline
Unknown
Training
103
2, 8(2), 10, 14, 15, 17, 20, 22, 26(3), 31,
33, 37, 48, 49, 50, 51, 52, 53, 56(2), 57,
59(2), 61(2), 62, 64(3), 66, 68(4), 70, 71,
72, 74, 75, 76, 77(2), 81, 82, 91(3), 92,
95, 101, 106(2), 113(3), 117, 124(3), 127,
133(4), 135(2), 136, 137, 140, 141(3),
142, 144, 145, 146, 147, 152, 154,
155(3), 161, 162(2), 165(2), 169(2), 173,
198, 203(9)
Recommended Recruitment Steps for
Journal Editor, CJNSE/RCJCÉ
Wired Learning Consultants
Core competencies
6
95, 117, 162, 167, 178(2) Table 2 Included documents from expanded scoping exercise (Continued)
Trainee Programs/Editorial Trainees
Springer
Core competencies
0
N/A
Confessions of a Journal Editor
The Chronicles of Higher Education
Core competencies
0
N/A
Ethics and the Psychiatry Journal Editor:
Responsibilities and Dilemmas
The Israel Journal of Psychiatry and
Related Sciences
Core competencies
9
20(2), 22, 36, 48, 64, 120, 140, 141
Editor Ethics 2.0 Code/Affirming Editors
University of North Carolina – Charlotte
Core competencies
2
20, 138
UOJM Editor Training: Results from the
2013 Editor Satisfaction Survey and
Highlights from 2013–2014 Training
Workshops
University of Ottawa Journal of Medicine
Core competencies
8
36, 61, 74, 79, 95, 123, 160, 175
FAME Guidelines
World Health Organization
Training
35
6(3), 10, 14, 15, 30, 34, 35, 39, 40(2),
47, 61, 72(3), 75, 79(2), 91, 92, 133, Table 2 Included documents from expanded scoping exercise (Continued) Research Journal Editor Position Description
Young Adult Library Service Associa
OTHER SOURCES
Medical/Scientific Editor (job posting)
Alexion Pharmaceuticals, Inc. Duties of Editors
Bioinfo Publications
What is Special about Science Editing? Biotext
What is Different about Science Editing? Emend Editing
What Exactly Does an Editor Do? Expanded scoping exercise Elsevier
Core competencies
7
110, 160, 162, 184, 185, 194, 203
Publishing Ethics Resource Kit (PERK)
Elsevier
Publisher search
0
N/A
European Respiratory Journal
Editor(s)-in-Chief
European Respiratory Society
Core competencies
6
162(2), 165, 167, 195, 178
Editor-in-Chief (position description)
International Society of Exposure
Science; Journal of Exposure Science
and Environmental Epidemiology
Core competencies
9
19, 35, 101, 138, 159, 165, 166, 170, 203
Editor-in-Chief (position description)
Journal of Family Planning and
Reproductive Health Care
Core competencies
19
8, 19(2), 36, 95, 116, 117, 121, 123(2),
127, 138, 159, 164, 167, 170, 178, 180,
181
Responsibilities of the Editor
Journal of Medical Internet Research
Core competencies
1
123
Responsibilities of the JNCI Editor‐in‐Chief
Journal of the National Cancer
Institute
Core competencies
8
95, 99, 127, 132, 138, 159, 174, 203
Responsibilities of Editors and Reviewers
Online Ethics Center for Engineering
and Science
Core competencies
0
N/A
Scientific Editing–A Wise Career Choice
Science Careers (from the journal Science)
Core competencies
0
N/A
Horses for Courses–Research Papers
versus Reviews
Science Careers (from the journal Science)
Core competencies
0
N/A
The Editors' World: Back to the Books
Science Careers (from the journal Science)
Core competencies
6
159(2), 165, 175, 178, 181
Bench to Page: An Editor's View of
Science Publishing
Science Careers (from the journal Science)
Core competencies
3
73, 164, 170
At the Gateway of Cutting-Edge Research
Science Careers (from the journal Science)
Core competencies
7
68, 108, 109, 122, 162, 163, 191
Translating Scientific Expertise into
Publishing Success
Science Careers (from the journal Science)
Core competencies
0
N/A
Journal Editors Get Twitter-Savvy
Science Careers (from the journal Science)
Core competencies
14
40, 71, 73, 90, 93, 95, 108, 164, 165,
174, 177, 185, 193, 196
Careers in Neuroscience/Career Paths:
Science Publishing
Society for Neuroscience
Core competencies
8
122, 164, 165, 166, 167(2), 178, 184
Academic Journal Editors’ Professionalism:
Perceptions of Power, Proficiency and
Personal Agendas
Society for Research Into Higher Education
Core competencies
9
33(2), 72, 73, 110, 121, 160, 167, 174
Editorial Guide
Springer
Publishers search
7
26, 65, 78, 82, 85, 138, 197 Springer Galipeau et al. Expanded scoping exercise BMC Medicine (2016) 14:16 Page 9 of 17 Table 2 Included documents from expanded scoping exercise (Continued)
Trainee Programs/Editorial Trainees
Springer
Core competencies
0
N/A
Confessions of a Journal Editor
The Chronicles of Higher Education
Core competencies
0
N/A
Ethics and the Psychiatry Journal Editor:
Responsibilities and Dilemmas
The Israel Journal of Psychiatry and
Related Sciences
Core competencies
9
20(2), 22, 36, 48, 64, 120, 140, 141
Editor Ethics 2.0 Code/Affirming Editors
University of North Carolina – Charlotte
Core competencies
2
20, 138
UOJM Editor Training: Results from the
2013 Editor Satisfaction Survey and
Highlights from 2013–2014 Training
Workshops
University of Ottawa Journal of Medicine
Core competencies
8
36, 61, 74, 79, 95, 123, 160, 175
FAME Guidelines
World Health Organization
Training
35
6(3), 10, 14, 15, 30, 34, 35, 39, 40(2),
47, 61, 72(3), 75, 79(2), 91, 92, 133,
138(2), 141, 143, 149, 159(2), 179, 189,
198, 203(2)
Research Journal Editor Position Description
Young Adult Library Service Association
Core competencies
11
35, 95(2), 117, 159, 160, 161, 162, 167,
174, 180,
OTHER SOURCES
Medical/Scientific Editor (job posting)
Alexion Pharmaceuticals, Inc. Core competencies
0
N/A
Duties of Editors
Bioinfo Publications
Core competencies
3
104, 141, 199
What is Special about Science Editing? Biotext
Core competencies
11
120, 124, 125, 129(2), 130, 131, 141,
165, 175, 193
What is Different about Science Editing? Emend Editing
Core competencies
13
57, 60, 117, 125, 126, 127, 129, 130,
131, 162, 165, 175, 193
What Exactly Does an Editor Do? Joseph Alpert
Training
11
39, 54, 68, 73, 86, 108, 138, 162,
198, 203(2)
Becoming a Journal Editor
PhD2Published
Core competencies
5
109, 167, 182, 190, 199
Public Knowledge Project School
Public Knowledge Project
Training
67
6(3), 14, 15(8), 20, 26(3), 30, 34, 35(3),
39, 46, 47, 50, 53(3), 57(2), 59, 61(2),
62, 68, 84, 85, 87, 91, 95, 104(2), 137(2),
138, 140, 141, 149, 160, 167(2), 197(2),
203(14)
What does an Editor (a Member of
Editorial Board) do Exactly in Journals? ResearchGate
Core competencies
0
N/A
What are the Role and Duties of a
Scientific Editor of an Academic
Peer-Review Journal? Expanded scoping exercise Joseph Alpert
Becoming a Journal Editor
PhD2Published
Public Knowledge Project School
Public Knowledge Project Core competencies
0
N/A
Core competencies
3
104, 141, 199
Core competencies
11
120, 124, 125, 129(2), 130, 131, 141,
165, 175, 193
Core competencies
13
57, 60, 117, 125, 126, 127, 129, 130,
131, 162, 165, 175, 193
Training
11
39, 54, 68, 73, 86, 108, 138, 162,
198, 203(2)
Core competencies
5
109, 167, 182, 190, 199
Training
67
6(3), 14, 15(8), 20, 26(3), 30, 34, 35(3),
39, 46, 47, 50, 53(3), 57(2), 59, 61(2),
62, 68, 84, 85, 87, 91, 95, 104(2), 137(2),
138, 140, 141, 149, 160, 167(2), 197(2),
203(14)
Core competencies
0
N/A
Core competencies
5
44, 72, 79, 159, 203
Core competencies
0
N/A
Core competencies
0
N/A
Training
103
2, 8(2), 10, 14, 15, 17, 20, 22, 26(3), 31,
33, 37, 48, 49, 50, 51, 52, 53, 56(2), 57,
59(2), 61(2), 62, 64(3), 66, 68(4), 70, 71,
72, 74, 75, 76, 77(2), 81, 82, 91(3), 92,
95, 101, 106(2), 113(3), 117, 124(3), 127,
133(4), 135(2), 136, 137, 140, 141(3),
142, 144, 145, 146, 147, 152, 154,
155(3), 161, 162(2), 165(2), 169(2), 173,
198, 203(9)
Core competencies
6
95, 117, 162, 167, 178(2)
Core competencies
0
N/A What does an Editor (a Member of
Editorial Board) do Exactly in Journals? ResearchGate
What are the Role and Duties of a
Scientific Editor of an Academic
Peer-Review Journal? ResearchGate
Job Description of an Editor-in-Chief
Study.com
So, you want to be a Science Writer
when you grow up…
The Black Hole
Ideas for a Topical Outline
Unknown Core competencies
0
N/A Number of competency statements extracted from the document p
y
b Corresponds to the item number from the list of competency-related statements (Table 3) Galipeau et al. BMC Medicine (2016) 14:16 Page 10 of 17 Page 10 of 17 Page 10 of 17 COPE, International Committee of Medical Journal
Editors) and the remaining documents were from a
variety of other sources. group of editors will collaborate to outline a set of
core competencies. Despite our finding that the competencies of editors
have been discussed in the published literature since the
mid-1950s, a recent systematic review [17] found no
comparative studies of the effectiveness of training for
editors. This is concerning, given the gate-keeping role
that scientific editors play as guardians of the scientific
record [23]. Expanded scoping exercise Trends in the number of publications annu-
ally would seem to indicate that, while the overall num-
ber of publications in this area has grown since the topic
first emerged in the literature, interest peaked around
2011 and is now waning. The trend, however, is more
reflective of editorial-type articles in journals, which
have declined since 2011, while the number of research-
based publications has remained relatively stable (yet
sparse) during the same timeframe. Environmental scan of core competencies for scien-
tific editors of biomedical journals The environmental
scan carried out for this project consisted of a total of 40
keyword searches (Additional file 1) that yielded 48 rele-
vant documents, of which 35 were deemed to meet the
eligibility criteria after screening in duplicate. These 35
documents
produced
426
competency-related
state-
ments (Table 2). Among the sample, 18 documents were
produced or published by journals, nine were from
associations and societies, six were from organizations
providing guidance to editors, and two were from
publishers. Search of networks, publishers, and meetings
A
search of networks, publishers, and meetings produced
an additional five documents, three of which were docu-
ments from publishers’ websites and two from the
EQUATOR Network (Table 2). We extracted seven
competency-related statements from these additional
documents. One possibility for the decline in total publications is
that organizations may believe these issues have already
been identified and they are working on training mate-
rials for them rather than publishing more research or
commentaries about them in medical journals. A possi-
bility for the continued lack of research in this area may
be that the focus of many major funding agencies is to
fund by disease (e.g. heart, kidney, cancer, diabetes). As
journal editing, and the field of journalology more
broadly, is a domain that spans across the spectrum of
research on diseases, it is often difficult to find appropri-
ate funding opportunities and even more difficult to
convince specific disease-based funding agencies that the
research merits their funding investment. Of the 64 documents in the expanded scoping exer-
cise, the European Association of Science Editors Edi-
tor’s Handbook (2nd Edition) [22] had the highest
number of competency-related statements (n = 166). The
median
number
of
competency-related
statements
across the scoping exercise documents was six. Collating and summarizing the data A major challenge that we anticipated as part of this
scoping review was that a large proportion of the evi-
dence may not be in the traditional research-based lit-
erature. For this reason, we placed a heavy emphasis on
extensively searching the grey and non-research-based
literature by including two related environmental scans
as part of the larger program on core competencies for
scientific editors of biomedical journals. However, it is
still possible that we may not have captured every aspect
of a biomedical editor’s work in our searches. In particu-
lar, some of the more tacit (e.g. difficult to describe) as-
pects of the work of editors may simply not be
documented, or may reside in documents that may not
necessarily be found in a database or Google search (e.g. resources residing behind membership or password-
protected webpages, paywalls). We expect that any crit-
ical missing items will be brought forth in the training
needs assessment, the Delphi, or the consensus meeting. Additionally, because of the subjective nature of this
type
of
data
extraction,
it
is
possible
that
some
competency-related statements were missed within the
included publications. However, we feel that the likeli-
hood of this was reduced as several members of the The combined 1,566 competency-related statements ori-
ginating from the 225 total documents were collated and
then de-duplicated, producing a list of 203 unique state-
ments (i.e. possible competencies of scientific editors)
(Table 3). The statements were organized into seven
categories that emerged from the data: (1) dealing with
authors; (2) dealing with peer reviewers; (3) journal pub-
lishing; (4) journal promotion; (5) editing; (6) ethics and
integrity; and (7) qualities and characteristics of editors. Discussion This scoping review identified 225 relevant publications,
spanning more than 60 years, and involving authors
from more than 20 countries. It produced a comprehen-
sive list of possible competency-related statements for
the scientific editor position within biomedical journals. This categorized list of statements will be used in a sub-
sequent Delphi exercise aiming to ask a broad spectrum
of scientific editors of biomedical journals to rate the
importance of each statement in relation to performing
their duties as a scientific editor. This data will then
help inform a consensus meeting in which a select Page 11 of 17 Galipeau et al. Discussion BMC Medicine (2016) 14:16 Scientific editors should:
1
Review study protocols and methods and encourage authors to make them publicly available
2
Ensure authors are aware of ethical authorship practices
3
Seek to help authors understand magnitude of effect
4
Assist potential authors in developing a spirit of inquiry
5
Develop wide acquaintance with potential authors
6
Demonstrate accountability to authors and ensure they are treated with fairness, courtesy, and objectivity
7
Provide constructive criticism to authors
8
Engage in mentorship and education of authors to help them produce work to best effect
9
Mediate sound communication between the comments of reviewers and responses of authors
10
Ensure publication decisions are clearly communicated to all authors
11
Interact with authors to confirm undisputed changes in authorship and act on any institutional findings concerning authorship disputes
12
Clarify the peer-review processes to authors
13
Negotiate manuscript publication delays with authors
14
Deal with authors who appeal against rejection
15
Ensure authors are informed about journal and article information and/or funding
16
Ensure that requests from authors that an individual not review their submission are respected, if these are well-reasoned
17
Engage in critical evaluation of authors’ manuscripts and the peer-review process itself
18
Provide active encouragement for revisions of manuscripts
19
Demonstrate experience as a competent author, academic, researcher, or reviewer
20
Demonstrate proficiency in dealing with author misconduct and other issues related to publication ethics
21
Work with publishers to defend author rights and pursue offenders
22
Act on concerns about plagiarism, data fabrication, or an authorship issue and follow-up with authors and then institutions
23
Request full disclosure of potential conflicts of interest by the authors
24
Support authors in dealing with breaches of copyright and plagiarism issues
25
Request appropriate documentation from authors when they submit manuscripts
II. Discussion Dealing with peer reviewers
26
Develop, facilitate, and monitor the peer review process
27
Knowledge of different types of peer review
28
Encourage and demonstrate awareness of new findings on peer review and publishing and how these influence their journal’s
processes
29
Review revised manuscripts
30
Provide guidance to peer reviewers
31
Ensure thorough statistical review
32
Ensure that peer review panels for individual papers are not biased
33
Evaluate and provide feedback to the reviewers on review quality
34
Ensure manuscript content is matched with the expertise of particular reviewers
35
Monitor and ensure the fairness, timeliness, thoroughness, and civility in the processing of manuscripts and in responding to
queries from authors and reviewers
36
Demonstrate knowledge of the workings of the peer review process
37
Train peer reviewers
38
Ensure reviewer comments are shared with all peer reviewers
39
Synthesize reviews and make ultimate editorial decisions in light of peer reviewers’ comments
40
Evaluate manuscripts in light of reviewers’ critiques and various selection criteria Page 12 of 17 Galipeau et al. BMC Medicine (2016) 14:16 Table 3 Competency-related statements a (Continued)
41
Demonstrate the ability to distinguish between objective peer reviewed research and reviews from opinion and the journal c
from advertising and other promotional content
42
Ensure reviewers who consistently produce discourteous, poor quality, or late reviews are removed from the journal’s pool of
reviewers
43
Ensure a decision is made on a manuscript when reviewers fail to submit a timely review
44
Ensure a very high standard of the referees, don’t accept sloppy reports from anyone
45
Demonstrate publication and reviewing skills and experience
46
Ensure that reviewers keep manuscripts, associated material, and the information they contain strictly confidential
47
Demonstrate sound judgment in the acceptance of research articles, editorials, and reviews that touch on current issues
III. Discussion Journal publishing
48
Demonstrate knowledge of marketing and advertising policies, including ethical issues
49
Demonstrate knowledge of the article embargo process
50
Demonstrate knowledge of indexing services
51
Demonstrate knowledge of reprint processes
52
Demonstrate knowledge of the specifications of the journal
53
Demonstrate knowledge of the goals of the journal
54
Demonstrate knowledge of formatting of layout for journal issues
55
Ensure the content of manuscripts submitted for publication is checked for accuracy
56
Demonstrate knowledge of the different parts, purposes, and characteristics of different types of journals
57
Demonstrate understanding of the editorial office and operations
58
Ensure that selected/published research is correct
59
Demonstrate knowledge about legal issues relating to the position of scientific editor
60
Be aware of how design can be used to improve the readability of a document
61
Demonstrate understanding of one’s responsibilities and rights as a journal editor
62
Demonstrate knowledge of the roles and responsibilities of the editorial staff
63
Identify and address issues related to data protection and confidentiality
64
Demonstrate knowledge of journal metrics and research impact
65
Demonstrate knowledge of online publishing and products
66
Demonstrate knowledge of the parts, purposes, and characteristics of audio and video clips
67
Demonstrate awareness of intellectual property issues and work with publisher to handle potential breaches
68
Demonstrate knowledge of technical-economical aspects of medical journal production
69
Explore and embrace innovative technologies
70
Maintain close contact with the latest trends in electronic media (e.g. tablets)
71
Engage in multimedia publishing practices
72
Act as a gatekeeper and guarantor of publications, checking both the quality and scope of research published in the journal
IV. Discussion Journal promotion
73
Maintain knowledge of important developments and trends in one’s own field
74
Demonstrate knowledge of history of journals and scientific publications
75
Demonstrate knowledge of national and regional variations between journals
76
Demonstrate knowledge of political and geopolitical issues
77
Demonstrate familiarity with associations and their educational resources
78
Stay on top of updates in one’s field
79
Demonstrate knowledge of, and work to maintain and improve the journal’s policies, vision, scope, content, processes, and g
80
Ensure decisions are based on the validity of the work and its importance to the journal’s readers Table 3 Competency-related statements a (Continued)
41
Demonstrate the ability to distinguish between objective peer reviewed research and
from advertising and other promotional content
42
Ensure reviewers who consistently produce discourteous, poor quality, or late reviews
reviewers
43
Ensure a decision is made on a manuscript when reviewers fail to submit a timely revi
44
Ensure a very high standard of the referees, don’t accept sloppy reports from anyone
45
Demonstrate publication and reviewing skills and experience
46
Ensure that reviewers keep manuscripts, associated material, and the information they
47
Demonstrate sound judgment in the acceptance of research articles, editorials, and rev
III. Discussion Journal publishing
48
Demonstrate knowledge of marketing and advertising policies, including ethical issues
49
Demonstrate knowledge of the article embargo process
50
Demonstrate knowledge of indexing services
51
Demonstrate knowledge of reprint processes
52
Demonstrate knowledge of the specifications of the journal
53
Demonstrate knowledge of the goals of the journal
54
Demonstrate knowledge of formatting of layout for journal issues
55
Ensure the content of manuscripts submitted for publication is checked for accuracy
56
Demonstrate knowledge of the different parts, purposes, and characteristics of differen
57
Demonstrate understanding of the editorial office and operations
58
Ensure that selected/published research is correct
59
Demonstrate knowledge about legal issues relating to the position of scientific editor
60
Be aware of how design can be used to improve the readability of a document
61
Demonstrate understanding of one’s responsibilities and rights as a journal editor
62
Demonstrate knowledge of the roles and responsibilities of the editorial staff
63
Identify and address issues related to data protection and confidentiality
64
Demonstrate knowledge of journal metrics and research impact
65
Demonstrate knowledge of online publishing and products
66
Demonstrate knowledge of the parts, purposes, and characteristics of audio and video
67
Demonstrate awareness of intellectual property issues and work with publisher to han
68
Demonstrate knowledge of technical-economical aspects of medical journal productio
69
Explore and embrace innovative technologies
70
Maintain close contact with the latest trends in electronic media (e.g. tablets)
71
Engage in multimedia publishing practices
72
Act as a gatekeeper and guarantor of publications, checking both the quality and scop
IV. Journal promotion
73
Maintain knowledge of important developments and trends in one’s own field
74
Demonstrate knowledge of history of journals and scientific publications
75
Demonstrate knowledge of national and regional variations between journals
76
Demonstrate knowledge of political and geopolitical issues
77
Demonstrate familiarity with associations and their educational resources
78
Stay on top of updates in one’s field
79
Demonstrate knowledge of, and work to maintain and improve the journal’s policies, v
80
Ensure decisions are based on the validity of the work and its importance to the journ
81
Ensure controversial topics (political, ethical) are dealt with
82
Stimulate others to write articles and editorials Page 13 of 17 Galipeau et al. Discussion BMC Medicine (2016) 14:16 Table 3 Competency-related statements a (Continued) Table 3 Competency-related statements a (Continued)
83
Engage in the promotion of scholarly research and best practices in conducting and reporting it
84
Entice leading researchers to submit to the journal
85
Serve as ambassador for the journal in establishing its visibility and image
86
Motivate physicians to read, ponder, and implement the information provided
87
Seek feedback/opinions on the journal
88
Enhance public understanding of science
89
Demonstrate understanding of who one’s constituency is
90
Demonstrate a responsibility to the scientific community
91
Hold paramount the interests of the particular journal’s readers
92
Engage in communication with the public
93
Engage with existing and new scientific communities
V. Editing
94
Demonstrate knowledge of policies for submission of manuscripts
95
Demonstrate broad and detailed knowledge of the skills needed to refine a piece of scientific work and shepherd it through to
publication
96
Demonstrate knowledge of typography
97
Demonstrate knowledge of and experience with online editing
98
Demonstrate knowledge of the fundamentals of editing various types of science copy
99
Enforce ICMJE authorship guidelines
100
Ensure logic and consistency of manuscripts
101
Demonstrate the ability to assess the quality of papers
102
Ensure papers selected are clinically relevant
103
Ensure papers selected have a clear story-line
104
Demonstrate the ability to select material for its merit, interest to readers, and originality alone
105
Ensure papers selected are suitable to the journal
106
Ensure papers selected for review are meaningful
107
Ensure manuscripts are triaged judiciously (for journals that use such a process)
108
Demonstrate the ability to form preliminary opinions on a submitted manuscript’s relevance
109
Demonstrate the ability to make fast, good decisions about papers
110
Demonstrate the ability to make difficult decisions
111
Demonstrate the ability to exercise excellent judgment
112
Handle manuscripts in the areas of one’s expertise and assist in finding persons qualified to handle papers in those areas outside
one’s expertise
113
Engage in and maintain interactions and good relations with media
114
Select, curate, and comment on articles for publication
115
Ensure alterations recommended based on peer reviewers’ comments can be justified
116
Demonstrate experience or familiarity with manuscript tracking software (e.g. Discussion ScholarOne, AllenTrack, PeerTrack, BenchPress)
117
Demonstrate aptitude in using technology (computers, Internet, e-mail, Manuscript Submission Systems) to perform his or her
editorial duties)
118
Possess a degree in medical editing or be trained as a journal editor
119
Demonstrate the ability to write editorials
120
Demonstrate working knowledge of the language in which the journal is published
121
Demonstrate skills in speed reading, skim reading, and critical reading
122
Demonstrate an aptitude for reading widely, deeply, and continually
123
Demonstrate experience and/or training in medical journal writing
124
Demonstrate understanding of the parts, purposes, and characteristics of tables, charts, graphs, and images Page 14 of 17 Galipeau et al. BMC Medicine (2016) 14:16 Table 3 Competency-related statements a (Continued) Table 3 Competency related statements
(Continued)
125
Demonstrate familiarity with scientific units, numerals, symbols, and nomenclature
126
Demonstrate familiarity with the presentation of data and data presentation problems
127
Demonstrate familiarity with the basic concepts of statistics
128
Demonstrate knowledge of literature reviews
129
Demonstrate familiarity with the principles of scientific investigation
130
Demonstrate familiarity with types of evidence
131
Demonstrate familiarity with scientific referencing
132
Demonstrate familiarity with clinical research design
133
Demonstrate knowledge of types of manuscripts
134
Be working towards a deeper understanding of multiple research epistemologies
135
Assist non-native speakers in dealing with language issues
VI. Ethics and integrity
136
Demonstrate knowledge of issues around registration (i.e. Discussion trials, systematic reviews, protocols)
137
Demonstrate knowledge of and adherence to the principles of editorial independence
138
Demonstrate expertise in ensuring the ethical integrity of publications
139
Identify and address allegations of fraud or plagiarism
140
Demonstrate understanding of privacy, confidentiality, and anonymity issues
141
Identify and address issues related to conflicts of interest
142
Identify and address issues related to industry-sponsored research
143
Separate decision-making from commercial considerations
144
Demonstrate knowledge of the ethical approval process for research involving humans and animals
145
Ensure the respect and privacy of patients described in clinical studies
146
Safeguard the rights of study participants and animals
147
Demonstrate understanding of issues related to dual-use research (research with multiple purposes or applications)
148
Identify and apply appropriate reporting guidelines
149
Guarantee access to, and long term preservation of, the published information
150
Encourage debate on important topics related to the journal
151
Promote higher standards of medical journalism
152
Identify and work to avoid publication bias
153
Demonstrate knowledge of COPE resources for editors, authors, and peer reviewers
154
Demonstrate knowledge of copyright issues
155
Demonstrate knowledge regarding problems with multiple publications (e.g. salami, duplicate, redundant)
156
Identify and address incongruities and bias in manuscripts
157
Recommend publication of papers that meet standards of scientific rigor
158
Identify and address issues related to image manipulation
VII. Qualities and characteristics of editors
159
Demonstrate experience and broad knowledge of the field(s) covered by the journal and of the people working in thos
160
Demonstrate the ability to work in a team
161
Delegate/divide the workload
162
Communicate clearly with others
163
Effectively summarize manuscripts in fields outside your experience
164
Possess a Doctorate or Master’s Degree in related content area
165
Demonstrate an academic education that includes science training or experience in a research environment
166
Demonstrate experience and aptitude in conflict resolution Page 15 of 17 Galipeau et al. BMC Medicine (2016) 14:16 research team are editors. Discussion Finally, due to the broad in-
clusion criteria and the decision to preserve the wording
used by authors to describe potential competencies as
much as possible, it is lik
tually be competencies
tasks, behaviors, and kn
Table 3 Competency-related statements a (Continued)
167
Demonstrate excellent organizational, project, and time management skills, including the ability
pressure
168
Maintain part time professional practice
169
Maintain membership in learned societies and editing-related associations
170
Be recognized as a distinguished scholar in one’s field
171
Maintain an active research portfolio/is employed in a research-oriented university or institute
172
Demonstrate past experience on an editorial board
173
Demonstrates competence as a practitioner in their field
174
Demonstrate strong interpersonal skills
175
Demonstrate good analytical skills
176
Demonstrate effective critical appraisal skills
177
Demonstrate the ability to achieve consensus among opinionated scientists
178
Demonstrate leadership skills
179
Demonstrate political and public relations sense
180
Demonstrate self-motivation
181
Demonstrate enthusiasm
182
Demonstrate tolerance and persistence
183
Demonstrate boldness
184
Demonstrate independent thinking
185
Maintain visibility and respect among peers and in the larger scientific community
186
Maintain rigid criteria
187
Demonstrate the ability to perpetuate or challenge master narratives
188
Exercise convictions with a positive attitude
189
Demonstrate a willingness to reconsider decisions
190
Demonstrate practicality
191
Demonstrate decisiveness
192
Demonstrate personal interest in medical ‘journalology’ or ‘editology’
193
Demonstrate an enjoyment of learning and a questioning mind
194
Demonstrate the desire to advance their field of study
195
Have access to a good academic network or have the potential to grow one
196
Demonstrate patience when dealing with authors and reviewers
197
Demonstrate knowledge of processes related to the editorial board
198
Respond promptly to complaints
199
Act with integrity and accountability
200
Engage with social media to reach out beyond the usual specialist audiences
201
Demonstrate knowledge of the parts, purposes, and characteristics of manuscripts
202
Demonstrate knowledge of open access models
Other potential competencies
203
Statements related specifically to the Editor-in-Chief position d
a The order in which the statements are presented is purely for purposes of organization and is not intended to conv
c Corresponds to the Item(s) columns in Tables 1 and 2
b Number of extracted competency-related statements across all data sources in the scoping review
d This item contains all statements pertaining only to potential competencies of Editors-in-Chief. Discussion Despite these poten
to this scoping review, we nevertheless wanted to account for them in our results as they did fit our inclusion criteria 169
Maintain membership in learned societies and editing-related associations
5
170
Be recognized as a distinguished scholar in one’s field
8
171
Maintain an active research portfolio/is employed in a research-oriented university or institute
5
172
Demonstrate past experience on an editorial board
1
173
Demonstrates competence as a practitioner in their field
2
174
Demonstrate strong interpersonal skills
5
175
Demonstrate good analytical skills
6
176
Demonstrate effective critical appraisal skills
4
177
Demonstrate the ability to achieve consensus among opinionated scientists
1
178
Demonstrate leadership skills
20
179
Demonstrate political and public relations sense
3
180
Demonstrate self-motivation
5
181
Demonstrate enthusiasm
4
182
Demonstrate tolerance and persistence
5
183
Demonstrate boldness
6
184
Demonstrate independent thinking
4
185
Maintain visibility and respect among peers and in the larger scientific community
4
186
Maintain rigid criteria
1
187
Demonstrate the ability to perpetuate or challenge master narratives
1
188
Exercise convictions with a positive attitude
1
189
Demonstrate a willingness to reconsider decisions
8
190
Demonstrate practicality
3
191
Demonstrate decisiveness
4
192
Demonstrate personal interest in medical ‘journalology’ or ‘editology’
3
193
Demonstrate an enjoyment of learning and a questioning mind
3
194
Demonstrate the desire to advance their field of study
15
195
Have access to a good academic network or have the potential to grow one
2
196
Demonstrate patience when dealing with authors and reviewers
4
197
Demonstrate knowledge of processes related to the editorial board
10
198
Respond promptly to complaints
7
199
Act with integrity and accountability
39
200
Engage with social media to reach out beyond the usual specialist audiences
22
201
Demonstrate knowledge of the parts, purposes, and characteristics of manuscripts
3
202
Demonstrate knowledge of open access models
2
Other potential competencies
203
Statements related specifically to the Editor-in-Chief position d
68
a The order in which the statements are presented is purely for purposes of organization and is not intended to convey any type of ranking
c Corresponds to the Item(s) columns in Tables 1 and 2
b Number of extracted competency-related statements across all data sources in the scoping review
d This item contains all statements pertaining only to potential competencies of Editors-in-Chief. Authors’ contributions
DM
i
d
f h
d DM conceived of the study, participated in its design and coordination, and
helped to draft the manuscript. JG participated in the design of the study,
coordinated the research, and was involved in title/abstract and full-text
screening, data extraction and verification, and writing of the manuscript. LS was involved in the design of the study, as well as participating in
title/abstract and full-text screening, and data extraction and verification. SS was involved in the design of the study. KDC was involved in data
extraction and verification. EW, SB, MC, HM, JD, MW, PG, VB, PT, and PB
all provided content expertise. All authors were involved in the analysis
and interpretation of data, revision of drafts of the manuscript, and reading and
approving the final manuscript. Abbreviations
COPE: Committee on Publication Ethics; WAME: World Association of
Medical Editors. COPE: Committee on Publication Ethics; WAME: World Association of
Medical Editors. Conclusion To our knowledge, this scoping review is the first at-
tempt to systematically identify possible competencies
of editors. On its own, the review will serve to in-
form readers on the extent and nature of existing lit-
erature in this area, as well as the breadth of skills,
abilities, tasks, knowledge, and training that may be
necessary to fulfill the position of scientific editor at
a biomedical journal. More importantly, the review is
part of a larger program to develop a minimum set
of core competencies for scientific editors of biomed-
ical journals. Acknowledgments
W th
k C
h We thank Cochrane, the Committee on Publication Ethics (COPE), the World
Association of Medical Editors (WAME), the European Association of Science
Editors (EASE), and the Council of Science Editors (CSE) for their partnership
in this scoping review. We thank Becky Skidmore for developing and
running the search strategy and Raymond Daniel for acquiring, uploading,
and managing records in DSR. We thank Jennifer Tetzlaff (JT) and Mona
Ghannad (MG) for their involvement in screening titles and abstracts. This
project is funded by Cochrane, Elsevier, and BioMed Central. The purpose of the 203 competency-related state-
ments generated here is to be the central tool used for a
Delphi exercise involving scientific editors of biomedical
journals from around the world. Subsequently, these
statements will serve to stimulate discussion at a con-
sensus meeting in which the goal will be for relevant
stakeholders to agree upon a minimum set of core
competencies for scientific editors of biomedical jour-
nals. This evidence-based approach will ultimately lay
the
groundwork
for
the
development
of
specific
competency-based training and certification for scien-
tific editors of biomedical journals [5]. The develop-
ment of core competencies and subsequent training
represent critical steps toward ensuring that the pub-
lication of biomedical research truly represents a hall-
mark of quality and trustworthiness, both within and
beyond the research community. Competing interests DM is supported by a University of Ottawa Research Chair. EW is a self-
employed consultant and provides training for editors, authors, and
reviewers. She has received payment for such training from a variety of
publishers, academic societies, universities, and commercial companies. She was also involved in developing the International Standards for
Editors and many of the COPE guidelines. SB, MC, and HM are part of the
Cochrane Central Editorial Unit. JD leads the Cochrane Collaboration’s test
evaluation activities. PG is a Coordinating Editor for the Cochrane Infectious
Diseases Group. MW participated in the creation and dissemination of a survey
to WAME members on medical journal editor professionalism in June
2015 and in the development of a series of sessions on medical journal
editor professionalism for the WAME International Conference for Medical
Journal Editors in New Delhi held in October 2015. JG, SS, LS, KDC, VB, PT,
and PB all have no competing interests to declare. Another limitation is that for feasibility purposes we
only considered English and French articles, which raises
the possibility that relevant information published in
another language was missed. Similarly, the databases
searched may not have included some journals from out-
side fiscally resourced countries. Discussion Despite these potential competencies not being directly relevant
to this scoping review, we nevertheless wanted to account for them in our results as they did fit our inclusion criteria 68 much as possible, it is likely that some items may not ac-
tually be competencies per se, but may instead describe
tasks, behaviors, and knowledge related to competencies. research team are editors. Finally, due to the broad in-
clusion criteria and the decision to preserve the wording
used by authors to describe potential competencies as much as possible, it is likely that some items may not ac-
tually be competencies per se, but may instead describe
tasks, behaviors, and knowledge related to competencies. Page 16 of 17 Galipeau et al. BMC Medicine (2016) 14:16 Additional files However, these items are still useful in describing im-
portant aspects of editors’ work and will therefore con-
tribute valuable information for the development of core
competencies. Additional file 1: Search strategies. (DOCX 23 kb)
Additional file 2: Non-research-based publications. (DOCX 49 kb) With the large number of competency statements and
our desire to create a manageable list for use down-
stream in our program of research, our efforts to remove
redundant and overlapping items in order to streamline
the list may also have led to the elimination of some nu-
ances between items that were subtly different from one
another. While we implemented measures to ensure
consistency in our methods (i.e. piloted forms, duplica-
tion of the classification exercise), ultimately there is a
degree of interpretation and selectivity embedded in this
process. Thus, our list of possible competencies may not
include all of the competencies of biomedical editors. As
noted above, the next phases of this project are designed
to elicit any missing items. Received: 2 December 2015 Accepted: 20 January 2016 References 1. Groves T. Enhancing the quality and transparency of health research. BMJ. 2008;337:a718. 2. Smith R. Medical journals and the mass media: moving from love and hate
to love. J R Soc Med. 2006;99(7):347–52. 3. Glasziou P, Altman DG, Bossuyt P, Boutron I, Clarke M, Julious S, et al. Reducing waste from incomplete or unusable reports of biomedical
research. Lancet. 2014;383(9913):267–76. 4. Kleinert S, Horton R. How should medical science change? Lancet. 2014;383:197–8. 4. Kleinert S, Horton R. How should medical science change? Lancet. 2014;383:197–8. 5. Moher D, Altman DG. Four proposals to help improve the medical research
literature. PLoS Med. 2015;12(9):e1001864. 6. World Association of Medical Editors. Policies and Resources. http://www. wame.org/policies-and-resources. Accessed 4 November 2014. 6. World Association of Medical Editors. Policies and Resources. http://www. wame.org/policies-and-resources. Accessed 4 November 2014. wame.org/policies-and-resources. Accessed 4 November 2014 7. Committee on Publication Ethics. http://publicationethics.org/. Accessed
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4 November 2014. 8. Journalology Blog. http://journalology.blogspot.ca/. Accessed 4 Nove 9. PSP Consulting. Editing medical journals - short course. http://www. pspconsulting.org/medical-short.shtml. Accessed 4 November 2014. 10. Council of Science Editors. Short Courses. http://www.resourcenter.net/Scripts/
4Disapi07.dll/4DCGI/events/2015/516-ShortCourses.html?Action=Conference_
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methodology. Implement Sci. 2010;5(1):1–9. 14. Levac D, Colquhoun H, O’Brien KK. Scoping
methodology. Implement Sci. 2010;5(1):1–9. 15. Sampson M, McGowan J, Cobo E, Grimshaw J, Moher D, Lefebvre C. An
evidence-based practice guideline for the peer review of electronic search
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evidence-based practice guideline for the peer review of electronic search
strategies. J Clin Epidemiol. 2009;62(9):944–52. 16. Peer Review Congress. http://www.peerreviewcongress.org/index.html. Accessed 8 November 2014. 16. Peer Review Congress. http://www.peerreviewcongress.org/index.html. Accessed 8 November 2014. 17. Galipeau J, Moher D, Campbell C, Hendry P, Cameron DW, Palepu A, et al. Author details
1 l
l
d 1Clinical Epidemiology Program, Ottawa Hospital Research Institute, Ottawa,
Canada. 2School of Medicine, Griffith University, Queensland, Australia. 3American Academy of Neurology, St. Paul, Minnesota, USA. 4Department of
Health Sciences, University of York, York, UK. 5School of Public Health and
Preventive Medicine, Monash University, Melbourne, Australia. 6Institute of
Applied Health Research, College of Medical and Dental Sciences, University
of Birmingham, Birmingham, UK. 7Liverpool School of Tropical Medicine,
Liverpool, UK. 8Cochrane Editorial Unit, London, UK. 9Department of
Medicine, University of Toronto, Toronto, Canada. 10School of Epidemiology,
Public Health and Preventive Medicine, Faculty of Medicine, University of
Ottawa, Ottawa, Canada. 11Sideview, Princes Risborough, UK. 12World
Association of Medical Editors, Chicago, USA. 13Council of Science Editors,
Colorado, USA. 14Cochrane Central Executive, London, UK. 15Department of
Psychology, University of Stirling, Stirling, UK. 16Cochrane Learning and
Support Department, London, UK. 17Department of Medicine Faculty of
Medicine, University of Ottawa, Ottawa, Canada. Page 17 of 17 Galipeau et al. BMC Medicine (2016) 14:16 References A
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and preventing scientific misconduct: strengths, weaknesses, opportunities,
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and preventing scientific misconduct: strengths, weaknesses, opportunities,
and threats. Med Law. 2007;26(3):545. Galipeau et al. BMC Medicine (2016) 14:16 Submit your next manuscript to BioMed Central
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https://openalex.org/W2094150788
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https://europepmc.org/articles/pmc3534526?pdf=render
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English
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Hyperemesis gravidarum in the Medical Birth Registry of Norway – a validity study
|
BMC pregnancy and childbirth
| 2,012
|
cc-by
| 4,610
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* Correspondence: ase.vigdis.vikanes@fhi.no
1Division of Epidemiology, Norwegian Institute of Public Health, Oslo,
Norway
2Department of Obstetrics and Gynaecology and Medical Faculty Division,
Akershus University Hospital, Lørenskog, Norway
Full list of author information is available at the end of the article Abstract Background: Valid registration of medical information is essential for the quality of registry-based research. Hyperemesis gravidarum (HG) is characterized by severe nausea and vomiting, weight loss and electrolyte
imbalance starting before 22nd gestational week. Given the fact that HG is a generally understudied disease
which might have short- and long- term health consequences for mother and child, it is of importance to know
whether potential misclassification bias influences the results of future studies. We therefore assessed the validity
of the HG-registration in the in Medical Birth Registry of Norway (MBRN) using hospital records. Methods: The sample comprised all women registered in MBRN with HG and who delivered at Ullevål and
Akershus hospitals in 1.1.-31.3.1970, 1.4.-30.6.1986, 1.7.-30.9.1997 and 1.10.-31.12.2001. A random sample of 10
women per HG case, without HG according to MBRN, but who delivered during the same time periods at the
same hospitals was also collected. The final sample included 551 women. Sensitivity, specificity, positive and
negative predictive values (PPV and NPV) were estimated using strict and less strict diagnostic criteria of HG,
indicating severe and mild HG, respectively. Hospital journals were used as gold standard. Results: Using less strict diagnostic criteria of HG, sensitivity, specificity, PPV and NPV were 83.9% (95% CI: 67.4-92.9)
96.0% (95% CI: 93.9-97.3), 55.3% (95% CI: 41.2-68.6) and 99.0% (95% CI: 97.7-99.6), respectively. For strict diagnostic
criteria, being hospitalised due to HG the corresponding values were 64% (95% CI: 38.8-87.2), 92% (95% CI:
90.2-94.6), 18.6% (95% CI: 10.2-31.9) and 99.0% (95% CI: 97.7-99.6). Conclusions: The results from our study are comparable to previous research on disease registration in MBRN,
and show that MBRN can be considered valid for mild HG but not for severe HG. Keywords: Hyperemesis gravidarum, Validity study, Medical Birth Registry of Norway Vikanes et al. BMC Pregnancy and Childbirth 2012, 12:115
http://www.biomedcentral.com/1471-2393/12/115 Open Access Hyperemesis gravidarum in the Medical Birth
Registry of Norway – a validity study Åse Vikanes1,2*, Per Magnus1, Siri Vangen1,3, Sølvi Lomsdal2,4 and Andrej M Grjibovski1,5 © 2012 Vikanes 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. © 2012 Vikanes 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. Vikanes et al. BMC Pregnancy and Childbirth 2012, 12:115
http://www.biomedcentral.com/1471-2393/12/115 Page 2 of 6 Page 2 of 6 registration among 25.761 women in MBRN was evaluated
using Patient Administrative Systems finding a sensitivity
of 91.8% and specificity of 91.8% [7]. Another study explor-
ing the validity of unexplained foetal deaths among 10.857
singleton pregnancies in MBRN using hospital records and
results from autopsies, reported a sensitivity of 78% and
specificity of 88% [8]. The sample also included a random selection of women
not registered with HG in MBRN, but who delivered
during the same time periods at the same hospitals; ten
women were selected per HG case. registration among 25.761 women in MBRN was evaluated
using Patient Administrative Systems finding a sensitivity
of 91.8% and specificity of 91.8% [7]. Another study explor-
ing the validity of unexplained foetal deaths among 10.857
singleton pregnancies in MBRN using hospital records and
results from autopsies, reported a sensitivity of 78% and
specificity of 88% [8]. Altogether 599 women delivering in Ullevål and
Akershus hospitals during the four time periods were
selected for the study. Among them, 53 women were
registered with HG in MBRN. Informed consent in
accordance with guidelines of the Ministry for Health
and Care Services was obtained from all women included
in the study sample. Altogether, 19 women did not give
their consent and were excluded. Furthermore, 29 hos-
pital records were missing resulting in exclusion of these
women. Our final study sample therefore comprised
551women among whom 48 women were registered with
HG in MBRN and 503 women were not (Figure 1). Hyperemesis gravidarum (HG) is characterised by
severe nausea and vomiting starting before the 22nd
gestational week, often leading to weight loss and nutri-
tional deficiencies [9,10]. The validity of the HG registra-
tion in MBRN has not yet been investigated, although
previous studies using the data from MBRN have found
the prevalence of HG to be similar to the prevalence
observed in neighbouring countries [11-13]. Whereas up
to 90% of all pregnant women report nausea or nausea
and vomiting (NVP), HG affects 0.3-3.2% [9,13]. Earlier
research has been influenced by the fact that HG and
the more common NVP have been studied as one and
the same condition; but so far we do not know if or how
these two conditions are related [14-18]. The hospital record was considered as gold standard
and checked manually. Vikanes et al. BMC Pregnancy and Childbirth 2012, 12:115
http://www.biomedcentral.com/1471-2393/12/115 In order to validate the HG
registration in MBRN we investigated all information
within the women’s hospital records including patient
journals, laboratory sheets with test results from blood
and urine samples and antenatal cards. An antenatal
card is a standardised form all pregnant women in
Norway receive at their first routine examination early
during the first trimester in pregnancy. The antenatal
card is later used as medical record for midwifes and
medical doctors during the entire pregnancy. After birth
the antenatal card is filed within the hospital record and
provides information from all examinations in preg-
nancy, such as the mother’s height, weight, blood pres-
sure, urine samples, the need of sick leave etc. The
antenatal card also provides information on any specific
symptom or complaint the pregnant woman might have,
such as HG. Additionally HG is a generally understudied disease,
which might have short and long term health conse-
quences for mother and child, such as increased risk of
rheumatic disease among mothers and increased risk of
testicular cancer and leukaemia among children [19-22]. Although MBRN in an international context provides an
extremely large dataset, it is important to know whether
potential systematic errors influence the results of previ-
ous and future studies using MBRN. Our aim was there-
fore to investigate the validity of the HG registration in
MBRN using hospital records as gold standard. Background and after pregnancy, although incomplete ascertainment
has been and still is a concern [4]. During the late 1950s and early 1960s Thalidomide was
prescribed to women suffering from nausea and vomit-
ing in early pregnancy, resulting in more than 10 000
limb deformities globally [1,2]. The Medical Birth Regis-
try of Norway (MBRN) was established in 1967 reflect-
ing the need for epidemiological surveillance of birth
defects [3]. Since then, MBRN has become a unique
source of perinatal data comprising several generations. Additionally, MBRN contains valuable information on
maternal diseases before pregnancy, during pregnancy Valid registration of medical information is essential
for the quality of registry-based research. Previous stu-
dies validating disease registration of in MBRN have
shown diverging results, depending on the condition
[4-8]. Engeland evaluated the validity of diabetes type 1
among 1.929 million women registered in MBRN using the
Norwegian Prescription Database and found a sensitivity of
90% and specificity of 100% [5]. For any type of diabetes
the corresponding values were 72% and 99%, although for
asthma the sensitivity was 51% and specificity 98%. Smaller
studies using hospital records as gold standard have
reported the sensitivity for maternal rheumatic disease in
MBRN to be 88% and for myasthenia gravis 99% [4,6]. Spe-
cificities were not reported. The obstetric sphincter tears Vikanes et al. BMC Pregnancy and Childbirth 2012, 12:115
http://www.biomedcentral.com/1471-2393/12/115 Methods In order to be registered with HG based on strict diag-
nostic criteria, indicating severe HG, the women had to
have been admitted to hospital due to HG. In addition
two out of the three following symptoms had to be
reported in the hospital record; weight loss, dehydration
or ketonuria. If the woman had been admitted to hos-
pital because of HG during the actual pregnancy, her
patient journal or laboratory sheets provided data on
symptoms such as nausea and vomiting, weight loss,
dehydration and ketonuria. Notification to MBRN is compulsory and is provided by
midwives and physicians attending the birth using a
standardised form [3]. From 1967 to 1998 pregnancy
outcomes were notified from the 16th gestational week,
after 1998 from the 12th gestational week. Maternal dis-
eases before and during pregnancy are also notified. Women with HG in MBRN were registered according to
International Classification of Diagnoses (ICD) [10]. From
1967 to 1998 HG was registered by the ICD-8 codes 638.0
(hyperemesis gravidarum with neuritis), 638.9 (hypere-
mesis gravidarum without mention of neuritis), and 784. 1
(nausea and vomiting) [11]. From 1999 and onwards HG
was registered by the ICD-10 codes O21.0 (mild hyperem-
esis gravidarum), O21.1 (hyperemesis gravidarum with
metabolic disturbances), and O21.9 (vomiting in preg-
nancy, unspecified) [13]. In order to be registered with HG based on less strict
diagnostic criteria, indicating mild HG, the women did
not have to have been admitted to hospital due to HG. If she was not admitted to hospital because of HG, her
antenatal card, which is filed within the hospital record,
contains the necessary health information. Since most
women experience some degree of nausea and vomiting
during pregnancy (NVP) we considered that the nausea
and vomiting worth describing on her antenatal card or
was mentioned in her patient journal would be more The sample consisted of all women registered with
HG in MBRN who delivered their babies at Ullevåla
and Akershusb hospitals during four time periods: 1.1-
31.3.1970, 1.4-30.6.1986, 1.7-30.9.1997 and 1.10-31.12.2001. Vikanes et al. Methods BMC Pregnancy and Childbirth 2012, 12:115
http://www.biomedcentral.com/1471-2393/12/115 Page 3 of 6 Page 3 of 6 53 women were registered in the Medical Birth Registry of Norway (MBRN) with
hyperemesis gravidarum (HG)
546 women not registered with HG in MBRN - randomly selected
(N=599)
19 women - no consent
29 women no hospital records
(N=48)
Final sample
48 women registered with HG in MBRN
503 not registered with HG in MBRN
(N=551)
Source population
Women delivering at Ullevål and Akershus University hospitals in
selected time periods from 1970 to 2001
Figure 1 Sampling frame. Source population Women delivering at Ullevål and Akershus University hospitals in
selected time periods from 1970 to 2001 53 women were registered in the Medical Birth Registry of Norway (MBRN) with
hyperemesis gravidarum (HG)
546 women not registered with HG in MBRN - randomly selected
(N=599) Final sample The results of validating the HG registration in MBRN
using hospital records are shown in Table 1 and Table 2. Table 1 reflects the
validity of the HG diagnosis
in MBRN by the use of strict diagnostic criteria indicat-
ing severe HG. Among the 48 women registered with
HG in MBRN, 9 women were hospitalised due to severe
HG. According to all hospital records, 14 women were
hospitalised due to severe HG, indicating that 5 were
not registered with HG in MBRN. The use of strict diag-
nostic criteria resulted in a sensitivity of 64.3% (95% CI:
38.8-83.7), specificity of 92.7% (90.2-94.6), PPV of 18.6%
(10.2-31.9) and NPV of 99% (97.7-99.6). pronounced than normal, and therefore should be regis-
tered as mild hyperemesis [14]. One hospital record was
missing when less strict diagnostic criteria were applied. Sensitivity, specificity, positive (PPV) and negative
(NPV) predictive values were calculated using hospital
records as gold standard. Wilson’s method was used to
calculate with 95% confidence intervals (CI) for the esti-
mates. All calculations were performed using Confidence
Intervals Analysis software. This study was approved by the Data Inspectorate,
the Ministry for Health and Care Services and the
Norwegian Scientific-Ethical Committees. Table 2 presents the corresponding results applying
less strict diagnostic criteria indicating mild HG. Among
the 48 women registered with HG in MBRN, 26 had the
disease according to the hospital records (Table 2). Methods The
use of less strict diagnostic criteria resulted in a sensitiv-
ity of 83.9% (95% CI: 67.4-92.9), specificity of 96.0%
(93.9-97.3), PPV of 55.3% (41.2-68.6) and NPV of 99.0%
(97.7-99.6). Discussion This is the first study to validate the registration of HG
in MBRN. When less strict diagnostic criteria, indicating
mild HG, were applied, the sensitivity was 83.9%, specifi-
city of 96.0%, PPV of 55.3% and NPV of 95.9%. These
results are comparable to previous research on disease
registration in MBRN, such as diabetes 1, showing that
HG is likewise valid for use in large epidemiologic stud-
ies when it comes to the mild form of the disease [4-6]. For strict diagnostic criteria, indicating severe HG, the
corresponding figures were 64.3%, 92.7%, 18.6% and
99.0%. The low PPV shows that MBRN is not valid for
severe HG. Thirty five percent of the women were ad-
mitted to hospital due to HG when less strict diagnostic
criteria were used, compared to 100% when strict criteria
were used. PPV reflects the probability of having HG in
accordance with the hospital record when registered
with HG in MBRN, and is depending on the prevalence
[13]. Since the registration of rare diseases is influenced
by false positive cases, the PPV is expected to be low
when the prevalence is low [23]. Table 2 Validity of HG registration in MBRN using
hospital records and less strict diagnostic criteria1
(N = 550)2
HG + in
hospital record
HG – in
hospital record
Total
HG + in MBRN
26
21
47
HG – in MBRN
5
498
503
Total
31
519
550
Sensitivity (26/31)
83.9%
95% CI: 67.4-92.9
Specificity (498/519)
96.0%
95% CI: 93.9-97.3
Positive predictive
value (26/47)
55.3%
95% CI: 41.2-68.6
Negative predictive
value (498/503)
99.0%
95% CI: 97.7-99.6
1 Less strict diagnostic criteria indicate mild HG. 2 One medical record went missing during the investigating period. Table 2 Validity of HG registration in MBRN using
hospital records and less strict diagnostic criteria1
(N = 550)2 In order to be able to differentiate between mild and
severe HG we therefore decided to distinguish between
strict and less strict diagnostic criteria, indicating severe
and mild HG. The use of the different diagnostic criteria
showed that the majority of women registered with HG
in MBRN suffered from milder forms and was not hos-
pitalised due to HG. Also the fact that the ICD 8 code
784.1 was included in our study as HG represents a
source of error. Results Sixty-nine percent of the hyperemesis patients were
registered by ICD 8 (1967–1998) and 31% by ICD 10
(1999–2006). Among the 48 women with HG in MBRN,
31 women were registered by the ICD 8 code 638.9, 2
women were registered by the ICD 8 code 784.1 and 15
women were registered by the ICD 10 code O21.9. Vikanes et al. BMC Pregnancy and Childbirth 2012, 12:115
http://www.biomedcentral.com/1471-2393/12/115 Vikanes et al. BMC Pregnancy and Childbirth 2012, 12:115
http://www.biomedcentral.com/1471-2393/12/115 Page 4 of 6 Table 1 Validity of HG registration in MBRN using
hospital records and strict diagnostic criteria1 (N = 551)
HG + in
hospital record
HG – in
hospital record
Total
HG + in MBRN
9
39
48
HG – in MBRN
5
498
503
Total
14
537
551
Sensitivity (9/14)
64.3%
95% CI: 38.8-83.7
Specificity (498/537)
92.7%
95% CI: 90.2-94.6
Positive predictive
value (9/48)
18.6%
95% CI: 10.2-31.9
Negative predictive
value (498/503)
99.0%
95% CI: 97.7-99.6
1 Strict diagnostic criteria indicate severe HG. Table 1 Validity of HG registration in MBRN using
hospital records and strict diagnostic criteria1 (N = 551) A major strength of this study is the sample represent-
ing all HG patients registered in MBRN among women
who delivered during four different time periods from
1970 to 2001 in two large hospitals; hospitals now serv-
ing 19% of all births in Norway (unpublished data from
MBRN). Although the two hospitals selected might not
be representative for all Norwegian hospitals, selection
bias is not very likely due to the national guidelines
for diagnosing and treating HG worked out by the
Norwegian Society for Gynecology and Obstetrics [24]. Furthermore, hospital records are commonly used as
“gold standard” when registry data are being validated
[4,6,8,25]. A limitation of our study might be the low
number of women included. 1 Strict diagnostic criteria indicate severe HG. Errors in information on HG in MBRN might have
occurred on three different levels; 1) by the midwife
or general practitioner filling in the antenatal card or
patient journal, 2) by the midwife or physician attending
the birth notifying MBRN, and 3) the registration at
MBRN. Even though diagnosing and treating HG in
Norway is carried out in accordance with national guide-
lines
worked
out
by
the
Norwegian
Society
for
Gynecology and Obstetrics, there is always a chance for
misdiagnosing due to inexact diagnostic criteria in clin-
ical practice [26]. Results For women who had not been hospita-
lized due to HG, the midwife or physician notifying
MBRN had to rely on the information written on the
woman’s antenatal card. Although the ICD 8 as well as
ICD 10 codes allow differentiating between mild and
severe HG, HG is not registered as such in MBRN. In
our study MBRN mainly registered HG as 638.9 in ICD
8 and only as O21.9 in ICD 10. Due to the exclusive use
of O21.9, describing unspecific vomiting in pregnancy,
Kari Klungsøyr who is responsible for their coding pro-
cedures in MBRN, was contacted. She confirmed that
there might be a potential for misclassification bias
during the first period after ICD 10 was introduced (per-
sonal communication). Exclusion of O21.9 from the ana-
lysis would have resulted in losing a third of the sample,
which is why we present our data without excluding
those coded as such. 2 One medical record went missing during the investigating period. 1 Less strict diagnostic criteria indicate mild HG.
2 Competing interests Competing interests
The authors declare that they have no competing interests. References 1. Franks ME, Macpherson GR, Figg WD: Thalidomide. Lancet 2004,
363:1802–1811. 1. Franks ME, Macpherson GR, Figg WD: Thalidomide. Lancet 2004,
363:1802–1811. 2. Mcbride WG: Thalidomide and congenital abnormalities. Lancet 1961,
278:1358. 2. Mcbride WG: Thalidomide and congenital abnormalities. Lancet 1961,
278:1358. 3. Irgens LM: The Medical Birth Registry of Norway. Epidemiological
research and surveillance throughout 30 years. Acta Obstet Gynecol
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research and surveillance throughout 30 years. Acta Obstet Gynecol
Scand 2000, 79:435–439. 4. Skomsvoll J, Ostensen M, Baste V, Irgens L: Validity of a rheumatic disease
diagnosis in the Medical Birth Registry of Norway. Acta Obstet Gynecol
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registration in pregnant women in the Medical Birth Registry of Norway. Acta Obstet Gynecol Scand 2009, 88:1083–1089. y
6. Hoff JM, Daltveit AK, Gilhus NE: Myasthenia gravis in pregnancy and birth:
identifying risk factors, optimising care. Eur J Neurol 2007, 14:38–43. 7. Baghestan E, Bordahl PE, Rasmussen SA, Sande AK, Lyslo I, Solvang I: A
validation of the diagnosis of obstetric sphincter tears in two Norwegian
databases, the Medical Birth Registry and the Patient Administration
System. Acta Obstet Gynecol Scand 2007, 86:205–209. 8. Rasmussen S, Albrechtsen S, Irgens LM, Dalaker K, Maartmann-Moe H,
Vlatkovic L, et al: Unexplained antepartum fetal death in Norway,
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AV AMG
d PM d
i AV, AMG and PM designed the study, whereas AV and SL collected the data. AV, PM, SV and AMG all contributed to drafting the manuscript; all involved
have approved the final version of the manuscript. Conclusions The results from our validity study show that MBRN
may be valid for mild HG, but not for severe HG. Fur-
thermore, the results are comparable to previous re-
search on disease registration in MBRN [4-6]. For future
studies on HG, MBRN is valid as database, although the
relatively large proportion of false positive cases might
influence the exposure-outcome associations in terms of
reducing associations closer to the null value. 13. Vikanes ÅV, Grjibovski A, Vangen S, Magnus P: Variations in prevalence of
hyperemesis gravidarum by country of birth: a study of 900,074 births in
Norway, 1967–2005. Scand J Public Health 2008, 36:135–142. 14. Verberg MF, Gillott DJ, Al Fardan N, Grudzinskas JG: Hyperemesis
gravidarum, a literature review. Hum Reprod Update 2005, 11:527–539. Author details
1 1Division of Epidemiology, Norwegian Institute of Public Health, Oslo,
Norway. 2Department of Obstetrics and Gynaecology and Medical Faculty
Division, Akershus University Hospital, Lørenskog, Norway. 3National Resource
Centre for Women’s Health, Department for Obstetrics and Gynecology, Oslo
University Hospital Rikshospitalet, Oslo, Norway. 4Department for Obstetrics
and Gynecology, Innlandet Hospital, Lillehammer, Norway. 5International
School of Public Health, Northern State Medical University, Arkhangelsk,
Russia. A Swedish study on HG’s effect on pregnancy out-
comes suggested that the exposure-outcome associations
were diluted due to the probability of registering milder
forms of HG since the HG diagnosis not being well
defined [27]. Our study shows that this “dilution” is the
case for MBRN since MBRN is valid for mild HG. When
it comes to differentiating between mild HG and the
more common NVP, our sample did not provide the
opportunity to investigate this. For future studies it will
be important to be able to differentiate between the
more common nausea and vomiting in pregnancy, mild
HG and severe HG necessitating hospitalisation. Severe
forms of HG in particular are found to be associated
with adverse pregnancy outcomes, such as preterm birth
and low birth weight [28-30]. Severe HG and excessive
vomiting in pregnancy has also been reported as risk fac-
tors for the development of childhood leukaemia and
testicular cancer as well as rheumatic disease among
mothers [20,22,31]. In utero exposure to HG has also
been linked to a 3.6-fold risk of psychological and behav-
ioural disorders in the offspring [32]. Received: 20 March 2012 Accepted: 22 October 2012
Published: 24 October 2012 Received: 20 March 2012 Accepted: 22 October 2012
Published: 24 October 2012 Acknowledgements
h
d
f This study was financially supported by form Research Council of Norway,
Norwegian Institute of Public Health and Akershus University Hospital. Vikanes et al. BMC Pregnancy and Childbirth 2012, 12:115
http://www.biomedcentral.com/1471-2393/12/115 Page 5 of 6 Page 5 of 6 excluding the two women registered with 784.1, the PPV
for HG in MBRN increased from 55.3% to 57.8%. Abbreviations
HG H HG: Hyperemesis gravidarum; ICD: International Classification of Diseases;
MBRN: Medical Birth Registry of Norway; NPV: Negative predictive value;
NVP: Nausea and vomiting in pregnancy; PPV: Positive predictive value. Previous
studies
validating
disease
registration
in
MBRN using hospital records as gold standard did only
report sensitivities and can therefore only partly be
compared to ours [4,6]. The sensitivity for HG is, how-
ever, comparable to the one for rheumatic disease as
well as myasthenia gravis [4,6]. Another study used the
Norwegian Prescription Database validating the registra-
tion of diabetes 1, all types of diabetes, epilepsy and
asthma in MBRN [5]. The validity for diabetes 1 in
MBRN was in line with our findings for mild HG with a
PPV of 56%. The validity for the registration of all types
of diabetes, epilepsy and asthma in MBRN was reflected
in lower sensitivities, specificities, PPV and NVP than
for mild HG. PPV for epilepsy and asthma was 37% and
46%, respectively. Discussion This is due to the fact that ICD code
784.1 is not related to nausea and vomiting during preg-
nancy in particular. The reason for including this ICD
code in our study, was to be comparable with a previous
publication on HG using data from MBRN [11]. When Vikanes et al. BMC Pregnancy and Childbirth 2012, 12:115
http://www.biomedcentral.com/1471-2393/12/115 Vikanes et al. BMC Pregnancy and Childbirth 2012, 12:115
http://www.biomedcentral.com/1471-2393/12/115 aOslo University Hospital Ullevål Hospital since 2009
bAkershus University Hospital since 2008 Vikanes et al. BMC Pregnancy and Childbirth 2012, 12:115
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a 15. Bashiri A, Neumann L, Maymon E, Katz M: Hyperemesis gravidarum:
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Reprod Biol 1995, 63:135–138. aOslo University Hospital Ullevål Hospital since 2009
bAkershus University Hospital since 2008 Page 6 of 6 Page 6 of 6 Vikanes et al. BMC Pregnancy and Childbirth 2012, 12:115
http://www.biomedcentral.com/1471-2393/12/115 Mullin PM, Bray A, Schoenberg F, Macgibbon KW, Romero R, Goodwin TM,
et al: Prenatal exposure to hyperemesis gravidarum linked to increased
risk of psychological and behavioral disorders in adulthood. Journal of
Developmental Origins of Health and Disease 2011, 2:200–204. doi:10.1186/1471-2393-12-115
Cite this article as: Vikanes et al.: Hyperemesis gravidarum in the
Medical Birth Registry of Norway – a validity study. BMC Pregnancy and
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Endogenous circadian reporters reveal critical consequences of diverse novel mutations in clock genes
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Endogenous circadian reporters reveal critical
consequences of diverse novel mutations in clock
genes Jiyoung Park
Florida State University
Kwangjun Lee
Florida State University
Hyeongseok Kim
Tech University of Korea
Heongsop Shin
Tech University of Korea
Choogon Lee
(
choogon.lee@med.fsu.edu
)
Florida State University Article License:
This work is licensed under a Creative Commons Attribution 4.0 International
License. d
ll Page 1/33 Page 1/33 Page 1/33 Abstract Adverse consequences from having a faulty circadian clock include compromised sleep quality and poor
performance in the short-term, and metabolic diseases and cancer in the long-term. However, our
understanding of circadian disorders is limited by the incompleteness of our molecular models and our
dearth of defined mutant models. Because it would be prohibitively expensive to develop live animal
models to study the full range of complicated clock mechanisms, we developed Per1-luc and Per2-luc
endogenous circadian reporters in a validated clock cell model, U2OS, where the genome can be easily
manipulated, and functional consequences of mutations can be accurately studied. Using these reporter
cells, we uncovered critical differences between two paralogs of Per and Cry, as well as working principles
of the circadian phosphotimer. Our system can be used as an efficient platform to study circadian sleep
disorders such as Familial Advanced Sleep Phase Syndrome (FASPS) and their underlying molecular
mechanisms. Introduction The circadian clock drives daily rhythms in behavior and physiology 1-5, and dysfunction or disruption of
the clock has been implicated in diverse disease states including sleep disorders 6-10. Decades of prior
work have revealed that the clock is built on a core transcriptional feedback loop that is cell autonomous,
involving transcriptional and post-translational regulation of the pacemaker Period (Per) genes 11,12. In
the feedback loop, the activator complex CLOCK:BMAL1 drives transcription of the pacemaker genes Per1
and Per2 (Per) along with many other clock-controlled genes. PER proteins form an inhibitory complex
that also contains CRYPTOCHROME (CRY) proteins and casein kinase CK1δ/ε proteins. The circadian
phase, e.g., onset of activity or sleep, is determined by the oscillations of this PER/CRY/CK1 complex 13-
15. The paralogs of Per and Cry genes share some common/redundant functions but also differ in function
and regulation. In the case of Per, the paralogs Per1 and Per2 (Per3 is considered nonessential) seem to
play redundant roles in the master clock tissue, the SCN, as knockouts of either gene produce little period
and phase alterations in behavioral rhythms 16,17. However, the regulation of Per1 and Per2 may differ
dramatically in peripheral tissues and cell culture. For example, Per1 transcription in cultured cells is
rapidly induced by high serum and forskolin, but Per2 transcription is marginally affected by these
signals 18. In mouse tissues, phases of Per1 transcript and protein rhythms are advanced relative to those
of Per2 19. Therefore, the circadian period and phase of PER protein oscillations in Per1 KO cells and Per2
KO cells may differ significantly, likely because of different kinetics in mRNA and protein profiles in these
peripheral cells. This is an unexplored critical issue because circadian properties such as phase of all
clock-controlled genes (ccgs) can be affected when only one Per is present compared to wt cells. In the
case of Cry, knockouts (KOs) of Cry1, Cry2, and Cry1/2 exhibit shortened rhythms, lengthened rhythms,
and arrhythmicity, respectively. Although it has been suggested that difference in binding affinity of two
CRYs to CLOCK:BMAL1 is the molecular underpinning for the opposite period alteration in Cry single KOs Page 2/33 Page 2/33 20,21, it has not been studied how two CRYs differentially affect the pacemaker genes Per1 and Per2. Period alteration in Cry KOs would be manifested ultimately through altered regulation of Per at
transcriptional and posttranscriptional levels. Introduction Another critical issue in the field is how PER phosphorylation by CK1δ and CK1ε can be extended to over
~12 hours, which is responsible for the prolonged circadian feedback loop. This is an unconventional
kinase-phosphorylation relationship, especially considering that PER and CK1 make an unusually stable
interaction through a dedicated domain in PER called CKBD (Casein Kinase Binding Domain) 19,22,23. A
series of phosphorylation steps across several serine residues in this domain seems to play an important
role in tuning phase and period 21,24,25. The Ser662Gly mutation in the CKBD of Per2 gene causes
advanced phase and shortened period in humans, leading to a condition called Familial Advanced Sleep
Phase Syndrome (FASPS) 14. Although the FASPS motif has been a main focus of studies of the CKBD
due to its defined role in the human sleep disorder, CKBD has other conserved motifs, and it has not been
studied how these motifs contribute to the phosphorylation-mediated timing system. Because the circadian clock is cell autonomous, genetic disruptions of the clock manifest similar
phenotypes at the behavioral and cellular levels, and cell culture has proven to be a valuable and valid
platform for characterizing the molecular biology of circadian rhythms 13,26,27. The endogenous clocks of
cultured cells—including mouse embryonic fibroblasts (MEFs) and human U2OS cells—can be precisely
measured in real time by introducing a luciferase (Luc) reporter gene under control of a clock promoter 26-
29. Across numerous studies, such cells have served as functional models for in vivo circadian clocks,
and results have been consistently validated in live animal models. Cell culture models are not only less
resource-consuming, but also more easily manipulated by chemicals and transgenes, which makes the
cell models more suitable for mechanistic studies. One key limitation in cell models today is the lack of
endogenous phase and period reporters other than the mPer2-Luc reporter in MEFs 26, which is a mouse,
not a human cell model. Exogenously transfected reporters like Per2- or Bmal1-promoter controlled Luc
are useful to an extent but do not accurately reflect the endogenous status of the clock. In this study, we generated human Per1-luc and Per2-luc endogenous knockin genes in U2OS cells,
to produce a human cell model with robust bioluminescence rhythms. When knockout phenotypes for
major clock genes were quantified by bioluminescence rhythms in these cells, they were consistent with
phenotypes of knockout mice. Introduction We further used these cells to uncover novel differences between the
paralogs of Per and Cry, and to gain critical insights into how timing cues are precisely generated by the
phosphotimer through dynamic interaction between PER and CK1δ/ε. Results have tested several established protocols as well as newer ones employing high fidelity CAS9 (HF CAS9)
and the paired Cas9D10A nickase approach to decrease off-target insertions 32-34, but they all produced have tested several established protocols as well as newer ones employing high fidelity CAS9 (HF CAS9)
and the paired Cas9D10A nickase approach to decrease off-target insertions 32-34, but they all produced
different off-target insertions and offered no meaningful advantage over wtCAS9 for on-target KI
generation. To streamline the selection process for positive clones after single-cell sorting, we employed a
dual reporter system that eliminates the need for molecular experiments during the selection process (Fig. 1a). To generate Per1 and Per2 reporter lines, U2OS cells were transfected with two plasmids, all-in-one
GFP-sgRNA-CAS9 35 and repair templates with luciferase-T2A-mRuby3 (Fig. 1a). If targeting is successful,
Luc-T2A-mRuby3 will be inserted between the last amino acid (AA) and stop codon in each Per gene (Fig. 1a). The T2A cleavage site was added because Luc alone has been proven not to disrupt PER function or
clock function in the mPer2Luc KI mouse 26 whereas this has not been proven for the bulkier Luc-mRuby3
dual tag. In the first selection after transfection, stable mRuby3-expressing cells were singly sorted into 96
well plates by FACS. Because we hypothesized mRuby3 expression would be low based on prior work
with the mPer2-GFP KI mouse 36, we selected a low range of red signal (Fig. 1b) for single cell sorting; this
tunability is a major advantage of FACS versus antibiotic selection. Subsequent bioluminescence
selection allowed us to select clones with robust rhythms without the need for expansion, genomic DNA
prep and PCR analysis from individual clones (Fig. 1c). We isolated 7 final clones for Per1 (4 hetero- and
3 homozygous KIs), and 6 final clones for Per2 (all heterozygotes). The 2nd selection produced ~30%
rhythmic clones. These final clones were further validated by junction PCR and sequencing (Fig. 1d and
Supplementary Fig. 1), and by immunoblotting with anti-PER and Luc antibodies (Fig. 1e and
Supplementary Fig. 2). Finally, single specific insertions were verified by showing a complete loss of
bioluminescence and mRuby3 signals when frame-shifting mutations were introduced in early exons in
Per genes (Supplementary Fig. 3). We confirmed that the KIs did not cause any disruption in clock function by comparing the molecular
rhythms between PER and PER-Luc proteins in heterozygote clones (Fig. 2). Results Generation of U2OS cells with endogenous Per1-luc and Per2-luc knockin genes and robust rhythms in
bioluminescence and clock proteins. The main challenge in developing reporter knockins (KIs) was that
the screening process could be very cumbersome due to a high number of off-target insertions 30,31. We Page 3/33 Page 3/33 have tested several established protocols as well as newer ones employing high fidelity CAS9 (HF CAS9)
and the paired Cas9D10A nickase approach to decrease off-target insertions 32-34, but they all produced
different off-target insertions and offered no meaningful advantage over wtCAS9 for on-target KI
generation. To streamline the selection process for positive clones after single-cell sorting, we employed a
dual reporter system that eliminates the need for molecular experiments during the selection process (Fig. 1a). To generate Per1 and Per2 reporter lines, U2OS cells were transfected with two plasmids, all-in-one
GFP-sgRNA-CAS9 35 and repair templates with luciferase-T2A-mRuby3 (Fig. 1a). If targeting is successful,
Luc-T2A-mRuby3 will be inserted between the last amino acid (AA) and stop codon in each Per gene (Fig. 1a). The T2A cleavage site was added because Luc alone has been proven not to disrupt PER function or
clock function in the mPer2Luc KI mouse 26 whereas this has not been proven for the bulkier Luc-mRuby3
dual tag. In the first selection after transfection, stable mRuby3-expressing cells were singly sorted into 96
well plates by FACS. Because we hypothesized mRuby3 expression would be low based on prior work
with the mPer2-GFP KI mouse 36, we selected a low range of red signal (Fig. 1b) for single cell sorting; this
tunability is a major advantage of FACS versus antibiotic selection. Subsequent bioluminescence
selection allowed us to select clones with robust rhythms without the need for expansion, genomic DNA
prep and PCR analysis from individual clones (Fig. 1c). We isolated 7 final clones for Per1 (4 hetero- and
3 homozygous KIs), and 6 final clones for Per2 (all heterozygotes). The 2nd selection produced ~30%
rhythmic clones. These final clones were further validated by junction PCR and sequencing (Fig. 1d and
Supplementary Fig. 1), and by immunoblotting with anti-PER and Luc antibodies (Fig. 1e and
Supplementary Fig. 2). Finally, single specific insertions were verified by showing a complete loss of
bioluminescence and mRuby3 signals when frame-shifting mutations were introduced in early exons in
Per genes (Supplementary Fig. 3). Results When comparing Per1 and
Per2 heterozygote KI clones (hereafter referred to as Per1Luc and Per2Luc) (Fig. 2a), PER1-Luc peaked 2-3
hrs earlier than PER2-Luc, which is consistent with the phases of PER rhythms in mouse peripheral
tissues 19. PER1-Luc signals were higher than those of PER2-Luc reporters (Fig. 2a, Supplementary Fig. 4a and 4b). When these PerLuc reporters were compared to the transgenic Bmal1-Luc reporter 27, they
showed almost antiphase oscillations, consistent with the natural antiphase oscillations of Per vs Bmal1
mRNA in vivo (Fig. 2b). The periods of Per1 and Per2 KI reporters were not different from each other (Fig. 2c and Supplementary Fig. 4c), but they were slightly different from that of the transgenic Bmal1-Luc
reporter. The mouse endogenous mPer2Luc reporter in MEFs produced similarly antiphase oscillations
compared to the Bmal1-Luc reporter in U2OS cells (Fig. 2d). Both PER1-Luc and PER2-Luc fusion proteins
showed similarly robust oscillations in abundance and phosphorylation as their wt counterparts in the
heterozygote clones (Fig. 2e and 2f). Robust oscillations of PER-Luc fusion proteins were also confirmed
by immunoblotting with anti-Luc antibody (Fig. 2e-g). Finally, their functionality was verified by
confirming that PER-Luc-containing clock complexes exhibited the same abundance and phosphorylation
rhythms as their wt PER-containing counterparts (Fig. 3a). Page 4/33 Page 4/33 We have previously shown that the level of PER phosphorylation highly correlates with the size of clock
protein complexes because PER hyperphosphorylation is induced by multimerization of PER
monomers 37. Studying our novel PER reporter cells uncovered interesting differences between PER1 and
PER2 at the posttranslational level. Although both PER1 and PER2 are much less stable than other clock
proteins 38, PER2 was significantly more unstable than PER1 (Fig. 3b and 3c). This was verified by
immunoblotting of time course samples and real-time bioluminescence (Fig. 3c). More significantly,
regarding the phosphotimer function of PER, PER1 phosphorylation occurs at a much higher rate than
that of PER2 (Fig. 3d). PER1 accumulated faster than PER2 after existing proteins were depleted by a
long cycloheximide treatment because PER1 was more stable (Fig. 3d and Supplementary Fig. 5a). These
data have important implications when either Per1 or Per2 is inactivated because although either one can
sustain the clock as the sole pacemaker, the resulting oscillations could be very different due to their
differences in these key parameters. Phase of circadian genes is reversed in Per1 knockout cells. Results However, in
Per1 KO cells, PER2 profile in abundance and phosphorylation was very different from that in wt cells, Page 5/33 Page 5/33 further supporting that the resetting mechanism for Per2 by zeitgebers is very different when Per1 is
present versus when it is absent. Cry genes affect circadian rhythms through posttranslational regulation of PER in addition to
transcriptional regulation of Per. CRY proteins are considered the main transcriptional inhibitors in the
circadian feedback loop 42, but circadian phase (timing of feedback inhibition) is determined by
cooperation between PER and CRY in the inhibitor complex 13,43. Like the Per paralogs, the two Cry genes
are not completely redundant. Cry1 and Cry2 KO mice show shortened and lengthened behavioral
rhythms (by <1hr) compared to wt mice, respectively, suggesting that they may have a slightly
antagonistic role in setting period 44,45. To measure how they affect Per-reporter rhythms, Cry1 and Cry2
genes were singly and doubly deleted in both Per1Luc and Per2Luc KI reporter cells. Deletion of Cry1 and
Cry2 in these cells produced period shortening and lengthening, respectively, consistent with phenotypes
in mice (Fig. 5a-d) 44,45. However, the amount of alteration was more dramatic in U2OS cells compared to
mice. Both PER1-Luc and PER2-Luc signals increased significantly in Cry1 KO cells but slightly decreased
in Cry2 KO cells, suggesting that CRY1 is the stronger inhibitor than CRY2, consistent with recent
studies 20,21. In line with the bioluminescence data, both PER1 and PER2 protein levels were elevated in
Cry1 KO cells (Fig. 5e). These data elegantly explain why Cry1 and Cry2 KO can cause period shortening
and lengthening, respectively. In Cry1 KO cells, PER threshold levels for feedback inhibition would be
reached earlier causing shortening of the feedback loop, while those would be delayed in Cry2 KO cells
resulting in period lengthening. Cry1/2 double-KO cells did not become completely arrhythmic
immediately, as opposed to immediate arrhythmicity in Cry1/2 double-KO mice. Instead, both PER1-Luc
and PER2-Luc were robustly rhythmic for one circadian cycle and then became arrhythmic (Fig. 5f and
5g). The first cycle is not included because it could be zeitgeber-driven rather than an endogenous
feedback loop-driven rhythm. PER1-Luc bioluminescence was higher than in wt cells, but PER2-Luc
signals were slightly lower compared to wt cells. Changes in PER abundance by Cry KO seems to directly
reflect changes in Per transcription in these mutant cells (Fig. 5h). Consistent with above data (Fig. Results Numerous lines of evidence indicate that the
phases of circadian behaviors such as wake and sleep timing (activity onset and offset) are determined
by phases of PER oscillations. For example, new wake/sleep cycles following transmeridian travel are
established by altered phases of Per genes through re-aligning of Per oscillations to the altered light
cycles 1,39,40. Per genes are the only clock genes that can be phase-shifted directly by light in the SCN. We
measured how period and phase of Per1 and Per2 genes are affected in non-light-responsive peripheral
clock cells (U2OS) by examining PER-Luc rhythms when the other paralog is absent. When Per2 was
knocked out in Per1Luc cells (Fig. 4a, 4b and 4d), the period and phase of PER1-Luc rhythms were little
changed. However, when Per1 was knocked out in Per2Luc cells (Fig. 4a, 4c and 4d), the phase of PER2-
Luc, but not the period, was dramatically altered, almost resulting in phase reversal. A similarly little
altered and dramatic phase shift in Per1 KO and Per2 KO U2OS cells, respectively, were observed with wt
Per paralogs using the transgenic Bmal1-Luc reporter (Fig. 4e and 4f), showing that the phase shift was
not an artifact of the PER-Luc fusion. The dramatically altered phase in PER2 rhythms in Per1 KO cells
was also confirmed by immunoblotting (Fig. 4g). This phase reversal is not a unique response to the use
of 50% horse serum (‘serum shock’) as the phase-setting stimulus (zeitgeber): similar responses of Per1
and Per2 KOs were observed by a different zeitgeber, forskolin (Supplementary Fig. 5b). These data
strongly suggest that the phase of all circadian genes, not just core clock genes, are phase-reversed in
peripheral clock cells when Per1 is deleted or inactivated. These data also suggest that Per1 phase is
predominant over Per2 in wt cells, and the resetting mechanism for Per2 is very different from that of
Per1 as suggested above (Fig. 2 and 3). Our data are consistent with previous studies by the Schibler
group showing that immediate transcriptional response to zeitgebers is very different between Per1 and
Per2 genes 18,41. As with mRNA levels during early hours after 2 hr serum shock 18, PER1 protein
increased more dramatically than PER2, but their phases were similar in wt cells (Fig. 4h). Results 3 and
4), Per1 transcription is more dramatically regulated than that of Per2 by Cry KO. High and low levels of
PER1 and PER2 in Cry double-KO cells, respectively, were confirmed by immunoblotting which also
revealed an additional difference between the PER paralogs (Fig. 5i and 5j). Hypophosphorylated
isoforms of PER1 and PER2 were much more pronounced in Cry double-KO cells. Hyperphosphorylated
species failed to accumulate after cycloheximide treatment (Supplementary Fig. 6a) suggesting that
hyperphosphorylated species cannot be generated or they are very unstable without CRY. Because
hyperphosphorylated PER species were readily detectable after calyculin A (CA) treatment
(Supplementary Fig. 6b), CRY seems to protect hyperphosphorylated species from dephosphorylation or
rapid degradation. Because PER, especially PER1, is more rapidly degraded in Cry double-KO cells after
CHX treatment (Supplementary Fig. 6a), these data suggest that the conversion of hypophosphorylated
to hyperphosphorylated PER species is normal, but they are unstable without CRY. Similar observations
were made in liver tissue between wt and Cry double KO mice 19 Hyperphosphorylation of PER was (Supplementary Fig. 6b), CRY seems to protect hyperphosphorylated species from dephosphorylation or
rapid degradation. Because PER, especially PER1, is more rapidly degraded in Cry double-KO cells after
CHX treatment (Supplementary Fig. 6a), these data suggest that the conversion of hypophosphorylated
to hyperphosphorylated PER species is normal, but they are unstable without CRY. Similar observations
were made in liver tissue between wt and Cry double-KO mice 19. Hyperphosphorylation of PER was Page 6/33 Page 6/33 restored when transgenic CRY was expressed in Cry double-KO cells, indicating that the phenotype is not
due to the genetic defect (Supplementary Fig. 6c). restored when transgenic CRY was expressed in Cry double-KO cells, indicating that the phenotype is not
due to the genetic defect (Supplementary Fig. 6c). C-terminus of CK1δ/ε is critical for rhythm generation through making stable interaction with their
substrate PER. There are seven CK1 isoforms in mammals, encoded by seven different genes 46. They are
very well conserved in the catalytic domain (AA1 to ~300), but highly divergent in C-terminal noncatalytic
domains (Supplementary Fig. 7). The circadian clock is completely compromised and PER
hyperphosphorylation is absent in CK1δ/ε double-KO cells 47. PER2 is not even noticeably
phosphorylated in the double-KO cells based on mobility shift. Results Although there is also evidence that CK1δ/
ε are not the only kinases that phosphorylate PER 48-50, the KO data indicate that other CK1 isoforms and
other kinases cannot replace CK1δ/ε as essential regulators of PER phosphorylation for the circadian
clock. Because the catalytic domains are highly conserved (Supplementary Fig. 7), the C-terminal regions
of CK1δ/ε are likely important for their function as circadian kinases. We began our studies of CK1δ/ε in
our reporter cells by confirming their circadian functionality: deleting both genes and measuring the effect
on bioluminescence rhythms. In our initial attempt to generate CK1δ/ε double-KO cells, we induced
frame-shift mutations in exon 4 of the CK1δ gene, and then targeted exon 2 in CK1ε in the resulting CK1δ
KO clones (Supplementary Fig. 7). However, no viable colonies were detected after single-cell sorting,
indicating that double KO leads to lethality, as has been reported in MEFs 47. To circumvent this issue and
test functionality of the C-terminus of CK1ε at the same time, frame-shifting mutations were introduced
in the non-essential C-terminal region in CK1ε (exon 8) (Supplementary Fig. 7). As expected, many viable
colonies were obtained. Since our anti-CK1δ/ε antibodies were raised against the non-conserved C-
termini, C-terminus-truncated CK1ε could not be detected on immunoblots (Fig. 6a). When CK1δ or ε was
deleted by targeting early exons, E4 and E2, respectively, circadian rhythms were significantly lengthened
by 1.5–2 hrs (Fig. 6b-d), consistent with genetic data in mice 51. Interestingly, frame-shifting mutations in
either exon 2 or exon 8 in the C-terminus of CK1ε produced similarly lengthened rhythms, suggesting that
the C-terminus of CK1ε plays an important role in the clockwork (Fig. 6c and 6d). Consistent with these
data, deletion of the C-terminus of CK1ε (exon 8) in a CK1δ KO background (thus creating double-mutant
cells) produced much longer periods than CK1δ single-KO cells in both Per1Luc and Per2Luc cells (Fig. 6e-g
and Supplementary Fig. 8a), further supporting the importance of C-terminal regions in CK1δ/ε for the
clockwork. One of the double mutants isolated based on period lengthening in Per1Luc CK1δ/ε double
mutant clones had deletion of two AAs plus a point mutation instead of a frame-shifting mutation
(Per1Luc CKKO-2) (Fig. 6g bottom panel and Supplementary Fig. 7). The mutant clone showed even longer
period than a C-terminal deletion mutant clone Per1Luc CKKO-1 (Fig. 6f, 6g and Supplementary Fig. 8a). Results In
all of these double mutant cells, PER could be hyperphosphorylated in a rhythmic manner albeit with a
delay. There was accumulation of hyperphosphorylated PER species (Fig. 6h), which was probably
induced by their increased stability (Fig. 6i). We believe the slowed hyperphosphorylation and thus
increased stability are caused by attenuated interaction between PER1 and C-terminus-deleted or mutated
CK1ε. PER interaction with the mutant CK1ε in the Per1Luc CKKO-2 clone was significantly attenuated C-terminus of CK1δ/ε is critical for rhythm generation through making stable interaction with their Page 7/33 Page 7/33 compared to wt CK1e when measured using transiently expressed proteins (Fig. 6j). Overall, these data
suggest that C-terminal regions of CK1δ/ε play an important role in promoting hyperphosphorylation in
PER for timely degradation through making stable interactions between enzyme and substrate. The circadian phosphotimer requires stable interaction between CK1δ/ε and PER. Two main events
regulated by the PER phosphotimer are nuclear entry and degradation of the PER-containing complex;
these events are separated by ~12 hours and define distinct circadian phases 19,38,47. Because PER
phosphorylation by CK1δ/ε is responsible for these events directly, the timing of PER phosphorylation
must occur over the same extended period, ~12 hrs. CK1δ and ε bind the substrate PER stably through a
dedicated ~220 AA domain called CKBD in PER 22,52, which is different from a typical transient kinase-
substrate relationship. Although many previous studies suggested that this domain (which, as discussed
earlier, contains the FASPS mutation S662G) plays an important role in setting period, how CKBD
contributes to the phosphotimer has been unsolved. While generating AA indels around S662 in CKBD by
CRISPR to study the significance of the domain, a mutant clone missing 2/3 of CKBD was isolated (Fig. 7a and Supplementary Fig. 8b). This clone exhibited a significantly lengthened rhythm, ~27.5 hrs (Fig. 7a
and 7b). The mutant PER2 levels were elevated compared to wt PER2. When Per1 was deleted in this
clone, rhythms were completely eliminated, and protein levels were further elevated, demonstrating that
the mutant PER2 is not functional, and the mutation is semi-dominant over Per1 (Fig. 7a). The mutant
PER2 was constitutively hyperphosphorylated, which was not dependent on PER1 (Fig. 7c and 7d). As
expected, its interaction with CK1δ/ε was dramatically attenuated (Supplementary Fig. 8c). Although
these data indicate that PER hyperphosphorylation is not dependent on stable interaction with CK1δ/ε,
when the mutant cell was treated with a specific CK1δ/ε inhibitor, PF670462, PER hyperphosphorylation
was inhibited in both wt and mutant PER2, demonstrating that hyperphosphorylation of the mutant PER2
does depend on CK1δ/ε (Fig. 7e and Supplementary Fig. 8d). Furthermore, hyperphosphorylation of the
mutant PER2 was more sensitive to the inhibitor; maximum inhibition was achieved at a much lower dose
of the inhibitor compared to wt PER2 (Fig. 7e and Supplementary Fig. 8d). Discussion A critical bottleneck in studying complex biological systems like the circadian clock is the lack of an
efficient in vivo-like platform where endogenous genes can be easily manipulated to test diverse
hypotheses in a time- and cost-effective manner. Historically, manipulation of endogenous clock genes in
cell culture models required first developing mutant mouse models from which cells such as MEFs were
then harvested 38,53,54. However, recent developments in CRISPR genome editing have created new
opportunities for generating cell culture models without first generating mutant mice. Several studies
including ours demonstrated that clock genes can be knocked out efficiently in culture using CRISPR 55-
59. One key limitation in cell models today is the lack of precise endogenous phase and period reporters
working like clock hands other than the mPer2-Luc reporter in MEFs. Fluorescence reporters have been
developed 60, but they are far less accurate than bioluminescence reporters and require heavy
deconvolution of data, especially when signal is barely above background. When we put mRuby3 before
luciferase to produce the PER-mRuby3 fusion protein, fluorescence signal was significantly lower than
non-fusion mRuby3 and barely above background when measured by FACS and microscopy. This is
probably due to decreased stability of mRuby3 when it is attached to the unstable PER. We were able to
observe robust rhythms for more than 10 days from our bioluminescence reporter cells compared to 2-3
days reported using fluorescence reporters 60. Fusion of a fairly large protein, luciferase, to PER did not seem to affect phosphorylation and stability of
PER, which suggests a dominant regulation of PER at the posttranslational level and can explain a wt-like
circadian phenotype in the mPer2Luc KI mouse. Although PER1 and PER2 are independently rhythmic in
abundance and phosphorylation and redundant in generation of circadian rhythms, their kinetics such as
stability and speed of phosphorylation differ significantly. Because PER protein phase and thus phase in
the feedback loop would be determined by these properties, it would be expected that phase of clock-
controlled genes would be different between Per1 and Per2 KO cells. Indeed, the phase of Bmal1-Luc was
reversed in Per1 KO, but not in Per2 KO cells, suggesting that Per1 phase is dominant over that of Per2 in
wt cells, probably due to higher abundance and leading phosphorylation kinetics. However, the circadian
phase of the master clock in the SCN is not altered in Per1 KO mice. C-terminus of CK1δ/ε is critical for rhythm generation through making stable interaction with their These data suggest that
hyperphosphorylation of PER by CK1δ/ε does not require stable interaction, but a functional
phosphotimer requires CKBD because it can allow slow and progressive phosphorylation. In the absence
of the stable interaction, PER phosphorylation by the kinases would be done in a transient manner like
typical kinase reactions. The mutant PER2 was predominantly nuclear as expected from their
phosphorylation status and significantly more stable than wt PER2 (Fig. 7f, 7g and Supplementary Fig. 9). When the mutant PER2 was treated with CA, extra hyperphosphorylated species were not detected,
suggesting that this mutant PER2 is defective in this extra phosphorylation, which is normally not
detected in a steady state because these extra hyperphosphorylated species are rapidly turned over by β-
TRCP-regulated proteasomal degradation (Fig. 7h) 38. Faster phosphorylation kinetics for the mutant
PER2 was confirmed with de novo PER2 after existing PERs was depleted (Fig. 7i). The lack of extra
hyperphosphorylation was reproduced with transiently expressed CK1δ and mutant PER2 (Fig. 7j). Page 8/33 Discussion Both Per1 and Per2 KO mice show
similar phases in behavioral rhythms in constant darkness after LD entrainment 17. We believe the
difference between peripheral and master clocks is due to different entrainment mechanisms. In the
master clock, both Per genes are rapidly induced by photic signals resulting in acute phase shifting 39. However, in peripheral cells, Per2 rhythm is not rapidly reset by zeitgebers because its transcription is not
acutely induced while Per1 rhythm is acutely reset by these signals through rapid induction of mRNA as
in SCN by photic signals 18. This has a significant implication in humans with a defective Per1 gene
because their peripheral clocks could be dissociated from the SCN clock. Because PER-Luc oscillates with a circadian period in Cry1/2 KO cells at least for one complete cycle
57,61, PER alone seems to act as a feedback inhibitor, but the clock cannot be sustained without CRY. PER
can directly interact with CLOCK:BMAL1 and repress the activity of the complex in in vitro reporter assays Page 9/33 Page 9/33 Page 9/33 supporting the inhibitory role 13,42. In β-Trcp mutant cells, the clock cannot be sustained because
hyperphosphorylated PER species are too stable in the nucleus 38. Similarly, it may be that the clock
cannot be sustained in Cry1/2 KO cells because hyperphosphorylated PER species are too unstable, and
CRY is necessary to extend nuclear presence of PER and PER-containing inhibitory complexes. Our data suggest that the progressive and controlled phosphorylation of PER is mediated by stable
interaction between the kinase and substrate resulting in slowing down kinase movement (processivity)
on PER for serial phosphorylation. The phosphotimer can be also modulated by mutations in CK1δ/ε. CK1s are considered anion- or phosphate-binding kinases because basic amino acids (AAs) in the anion-
binding pockets interact with anions or phosphate groups on substrates 62,63. CK1s depend on negative
charges or prior phosphorylation on a substrate for processive phosphorylation as in a typical consensus
motif pSxxS 25,62,63, which appears throughout the entire PER1 and PER2 protein, not just the FASPS
domain, yet current working models of the clockwork focus on only two motifs, FASPS and a degron
motif 25,64. CK1s have several substrate-facing, anion-binding pockets; thus the more PER is
phosphorylated in CKBD, the more robustly PER will hold CK1, resulting in slower processivity. In vivo,
CK1δ/ε are predominantly copurified only with hyperphosphorylated PER species 19, supporting the
model. Discussion Further, charge inversion mutations such as tau (R178C) and K224D in one of the anion-binding
pockets of CK1 accelerate the phosphotimer and the clock64,65. Consistent with this model, deletion of the
whole CKBD resulted in typical rapid kinase reactions, leading to constitutive hyperphosphorylation. This
level of phosphorylation was enough for nuclear entry, but not for degradation as shown in Fig 7. The
PER phosphotimer seems to have two distinctive phases, one for nuclear entry and the other for
degradation, matching with onset and offset sleep phases. CKBD is required for the second phase of the
phosphotimer. It is not clear whether CKBD itself contains a critical phospho-degron or whether stable
CK1 binding to PER is required for phosphorylation in a degron somewhere else in PER. The phosphotimer is at the heart of the circadian clock; it is the basis for how a cell can measure time
precisely over timeframes much longer than typical cellular processes. Our studies revealed many critical
properties of this timer using diverse clock mutants. In addition, our human reporter cell model could
provide direct insights into pathogenicity of many period- and phase-altering mutations in CK1 and PER
and serve as an efficient platform to test diverse hypotheses developed via human genetics and
biochemical studies. Acknowledgements We thank Dennis Chang for assistance with manuscript revisions and Robert Tomko for gel filtration
chromatography. We thank Jonathan M. Philpott and Carrie L. Partch for critical reading of the
manuscript and providing feedback. This work was supported by NIH R01 GM131283 awarded to C.L. and by Korea Institute for Advancement of Technology (KIAT, P0002007, HRD Program for Industrial
Innovation) grant to H.S. Page 10/33 Page 10/33 Author contributions C.L., H.S. and J.P conceived the project. All authors produced and interpreted experimental data. C.L., J.P
and K.L wrote the paper. All authors provided feedback on the paper. Materials and Correspondence Correspondence and material requests should be addressed to C.L. Correspondence and material requests should be addressed to C.L. Competing interests The authors declare no competing interests. The U2OS cell line was purchased from ATCC (#HTB-96). U2OS-Bmal1-Luc wt and Per2 knockout (KO) (E5-2) lines were described previously 57. U2OS-Bmal1-Luc;
Per1 KO clones were selected from the previous study 57, and #E6-2-4 was used for this study. mPer2Luc knockin (KI) reporter MEFs were described previously 26. HEK293a (ThermoFisher #70507) was used in Fig 6J. All cells were maintained at 37 °C, 5% CO2 in DMEM, supplemented with 10% FBS. Cell lines The U2OS cell line was purchased from ATCC (#HTB-96). The U2OS cell line was purchased from ATCC (#HTB-96). Generation of Per KI cell lines Generation of Per KI cell lines For all CRISPR-induced mutations, sgRNAs were selected via CHOPCHOP (https://chopchop.cbu.uib.no),
cloned into pAdTrack-Cas9-DEST, and tested for efficiency by T7E1 assay as described previously (Jin et
al., 2019). Wild-type (wt) U2OS cells were transfected with all-in-one pAdTrack-Cas9-DEST plasmids (sgRNA
sequence described in Supplementary Table 1 and T7E1 primers described in Supplementary table 2) and
linear repair templates (Supplementary Fig 10 and 11) using jetOPTIMUS according to the manufacturer’s
protocol (jetOPTIMUS transfection reagent, Polyplus). The linear templates were prepared from Per-Luc-
T2A-Ruby_pUC19 plasmid (Supplementary Fig 12) by PCR amplification. Cloning primers for the Page 11/33 Page 11/33 Page 11/33 Page 11/33 plasmids are described in Supplementary Table 3 and Supplementary Figure 12. Assembly of these
amplicons were done using NEB HiFi DNA Assembly Master Mix according to the manufacturer’s
protocol. The transfected cells were maintained and expanded for 10 days before they were subjected to
trypsinization and FACS sorting using BD FACSAria SORP equipped with an Automated Cell Deposition
Unit (ACDU) for mRuby3-positive single-cell sorting into 96 well plates as well as bulk sorting. The single
cells were expanded for 2 weeks and split into one well in a regular 24-well plate and one well in a black-
wall 24-well plate (PerkinElmer #1450-605, Arkon, OH, USA). The bulk sorted cells were also added into
the black-wall plate before it was set up into Lumicycle 96. Several clones with robust circadian
bioluminescence rhythms were selected for each Per gene and subjected to junction PCR,
immunoblotting with anti-PER1 (GP62), anti-PER2 (hP2-GP49) and anti-Luciferase (Luc) antibodies, and
Sanger sequencing. For Luc immunoblotting, novel polyclonal anti-Luc antibodies were generated by
Cocalico Biologicals, Inc (449 Stevens Rd, Reamstown, PA) in guinea pigs using Luc AA 300-550 peptide. The antisera were validated against transfected Luc and Luc KI cells. GP77 was used in this study. To
verify a single insertion of the dual reporter, frameshift mutations were introduced in early exons in these
clones using all-in-one CRISPR adenovirus as described previously (Jin et al) (Fig S3). Disappearance of
mRuby3 signal was confirmed by fluorescence microscopy and by FACS and bioluminescence in the
Lumicycle 96. Final positive clones are summarized in Supplementary Table 1. Generation of Per KI cell lines The transfected cells were maintained and expanded for 10 days before they were subjected to
trypsinization and FACS sorting using BD FACSAria SORP equipped with an Automated Cell Deposition
Unit (ACDU) for mRuby3-positive single-cell sorting into 96 well plates as well as bulk sorting. The single
cells were expanded for 2 weeks and split into one well in a regular 24-well plate and one well in a black-
wall 24-well plate (PerkinElmer #1450-605, Arkon, OH, USA). The bulk sorted cells were also added into
the black-wall plate before it was set up into Lumicycle 96. Several clones with robust circadian
bioluminescence rhythms were selected for each Per gene and subjected to junction PCR, immunoblotting with anti-PER1 (GP62), anti-PER2 (hP2-GP49) and anti-Luciferase (Luc) antibodies, and
Sanger sequencing. For Luc immunoblotting, novel polyclonal anti-Luc antibodies were generated by
Cocalico Biologicals, Inc (449 Stevens Rd, Reamstown, PA) in guinea pigs using Luc AA 300-550 peptide. The antisera were validated against transfected Luc and Luc KI cells. GP77 was used in this study. To
verify a single insertion of the dual reporter, frameshift mutations were introduced in early exons in these
clones using all-in-one CRISPR adenovirus as described previously (Jin et al) (Fig S3). Disappearance of
mRuby3 signal was confirmed by fluorescence microscopy and by FACS and bioluminescence in the
Lumicycle 96. Final positive clones are summarized in Supplementary Table 1. Mutations of clock genes in Per1Luc and Per2Luc reporter cells Genes
Targets
sgRNA Sequences (PAM)
Selected clones
Per2
Exon 6
ATCTCTTTTACAGTGAAATA(TGG)
Per2-E6-7, 55
Per2
Exon 17
GCCGGGCAAGGCAGAGAGTG(TGG)
Per2-E17-17 For Fig 4B, single Per2 KO clones were isolated by using Adv-Per2-E6 as described above. Clone #Per2-E6-
55 was used. To generate in-frame indels in Per2 exon 17, the Adv-Per2-E17 was infected into the Per2Luc
reporter cells and mRuby3-positive cells were sorted into 96-well plates by FACS. These cells are either wt
or in-frame mutant cells because mRuby3 will be eliminated by out of frame mutations. One of the
mutants was E17-17 which is missing 2/3 of CKBD. To delete Per1 in this clone, Adv-Per1-E6 was infected
into the cells and Per1 KO was confirmed by immunoblotting. Mutations of clock genes in Per1Luc and Per2Luc reporter cells Heterozygous KI clones, H10 for Per1Luc and LH1 for Per2Luc, were used to generate mutations in other
clock genes. The mRuby3-expressing reporter cells were transfected with all-in-one pAdTrack-Cas9-DEST
plasmids expressing GFP. In Fig S3, the reporter cells were infected with all-in-one CRISPR adenovirus
to induce indels in the majority of the cells with near 100% efficiency as described previously. For clonal
isolation of clock mutant cells, GFP-positive cells were sorted by FACS into 96-well plates, and these
clones were further selected based on alterations in period and/or phase in bioluminescence rhythms. The sgRNA sequence and final clones are summarized in Supplementary Table 1. In each project, the
majority of putative mutant clones showed a similar degree of period lengthening or shortening in
bioluminescence screening. Some of these mutant clones were fully characterized by immunoblotting
and sequencing. Per mutant clones in PerLuc reporter cell lines Per mutant clones in PerLuc reporter cell lines Page 12/33 Page 12/33 Page 12/33 All-in-one adenovirus expressing CAS9 and Per1-E6 sgRNA used to generate indels in the Per1 gene were
described previously 57. For Fig 4C, single Per1 KO clones were isolated by FACS followed by
immunoblotting and a clone #Per1-E6-11 was used. To generate all-in-one adenovirus targeting Per2 exon
6 or exon 17, the following sgRNA sequence was cloned into the pAdTrack-Cas9-Dest and adenoviruses
were packaged as described previously 57. Genotyping For Per KI clones, KI and non-KI alleles from each clone were PCR-amplified using the primers described in
Supplementary Table 4 and Supplementary Fig 13, and the amplicons were sequenced using primers
described in Supplementary Table 5. For mutant clones with indels, amplicons of the target regions were prepared using the surveyor primers
(Supplementary Table 2) and sequenced. In the majority of the clones, two different Sanger sequencing
traces were mixed due to different indels in two alleles. These results were deconvoluted by a computer
algorithm called DECORD v3 (https://decodr.org/) into two separate traces. Accuracy of the
deconvolution was confirmed by TA cloning of several amplicons from several mutant clones. Frameshift
mutations in one or both alleles were also confirmed by immunoblotting. Bioluminescence recording Page 13/33 Cells were plated into 24-well plates or 35 mm dishes to be approximately 90% confluent 24 hours prior to
the start of the experiment. Immediately before the start of the experiment, cells were given a two-hour
serum shock with 50% horse serum in DMEM or 10mM forskolin in DMEM (Fig S5), washed with
phosphate-buffered saline (PBS) and fresh DMEM supplemented with 1% FBS, 7.5mM sodium
bicarbonate, 10 mM HEPES, 25 U/ml penicillin and 25 μg/ml streptomycin. Also 0.1mM luciferin was
added. The plates were sealed with cellophane tape and the dishes with cover glass with vacuum grease,
and placed into a Lumicycle 96 or 32 (Actimetrics, Wilmette, IL). For all bioluminescence experiments, the
results were reproduced in at least two independent experiments. Real-time levels, period, and phase of
the bioluminescence rhythms were evaluated using the Lumicycle software (Actimetrics). Drug treatments in cells Cells were seeded in 60-mm dishes for immunoblots or 24-well plates for bioluminescence monitoring to
be 90% confluent 24 hours prior to the experiment. For cycloheximide treatment, 8 μg/ml was added to
cells and cells were collected at specified times after the treatment or placed into Lumicycle 96 for
bioluminescence monitoring. For CHX washout experiments, cells were treated with CHX for 8 hrs
followed by the normal DMEM, and cells were harvested at the indicated times after the CHX treatment. PF670462 was purchased from Sigma. 20 nM Calyculin A (CA, EMD chemicals) were used for cells. Immunoblotting, gel filtration and Immunoprecipitation The cells in 6 cm dishes were harvested and flash-frozen on dry ice. Protein extraction and
immunoblotting were performed as previously described 66. Briefly, cells were homogenized at 4°C in
70 ml extraction buffer (EB) (0.4M NaCl, 20mM HEPES (pH 7.5), 1mM EDTA, 5mM NaF, 1 mM
dithiothreitol, 0.3% Triton X-100, 5% glycerol, 0.25mM phenylmethylsulfonyl fluoride, 10mg of aprotinin
per ml, 5mg of leupeptin per ml, 1mg of pepstatin A per ml). Homogenates were cleared by centrifugation
12 min, 12,000g at 4°C. Supernatants were mixed with 2x sample buffer and boiled. Proteins were
separated by electrophoresis through SDS polyacrylamide gels and then transferred to nitrocellulose
membranes. Membranes were blocked with 5% non-fat dry milk in TBS-0.05% Tween-20 (TBST),
incubated with primary antibodies overnight followed by incubation with secondary antibodies for 1 h. The blots were developed using an enhanced chemiluminescence substrate (WestFemto, ThermoFisher
Scientific). Page 14/33
Antibodies to clock proteins were generated as previously reported 19,22. CLK-1-GP, BM1-2-GP, C1-GP
(CRY1), C2-GP (CRY2), CK1d-GP and CK1e-GP antibodies were used at 1:1,000 dilution in 5% milk–TBST Page 14/33 Page 14/33 olution. Rabbit anti-ACTIN antibody (Sigma, A5060) was used at 1:2,000. Cell extracts from wt U2OS and a heterozygous Per1Luc reporter cells were fractionated by Superose 6
column (Cytiva, Marlborough, MA) and immunoblotted for PER1 and PER2 as described previously 37. Fractions after the void volume of the column are shown. Immunoprecipitation was performed as described previously 19. Briefly, protein extracts from cells
harvested from 10 cm dishes were prepared as described above. 10% of the initial protein extract was
saved for the input. 20μL Protein G Sepharose 4 Fast Flow beads (GE Healthcare) per reaction was
equilibrated with 500μL of EB for 15 minutes on a rotating wheel. The beads were centrifuged at 3000
rpm for 15 seconds and the supernatant was removed. This wash step was repeated three additional
times. After the final wash, two volumes of EB were added to the beads. To pre-clear the extracts, 10μL of
the equilibrated bead solution were added to the extracts and incubated for 30 minutes on a rotating
wheel at 4°C. The samples were centrifuged at 12,000 rpm for 5 minutes at 4°C and then the pre-cleared
extract was transferred to a fresh tube. Then 0.1μg of affinity-purified antibody and 10 ml of the
equilibrated beads were added to the tube. Quantitative RT–PCR Total RNAs were extracted from U2OS wt and mutant cells using RNeasy Mini Kit (QIAGEN, Germantown,
MD). After cDNAs were synthesized from 1 mg of total RNAs using qScript cDNA SuperMix (Quantabio,
Beverly, MA 01915), real-time PCR was performed using SYBR Green according to the manufacturer’s
protocol (PowerTrack SYBR Green Master Mix, Thermo Fisher Scientific) and primers described in the
table below. RNA expression levels were calculated from three experiments. Immunoblotting, gel filtration and Immunoprecipitation This mixture was incubated at 4°C on a rotating wheel for four
hours. The tubes were centrifuged at 3000 rpm for fifteen seconds, and the supernatant was removed. 1mL of EB was added to the tube and incubated on a rotating wheel at 4°C for twenty minutes. The
samples were centrifuged at 3000 rpm for 15 seconds, and the supernatant was removed. This step was
repeated three more times to completely wash the beads. After the final wash, the majority of the EB was
removed and 30μL of 1X sample buffer was added. The samples were boiled at 95°C for 3 minutes. Quantitative RT–PCR Statistics In this study, asterisks indicate significant p-values as follows: *, p<0.05; **, p<0.01; ***, p<0.001. Data
across multiple experiments are shown as mean SD in all graphs except Fig 5H where mean+/-SEM is
shown. One-way ANOVA and Dunnett’s post-hoc tests were used in Fig 5H. Student’s t-test was used to
compare between wt and mutant cells. One-way ANOVA and Dunnett’s post-hoc tests were used for Fig
2C. Adenoviral vectors and plasmids Adenoviral vectors and plasmids To compare subcellular localization between wt PER2 and PER2DCKBD (Per2-E17-17) (Fig 7F), inducible
adenoviruses expressing wt hPER2-Venus or hPER2DCKBD-Venus were generated by replacing the mouse
Per2 cDNA in the previous inducible tetO-Per2; CMV-rtTA-pAdTrack plasmid 37 with wt hPer2 or hPer2
cDNA missing the same CKBD sequence: pAdTrack-iPer2-Venus and pAdTrack-iPer2DCKBD-Venus. Adenoviruses were packaged as described previously 57. For Fig 6J, human CK1ε cDNA was cloned into pUC19 plasmid along with CMV promoter, SV40 polyA
sequence, and GFP. The T2A cleavage sequence was inserted between CK1ε and GFP resulting in CMV-
CK1ε-T2A-GFP-SV40 polyA in pUC19 or pUC19_CK1ε-T2A-GFP. Expression and cleavage of the fusion
protein will generate CK1ε (Fig S7) with additional 18 AAs at the C-terminus (LGGRGSLLTCGDVEENPG). The C-terminal mutant CK1ε was generated by deleting sequence encoding two missing AA (RE) in the
CK1ε mutant clone. pcDNA-Per1, Per2 and CK1d plasmids were described previously 13. For Fig 7J, equal amounts of pAdTrack-iPer2-Venus and pAdTrack-iPer2DCKBD-Venus plasmids were
transfected into wt U2OS cells. On the following day, different titers of adenovirus expressing CK1δ were
infected into the transfected cells; 0, 0.005, 0.05, 0.5, 5 and 50 MOI. Adenovirus expressing wt CK1δ was
generated by cloning cDNA encoding mouse CK1δ into pAdTrack plasmid and packaging in 293A cells as
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effects on circadian rhythmicity. PLoS Genet 4, e12, doi:10.1371/journal.pgen.0040012 (2008). 50 Chiu, J. C., Ko, H. W. & Edery, I. NEMO/NLK phosphorylates PERIOD to initiate a time-delay
phosphorylation circuit that sets circadian clock speed. Cell 145, 357-370, doi:10.1016/j.cell.2011.04.002
(2011). 51 Etchegaray, J. P. et al. Casein kinase 1 delta regulates the pace of the mammalian circadian clock. Mol Cell Biol 29, 3853-3866, doi:10.1128/MCB.00338-09 (2009). 52 Eide, E. J. et al. 10.1016/j.cell.2008.08.022 (2008). Mol Cell 81, 1133-1146, doi:10.1016/j.molcel.2021.01.006 (2021). 64 Narasimamurthy, R. & Virshup, D. M. The phosphorylation switch that regulates ticking of the
circadian clock. Mol Cell 81, 1133-1146, doi:10.1016/j.molcel.2021.01.006 (2021). 65 Shinohara, Y. et al. Temperature-Sensitive Substrate and Product Binding Underlie Temperature-
Compensated Phosphorylation in the Clock. Mol Cell 67, 783-798 e720, doi:10.1016/j.molcel.2017.08.009
(2017). 66 D'Alessandro, M. et al. A tunable artificial circadian clock in clock-defective mice. Nat Commun 6,
8587, doi:10.1038/ncomms9587 (2015). 10.1016/j.cell.2008.08.022 (2008). Control of mammalian circadian rhythm by CKIepsilon-regulated proteasome-
mediated PER2 degradation. Mol Cell Biol 25, 2795-2807 (2005). 53 Xu, H. et al. Cryptochrome 1 regulates the circadian clock through dynamic interactions with the
BMAL1 C terminus. Nat Struct Mol Biol 22, 476-484, doi:10.1038/nsmb.3018 (2015). 54 Park, N. et al. A Novel Bmal1 Mutant Mouse Reveals Essential Roles of the C-Terminal Domain on
Circadian Rhythms. PLoS One 10, e0138661, doi:10.1371/journal.pone.0138661 (2015). 55 Korge, S., Grudziecki, A. & Kramer, A. Highly Efficient Genome Editing via CRISPR/Cas9 to Create
Clock Gene Knockout Cells. J Biol Rhythms 30, 389-395, doi:10.1177/0748730415597519 (2015). 56 Lu, C. et al. Role of circadian gene Clock during differentiation of mouse pluripotent stem cells. Protein Cell 7, 820-832, doi:10.1007/s13238-016-0319-9 (2016). 57 Jin, Y. H. et al. Streamlined procedure for gene knockouts using all-in-one adenoviral CRISPR-Cas9. Sci Rep 9, 277, doi:10.1038/s41598-018-36736-y (2019). 58 Matsu-Ura, T., Baek, M., Kwon, J. & Hong, C. Efficient gene editing in Neurospora crassa with CRISPR
technology. Fungal Biol Biotechnol 2, 4, doi:10.1186/s40694-015-0015-1 (2015). 59 Kim, B. et al. Multiplexed CRISPR-Cas9 system in a single adeno-associated virus to simultaneously
knock out redundant clock genes. Sci Rep 11, 2575, doi:10.1038/s41598-021-82287-0 (2021). 60 Gabriel, C. H. et al. Live-cell imaging of circadian clock protein dynamics in CRISPR-generated knock-
in cells. Nat Commun 12, 3796, doi:10.1038/s41467-021-24086-9 (2021). Page 21/33
61 Putker, M. et al. CRYPTOCHROMES confer robustness, not rhythmicity, to circadian timekeeping. EMBO J 40, e106745, doi:10.15252/embj.2020106745 (2021). w, H. et al. Phosphate groups as substrate determinants for casein kinase I action. J Biol Chem
4-14269 (1990). ando, A., Ruzzene, M. & Pinna, L. A. Casein kinase: the triple meaning of a misnomer. Biochem J
56, doi:10.1042/BJ20140178 (2014). mamurthy, R. & Virshup, D. M. The phosphorylation switch that regulates ticking of the
clock. Mol Cell 81, 1133-1146, doi:10.1016/j.molcel.2021.01.006 (2021). hara, Y. et al. Temperature-Sensitive Substrate and Product Binding Underlie Temperature-
ted Phosphorylation in the Clock. Mol Cell 67, 783-798 e720, doi:10.1016/j.molcel.2017.08.009
sandro, M. et al. A tunable artificial circadian clock in clock-defective mice. Nat Commun 6,
0.1038/ncomms9587 (2015). 62 Flotow, H. et al. Phosphate groups as substrate determinants for casein kinase I action. J Biol Chem
265, 14264-14269 (1990). 63 Venerando, A., Ruzzene, M. & Pinna, L. A. Casein kinase: the triple meaning of a misnomer. Biochem J
460, 141-156, doi:10.1042/BJ20140178 (2014). 64 Narasimamurthy, R. & Virshup, D. M. The phosphorylation switch that regulates ticking of the
circadian clock. 66 D'Alessandro, M. et al. A tunable artificial circadian clock in clock-defective mice. Nat Commun 6,
8587, doi:10.1038/ncomms9587 (2015). Figures Page 22/33 Figure 1
A streamlined selection process using a tandem reporter system successfully and rapid Figure 1
A streamlined selection process using a tandem reporter system successfully and rapidly
knockin (KI) reporter cells. a. KI schematics using a tandem reporter, fluorescence plus bio
In heterozygote KI clones, the non-KI alleles had minor mutations near the stop codon du
i h
KI (Fi
S1) b C ll
i
R b 3 i
l
l
d i
b lk Page 23/33
Figure 1
A streamlined selection process using a tandem reporter system successfully and rapidly identified
knockin (KI) reporter cells. a. KI schematics using a tandem reporter, fluorescence plus bioluminescence. In heterozygote KI clones, the non-KI alleles had minor mutations near the stop codon due to indels
without KI (Figure S1). b. Cells expressing mRuby3 in a low range were selected in bulk and singly sorted
into 96-well plates by FACS. c. The bulk sorted cells and single clones were set up in Lumicycle 96. Note Figure 1 Page 23/33
A streamlined selection process using a tandem reporter system successfully and rapidly identified
knockin (KI) reporter cells. a. KI schematics using a tandem reporter, fluorescence plus bioluminescence. In heterozygote KI clones, the non-KI alleles had minor mutations near the stop codon due to indels
without KI (Figure S1). b. Cells expressing mRuby3 in a low range were selected in bulk and singly sorted
into 96-well plates by FACS. c. The bulk sorted cells and single clones were set up in Lumicycle 96. Note Page 23/33 Page 23/33 that red and blue traces represent specific Per1Luc and Per2Luc KI clones, respectively. d. Three-primer PCR
using two primers binding outside of the homologous arms and a primer binding to luciferase was done
to screen for on-target KI. e. On-target KI (Ho = homozygous, Het = heterozygous) was confirmed by
immunoblotting using anti-PER and anti-Luc antibodies (Supplementary Fig. 2). * non-specific band. Figure 2 Page 24/33 Endogenous PerLuc reporters exhibit robust oscillations without disrupting the clock mechanisms. a. Endogenous PerLuc reporters exhibit robust oscillations without disrupting the clock mechanisms. a. g
p
p
g
Bioluminescence rhythms are shown for representative heterozygous Per1Luc and Per2Luc clones, using a
similar number of cells. Note that PER1-Luc peaks earlier than PER2-Luc. Three replicates are shown for
each clone. Homozygous Per1Luc clones showed higher signals than heterozygous clones demonstrating
that reporter cells are highly quantitative (Supplementary Fig.4a). More clones are shown in
Supplementary Fig. 4b and 4c. b. Per1Luc and Per2Luc exhibited antiphase oscillations to transgenic
Bmal1 promoter-Luc reporter in U2OS cells. c. Circadian period of Per1Luc and Per2Luc reporters is not
significantly different. Representative of three experiments. Mean+/-SD (n=3, two-tailed t-test). *<0.05; **
<0.01; ***<0.001. d. mPer2Luc MEFs showed antiphase oscillations to Bmal1-Luc U2OS cells. e-g. PER-Luc
fusion proteins showed robust oscillations in abundance and phosphorylation almost identical to their wt
counterparts. Bioluminescence rhythms are shown for representative heterozygous Per1Luc and Per2Luc clones, using a
similar number of cells. Note that PER1-Luc peaks earlier than PER2-Luc. Three replicates are shown for
each clone. Homozygous Per1Luc clones showed higher signals than heterozygous clones demonstrating
that reporter cells are highly quantitative (Supplementary Fig.4a). More clones are shown in
Supplementary Fig. 4b and 4c. b. Per1Luc and Per2Luc exhibited antiphase oscillations to transgenic
Bmal1 promoter-Luc reporter in U2OS cells. c. Circadian period of Per1Luc and Per2Luc reporters is not
significantly different. Representative of three experiments. Mean+/-SD (n=3, two-tailed t-test). *<0.05; **
<0.01; ***<0.001. d. mPer2Luc MEFs showed antiphase oscillations to Bmal1-Luc U2OS cells. e-g. PER-Luc
fusion proteins showed robust oscillations in abundance and phosphorylation almost identical to their wt
counterparts. Page 25/33 Figure 3
PER1 and PER2 are significantly different in key parameters. a. Reporter KI did not disrupt endogenous
complex formation. * non-specific band. Representative of two experiments. b, c. PER1 was significantly
more stable than PER2. Representative of three experiments. Mean of three traces are shown (n=3, two-
tailed t-test). p<0.001. d. PER1 phosphorylation and accumulation were significantly faster than those of
PER2 (p<0.001, see Supplementary Fig. 5a for quantitative analysis). Existing PER was depleted by CHX
treatment for 8 hrs (post) PER1 and PER2 are significantly different in key parameters. a. Reporter KI did not disrupt endogenous
complex formation. * non-specific band. Representative of two experiments. b, c. PER1 was significantly
more stable than PER2. Representative of three experiments. Mean of three traces are shown (n=3, two-
tailed t-test). p<0.001. d. Endogenous PerLuc reporters exhibit robust oscillations without disrupting the clock mechanisms. a. PER1 phosphorylation and accumulation were significantly faster than those of
PER2 (p<0.001, see Supplementary Fig. 5a for quantitative analysis). Existing PER was depleted by CHX
treatment for 8 hrs (post). Page 26/33 Page 26/33 Fi
4 Figure 4 Figure 4 Per2 phase is reversed in the absence of the Per1 gene in peripheral cells. a. Per KO was screened by
immunoblotting. b,c. Bioluminescence rhythms of representative Per2 KO in Per1Luc and Per1 KO in
Per2Luc cells are shown. Two traces are shown for each clone. n=3 total. d. Circadian period was not
significantly different between Per KO and wt reporter cells. Mean+/-SD (n=3, two-tailed t-test). Representative of three experiments. e, f. Per KO in Bmal1-Luc U2OS cells showed similar results. Two
traces are shown for each clone. g. Antiphase oscillations of PER2 between wt and Per1 KO cells were Page 27/33 Page 27/33 confirmed by immunoblotting for PER2. h. Early response of PER2 to horse serum treatment (an
established zeitgeber for cultured cells) was dramatically different between wt and Per1 KO cells. Cells
were given a 2-hour serum shock and harvested continuously for another 14 hours. Note that PER2
oscillation seems to be coupled to that of PER1 in wt cells. Representative of two experiments. Figure 5 Figure 5 Figure 5 Page 28/33 Deletion of Cry genes in the reporter cells reveals functional difference between two CRYs. a. Cry KOs
were screened by altered bioluminescence rhythms and confirmed by immunoblotting. b-d. Cry KO
phenotypes were consistent with mouse genetics data. Each trace represents a different clone and is
representative of three traces. Mean+/-SD of two clones each for Cry1 and Cry2 KO in Per2Luc is shown in
d (n=3 per clone, two-tailed t-test) . e. PER protein levels were elevated in Cry1 KO cells. f, g. PER1-Luc and
PER2-Luc signals were higher and lower, respectively in PerLuc reporter cells. A normalized
bioluminescence graph is shown in f to compare apparent period between wt and Cry1/2 double KO cells. h. Modulations of PER proteins by Cry KO are explainable by modulations in Per mRNA levels. Note that
Per1 mRNA levels were elevated in Cry1 and Cry1/2 KO cells but those of Per2 mRNA were significantly
elevated only in Cry1 KO cells. Mean+/-SEM of three RT-qPCR experiments (one-way ANOVA and
Dunnett’s post-hoc tests). i. Modulation of PER proteins in Cry1/2 KO cells was confirmed by
immunoblotting. j. PER proteins are constitutively hypophosphorylated in Cry1/2 double-KO cells. *
nonspecific band. Deletion of Cry genes in the reporter cells reveals functional difference between two CRYs. a. Cry KOs
were screened by altered bioluminescence rhythms and confirmed by immunoblotting. b-d. Figure 4 Cry KO
phenotypes were consistent with mouse genetics data. Each trace represents a different clone and is
representative of three traces. Mean+/-SD of two clones each for Cry1 and Cry2 KO in Per2Luc is shown in
d (n=3 per clone, two-tailed t-test) . e. PER protein levels were elevated in Cry1 KO cells. f, g. PER1-Luc and
PER2-Luc signals were higher and lower, respectively in PerLuc reporter cells. A normalized Page 29/33 Figure 6
C-termini of CK1δ/ε play important roles in rhythm generation. a. Deletion of CK1δ/ε w Page 30/33
Figure 6
C-termini of CK1δ/ε play important roles in rhythm generation. a. Deletion of CK1δ/ε was screened by
altered bioluminescence rhythms and confirmed by immunoblotting. * nonspecific band. Note that CK1e
is detected in double KO #2 in Per1Luc reporter cell due to an in-frame mutation in one allele (Per1Luc
CKKO-2) (see Supplementary Fig. S7). b-d. Single KOs of CK1δ and CK1ε and C-terminus deletion of
CK1ε all lead to lengthened rhythms. Two representative clones for each KO are shown in d. Mean+/-SD Figure 6
C-termini of CK1δ/ε play important roles in rhythm generation. a. Deletion of CK
altered bioluminescence rhythms and confirmed by immunoblotting. * nonspecif
is detected in double KO #2 in Per1Luc reporter cell due to an in-frame mutation i
CKKO 2) (see Supplementary Fig S7) b d Single KOs of CK1δ and CK1ε and C Figure 6
f
δ
l
l
h
h Figure 6 Page 30/33
C-termini of CK1δ/ε play important roles in rhythm generation. a. Deletion of CK1δ/ε was screened by
altered bioluminescence rhythms and confirmed by immunoblotting. * nonspecific band. Note that CK1e
is detected in double KO #2 in Per1Luc reporter cell due to an in-frame mutation in one allele (Per1Luc
CKKO-2) (see Supplementary Fig. S7). b-d. Single KOs of CK1δ and CK1ε and C-terminus deletion of
CK1ε all lead to lengthened rhythms. Two representative clones for each KO are shown in d. Mean+/-SD Page 30/33 Page 30/33 (n=3 per clone, two-tailed t-test). e-g, CK1δ/ε double-mutant clones showed dramatically lengthened
rhythms. Note that CKKO-2 in f represents the double-mutant clone with the in-frame mutation in C-
terminus instead of a complete knockout via a frame-shifting mutation. Mean+/-SD (n=3 per clone, two-
tailed t-test). h. Hyperphosphorylated PER species were pronounced in CK1 double mutant cells. CKKO-1
clone in Per1Luc reporter cell is shown. * nonspecific band. i. PER, especially PER1 was stabilized in
double mutant clones. CKKO-1 clone in Per1Luc reporter cell is shown. j. PER interaction with a mutant
CK1ε with two AA deletion (RE) in C-terminus was significantly attenuated. PER was co-expressed with wt
CK1e or mut CK1e and subjected to coimmunoprecipitation (coIP). Note that small amounts of
transgenic PER were copurified with endogenous CK1e by the anti-CK1e antibody. Transgenic CK1e is
larger than the endogenous counterpart by 18 AAs. (n=3 per clone, two-tailed t-test). e-g, CK1δ/ε double-mutant clones showed dramatically lengthened
rhythms. Note that CKKO-2 in f represents the double-mutant clone with the in-frame mutation in C-
terminus instead of a complete knockout via a frame-shifting mutation. Mean+/-SD (n=3 per clone, two-
tailed t-test). h. Hyperphosphorylated PER species were pronounced in CK1 double mutant cells. CKKO-1
clone in Per1Luc reporter cell is shown. * nonspecific band. i. PER, especially PER1 was stabilized in
double mutant clones. CKKO-1 clone in Per1Luc reporter cell is shown. j. PER interaction with a mutant
CK1ε with two AA deletion (RE) in C-terminus was significantly attenuated. PER was co-expressed with wt
CK1e or mut CK1e and subjected to coimmunoprecipitation (coIP). Note that small amounts of
transgenic PER were copurified with endogenous CK1e by the anti-CK1e antibody. Transgenic CK1e is
larger than the endogenous counterpart by 18 AAs. Page 31/33 Figure 7
CKBD allows for slow phosphorylation kinetics essential for the phosphotimer. a, b. Figure 6 A P
lacking the CKBD showed lengthened rhythms in a Per1 wt background but arrhythmici Page 32/33
Figure 7
CKBD allows for slow phosphorylation kinetics essential for the phosphotimer. a, b. A Per2 mutant clone
lacking the CKBD showed lengthened rhythms in a Per1 wt background but arrhythmicity in Per1 KO cells. Note that PER2-Luc signals were elevated in Per1 wt and further elevated in the Per1 KO background. A
similar number of cells was used. A smaller size mutant protein was confirmed by sequencing of cDNA
(Supplementary Fig. 8a) and immunoblotting (bottom panel in a). c. The mutant PER2 seemed to be CKBD allows for slow phosphorylation kinetics essential for the phosphoti Page 32/33
CKBD allows for slow phosphorylation kinetics essential for the phosphotimer. a, b. A Per2 mutant clone
lacking the CKBD showed lengthened rhythms in a Per1 wt background but arrhythmicity in Per1 KO cells. Note that PER2-Luc signals were elevated in Per1 wt and further elevated in the Per1 KO background. A
similar number of cells was used. A smaller size mutant protein was confirmed by sequencing of cDNA
(Supplementary Fig. 8a) and immunoblotting (bottom panel in a). c. The mutant PER2 seemed to be Page 32/33 Page 32/33 constitutively hyperphosphorylated based on mobility shift on the immunoblot. d. Hyperphosphorylation
was confirmed by phosphatase treatment. Two exposures are shown. Representative of two experiments. e. Phosphorylation of the mutant PER2 is mediated by CK1δ/ε. Lower doses were used in Supplementary
Fig. 8d. f. The mutant PER2 was constitutively nuclear. Wt PER2 was detectable in both cytoplasm and
nucleus. When wt PER2 was expressed in high amounts, they were predominantly cytoplasmic
(Supplementary Fig. 9) 37. g. The mutant PER2 was significantly more stable than wt PER2. h. The
mutant PER2 was not additionally phosphorylated by CA treatment (see extra hyperphosphorylation in
Supplementary Fig. 6b). i. Phosphorylation of de novo mutant PER2 was significantly advanced
compared to its wt counterpart. Time ‘0 hr’ represents 8 hrs after CHX treatment right before washout. Note that a significant amount of the mutant PER2 still existed after CHX treatment. j. The mutant PER2
is less phosphorylated in vitro by CK1δ compared to wt PER2. Increasing amounts of CK1δ were co-
expressed with a fixed amount of PER2. constitutively hyperphosphorylated based on mobility shift on the immunoblot. d. Hyperphosphorylation
was confirmed by phosphatase treatment. Two exposures are shown. Representative of two experiments. e. Figure 6 Phosphorylation of the mutant PER2 is mediated by CK1δ/ε. Lower doses were used in Supplementary
Fig. 8d. f. The mutant PER2 was constitutively nuclear. Wt PER2 was detectable in both cytoplasm and
nucleus. When wt PER2 was expressed in high amounts, they were predominantly cytoplasmic
(Supplementary Fig. 9) 37. g. The mutant PER2 was significantly more stable than wt PER2. h. The
mutant PER2 was not additionally phosphorylated by CA treatment (see extra hyperphosphorylation in
Supplementary Fig. 6b). i. Phosphorylation of de novo mutant PER2 was significantly advanced
compared to its wt counterpart. Time ‘0 hr’ represents 8 hrs after CHX treatment right before washout. Note that a significant amount of the mutant PER2 still existed after CHX treatment. j. The mutant PER2
is less phosphorylated in vitro by CK1δ compared to wt PER2. Increasing amounts of CK1δ were co-
expressed with a fixed amount of PER2. Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. ParketalSupplInfoNatComm070922submitted.docx Supplementary.docx Supplementary.docx Page 33/33
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Shining a Light on Photoresponsive Type III Porous Liquids
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Shining a Light on Photoresponsive Type III Porous Liquids
Michael. C. Brand,a Nicola Rankin,a Andrew I. Cooper,a Rebecca L. Greenawaya,b* a Department of Chemistry, Materials Innovation Factory, University of Liverpool, 51 Oxford Street,
Liverpool, L7 3NY, UK. b Department of Chemistry, Molecular Sciences Research Hub, Imperial College London, 82 Wood
Lane, London, W12 0BZ, UK. b Department of Chemistry, Molecular Sciences Research Hub, Imperial College London, 82
Lane, London, W12 0BZ, UK. Email: r.greenaway@imperial.ac.uk Email: r.greenaway@imperial.ac.uk Email: r.greenaway@imperial.ac.uk Porous materials are the subject of extensive research because of potential
applications in areas such as gas adsorption and molecular separations. Until recently,
most porous materials were solids, but there is now an emerging class of materials
known as porous liquids. The incorporation of intrinsic porosity or cavities in a liquid
can result in free-flowing materials that are capable of gas uptakes that are significantly
higher than conventional non-porous liquids. A handful of porous liquids have also
been investigated for gas separations. Until now, the release of gas from porous liquids
has relied on molecular displacement (e.g., by adding small solvent molecules),
pressure or temperature swings, or sonication. Here, we explore a new method of gas
release that involves photoisomerisable porous liquids comprising a photoresponsive
MOF dispersed in an ionic liquid. This results in the selective uptake of CO2 over CH4,
and allows gas release to be controlled by using UV light. 1 1 Introduction There are already excellent examples of what conventional (non-porous) liquid solvents can
achieve in industrial processes such as ‘wet-scrubbing’ for CO2 capture, which involves
aqueous amine solutions to chemically absorb CO2.23 However, this typically requires large
amounts of energy to regenerate the liquid.24 Porous liquids, on the other hand, might be
adapted into existing infrastructure that uses conventional flow processes, while also lowering
the energy penalty of solvent regeneration by exploiting physisorption rather than
chemisorption.25,26 In addition, the incorporation of discrete molecules or frameworks into a
liquid also adds the potential for shape and size specificity, allowing porous liquids to be
tailored towards target separations and applications.27,28 2 2 Figure 1 a) Illustration of the components used to form a photoresponsive Type III porous liquid from
the MOF Zn(AzDC)(4,4‘-BPE)0.5 and the ionic liquid [BMIM][NTf2]; b) Illustration of the uptake and
release cycle for CO2 in the photoresponsive porous liquid. Figure 1 a) Illustration of the components used to form a photoresponsive Type III porous liquid from
the MOF Zn(AzDC)(4,4‘-BPE)0.5 and the ionic liquid [BMIM][NTf2]; b) Illustration of the uptake and
release cycle for CO2 in the photoresponsive porous liquid. To date, porous liquids have been mostly studied for their ability to absorb gas, and only a
handful of materials have been explored for gas selectivity. So far, little research has gone
into understanding the gas release mechanisms of porous liquids. Several methods for
controlled gas release from porous liquids have been reported, each providing its own
challenges. As for porous solids, the most common gas release mechanisms are pressure
and temperature swings.11,29,30 This significantly limits the solvent choice in a porous liquid,
restricting us to solvents that have a low or (ideally) near-zero vapour pressure.11,31 There is
also a significant parasitic energy penalty with pressure and temperature swings. Moreover,
some materials might not be stable to temperature swings; for example, it was reported that
some porous liquids are unable to withstand temperature displacement over multiple cycles.11
An alternative method to release gases is based on guest exchange by chemical
displacement, where the addition of a small molecule with a more favourable binding in the
cavity is used to displace the gaseous guest from the pores.11,32,33 While effective in the
laboratory, this method has very limited practical appeal because the displacing molecule is
harder to remove than the gas. Introduction Porous liquids are a class of porous material that has recently caught the attention of
researchers. First envisaged in 2007 by James and co-workers,1 they later became a reality
in 2015 where the first porous liquids were realised in the laboratory.2,3 These liquids differ
from traditional solvents by having intrinsic micropores, allowing these materials to retain
permanent porosity that leads to increased gas solubility and, in some cases, gas selectivity;
that is, properties that are difficult to achieve with conventional non-porous liquids.4–7 Porous
liquids can be classified by the following categories: Type I porous liquids are discrete liquid
hosts where the liquid component has shape-persistent molecular porosity;8,9 Type II porous
liquids are prepared by dissolving discrete porous molecules, such as porous organic cages
(POCs), in a solvent that is excluded from the pores;10,11 Type III porous liquids are dispersions
of porous solid particles, such as metal organic frameworks (MOFs), in a fluid where the
solvent is excluded from the pores;12,13 Type IV porous liquids are materials with extended
connectivity in three dimensions that retains porosity in the liquid state.14,15 Since their
conceptualisation, there have now been reports of all four types of porous liquids.9,14,16–18 While porous solids are applicable in a wide range of applications, including molecular
separations and catalysis,19–21 liquids have potential differentiable advantages because they
can flow and might also promote more rapid heat transfer and dissipation.22 By contrast, the
porosity per unit volume for porous liquids is thus far much lower than for porous solids. As
such, porous liquids might allow for new, different processes, rather than simply replacing
porous solids. Introduction Lastly, sonication has been used to release gaseous guests
from porous liquids.30 While this method is a non-invasive alternative to chemical
displacement, the relative parasitic energy costs are as yet unclear and more problematically, To date, porous liquids have been mostly studied for their ability to absorb gas, and only a
handful of materials have been explored for gas selectivity. So far, little research has gone
into understanding the gas release mechanisms of porous liquids. Several methods for
controlled gas release from porous liquids have been reported, each providing its own
challenges. As for porous solids, the most common gas release mechanisms are pressure
and temperature swings.11,29,30 This significantly limits the solvent choice in a porous liquid,
restricting us to solvents that have a low or (ideally) near-zero vapour pressure.11,31 There is
also a significant parasitic energy penalty with pressure and temperature swings. Moreover,
some materials might not be stable to temperature swings; for example, it was reported that
some porous liquids are unable to withstand temperature displacement over multiple cycles.11 3 sonication may be undesirable for large scale processes (e.g., cavitation can damage metal
containment vessels).34 Here, we investigate the formation of photoresponsive type III porous liquids. We exploited
well-known photochromic molecules, azobenzene and 1,2-bis(4-pyridyl)ethylene, both of
which have been incorporated into a zinc-based MOF (Figure 1a) that is capable of efficient
and reversible cis to trans photoisomerisation when exposed to UV or visible light (trans: max
≈ 370 nm, cis max ≈ 460 nm). This system was previously reported by Chen et al. and later
studied by Lyndon et al. for its dynamic photoswitching in the solid state; specifically a change
in CO2 uptake was reported upon photoswitching.35,36 By dispersing this photoresponsive MOF
in an ionic liquid as the size-excluded solvent, we have developed a new Type III porous liquid,
creating what we believe to be the first proof-of-concept photoresponsive porous liquid that is
capable of selective gas uptake and UV light-triggered release (Figure 1b). Results & Discussion First, we built a suitable photoreactor that would allow us to carry out sorption measurements
while irradiating the sample (Figure S1). For this, we designed a reactor that could
accommodate a gas sorption tube and light source, with a stirrer plate positioned underneath
the porous liquid sample during the gas sorption experiment (Figure S2). This design was
subsequently 3D printed using a resin printer. The first prototypes used an LED light source
that did not produce enough power output and that also caused an increase in the internal
temperature in the reactor. This was mitigated by using an external light source in tandem with
fans mounted to either side of the reactor, with small passages to create a push-pull airflow
through the reactor. The temperature was monitored over the course of several experiments,
and it remained in the range 28.5±0.5 °C, regardless of whether the light source (365 nm) was
switched on (Figure S3). The CAD design and 3D print files are available at
github.com/GreenawayLab/Sorption-Photoreactor. The photoresponsive MOF Zn(AzDC)(4,4‘-BPE)0.5 was chosen based on previous reports that
it exhibits a decrease in gas uptake when exposed to UV light: a difference of 64% was found
under dynamic conditions and 42% under static conditions.36 In addition, this material was
noted for having selective adsorption for carbon dioxide over methane. In our hands, the CO2
uptake of this MOF varied considerably between batches, ranging from 277–766 µmol/g at 1
bar / 301 K, even when the synthesis and activation was carried out on the same scale (Figure
S5). For the batch with the largest CO2 uptake, a reduction in CO2 uptake of 31% was achieved 4 4 under static irradiation conditions at 365 nm with 100% exposure in the 3D printed reactor
(CO2 uptake, 1 bar: 766 µmol/g, ambient; 526 µmol/g under UV irradiation). Next, a suitable solvent was selected to produce a Type III porous liquid. To date, a wide
variety of liquids have been used, including organic solvents, oils, and ionic liquids.37,38 For
this study, we screened a small number of liquids that have previously been reported as being
size-excluded for POCs and MOFs, including silicone oil AR 20, and the ionic liquids 1-
butylpyridinium bis(trifluoromethanesulfonyl)imide ([BPy][NTf2]), 1-ethyl-3-(hydroxymethyl)
pyridinium
ethyl
sulfate
([EtHPy][EtS]),
and
1-butyl-3-methylimidazolium
bis(trifluoromethylsulfonyl)imide ([BMIM][NTf2]). Results & Discussion Ionic liquids are good candidates for porous
liquids due to their low vapour pressures, compatibility with organic and inorganic materials,
and their ability to be tailored to suit different applications. To screen these solvents for size-
exclusion from the MOF, 12.5 wt. % dispersions were prepared, and the CO2 uptake was
measured under ambient conditions (i.e., with no exposure to UV or blue light) while stirring
at 300 rpm (Figure S4). While several of these combinations showed enhanced CO2 uptake
compared to the neat liquids, which indicated the successful formation of Type III porous
liquids, silicone oil AR 20 and [BMIM][NTf2] demonstrated the highest CO2 uptakes when
compared to the neat liquids. [BMIM][NTf2] was selected due to the larger increase in CO2
uptake on going from the neat liquid to the 12.5 wt. % dispersion. The porous liquid also
demonstrated excellent CO2/CH4 selectivity, whereas the silicone oil had a CH4 uptake that
was four times greater than the neat ionic liquid [BMIM][NTf2]. In addition, for the specific
sample of MOF and batch of ionic liquid used in this screen, the CO2 uptake in the 12.5 wt. %
dispersion corresponded to 74% of the theoretical total uptake, based on the calculated CO2
uptake from the proportion of the MOF (749 µmol/g) combined with the uptake of the neat
liquid (83 µmol/gL); that is, the porous liquid expresses 74% of the total uptake that would be
expected assuming a linearly additive system upon mixing. 5 5 Figure 2 a) CO2 adsorption isotherms for Zn(AzDC)(4,4‘-BPE)0.5 [BMIM][NTf2] porous liquid and neat
[BMIM][NTf2] ionic liquid under ambient conditions (filled) and UV light (emtpy); b) Measured gas uptake
across a range of concentrations of porous liquid, under ambient conditions (blue squares) and constant
irradiation (purple circles), with the corresponding linear fit lines. Figure 2 a) CO2 adsorption isotherms for Zn(AzDC)(4,4‘-BPE)0.5 [BMIM][NTf2] porous liquid and neat
[BMIM][NTf2] ionic liquid under ambient conditions (filled) and UV light (emtpy); b) Measured gas uptake
across a range of concentrations of porous liquid, under ambient conditions (blue squares) and constant
irradiation (purple circles), with the corresponding linear fit lines. To investigate the photoresponsive nature of the porous liquid formed with ionic liquid
[BMIM][NTf2], we prepared a series of 5, 12.5 and 20 wt. % samples (based on the MOF
loading) directly in gas sorption tubes and measured the CO2 uptake. Results & Discussion First, the batch of MOF
with the highest CO2 uptake (766 µmol/g) was investigated. We observed a proportionate
increase in CO2 uptake upon increasing the wt. % loading of the MOF in the porous liquid
(Figure 2; Table S2). The CO2 uptake measurements were then repeated for each porous
liquid sample while exposing them to UV light (365 nm) for the full duration of the sorption
experiment. All samples demonstrated a consistent decrease in CO2 uptake at 1 bar, with
decreases of 17%, 16%, and 16% observed for samples with MOF loadings of 5, 12.5 and 20
wt. %, respectively (Figure 2b). 6 A decrease in CO2 solubility was also observed in the neat ionic liquid [BMIM][NTf2] upon UV
irradiation (63 µmol/gL, ambient; 50 µmol/gL, UV irradiation). Since the temperature in the
photoreactor remained constant during the sorption measurements, it is unlikely that this was
the cause of decrease in CO2 solubility. To our knowledge, there are few studies investigating
the effects of UV irradiation on ionic liquids. However, a report by Rao et al.39 notes changes
in the UV-vis and fluorescence spectra on a similar ionic liquid, [BMIM][BF4], but concludes
that there is very little change in the bulk of the material. For [BMIM][NTf2], changes are
observed in the UV-vis spectra on irradiation (Figure S17), which may be responsible for the
observed changes in gas solubility, but no structural changes were observed in the 1H NMR
spectra (Figure S18). In addition, the difference in CO2 uptake upon irradiation in the ionic
liquid (13 µmol/gL) was lower than for the porous liquids at higher MOF loadings (12.5 wt. %,
23 µmol/gPL; 20 wt. %, 29 µmol/gPL), confirming an additional contribution from the
incorporated MOF. Given the observed difference in CO2 uptake across the different batches of MOF (Figure S5),
we were interested to see whether there was a direct correlation with the resulting uptake in
the subsequent porous liquid. Therefore, 5, 12.5, and 20 wt. % porous liquid samples
incorporating two further batches of MOF with CO2 uptakes of 457 µmol/g (Figure S6-S7,
Table S1) and 277 µmol/g (Figure S12-S13, Table S3) were investigated. As might be
expected for a Type III porous liquid where the solid pore carrier is simply dispersed, there
was a linear trend between the measured uptake in the solid MOF and the resulting uptake in
the porous liquid under ambient conditions (Figure S15). Results & Discussion Similarly, the reduction in CO2
uptakes was consistent under UV irradiation across the different samples (Figure S7, S13). The porous liquids investigated (using the three different batches of MOF with uptakes of 277,
457 and 766 µmol/g), both under ambient conditions and under UV irradiation, demonstrated
at least 82% of the theoretical calculated total uptakes. The MOF also retained its bulk
crystallinity after being dispersed in the ionic liquid and after gas uptake measurements under
both ambient conditions and under UV irradiation (Figure S8, S11, S14). We next investigated the cyclability and reproducibility of CO2 uptake in the photoresponsive
porous liquid on switching between UV (365 nm) and blue (450 nm) light (Figure 1b). A 12.5
wt. % porous liquid sample was prepared and cycled through 10 experiments where it was
continually exposed to UV light for the ‘on’ experiments, before being exposed to blue (450
nm) light for 10 minutes and then starting the ‘off’ experiment (Figure 3). Overall, the average
CO2 uptake was 97.9±2.0 mol/gPL after the porous liquid was exposed to blue light, compared
to 90.4±1.6 mol/gPL when the porous liquid was exposed to UV light – this is a decrease of
7.5 mol/gPL, equating to an 8% reduction of CO2 in the porous liquid when exposed to UV
light. While this was lower than the previously observed 16%, we believe this is due to partial 7 saturation since the sample was not left to degas for 24 hours, unlike the initial experiments,
but rather was only degassed for 60 minutes between measurements. Hence the sample is
likely still partially saturated with gas leading to a lower CO2 capacity and smaller difference
when the sample is exposed to UV light. Figure 3 Recycling study of the 12.5 wt. % porous liquid for CO2 uptake with UV light on (365 nm,
purple circles) and light off (exposed to 450 nm for 10 minutes, blue squares). Figure 3 Recycling study of the 12.5 wt. % porous liquid for CO2 uptake with UV light on (365 nm,
purple circles) and light off (exposed to 450 nm for 10 minutes, blue squares). We also studied the gas selectivity of this porous liquid. First, the CH4 uptake was investigated
in the neat ionic liquid and in the 20 wt. % porous liquids formed using the three different
batches of MOF. Results & Discussion Little to no CH4 uptake was observed, and all of the porous liquid uptakes
were well below that of the theoretical gas uptakes (Table S1, S2 and S3). For the MOF with
the highest uptake in the solid state, there was a very small increase in CH4 uptake from 4.0
mol/gPL in the neat ionic liquid to 13.4 mol/gPL in the porous liquid; this demonstrates a 13-
fold increase of CO2 over CH4 for the 20 wt. % porous liquid, which we believe is the highest
CO2/CH4 selectivity of any reported porous liquid to date. We ascribe the lack of CH4 uptake
in the porous liquid to the very low solubility of methane in the ionic liquid carrier solvent and
the preferential CO2 uptake observed in the photoresponsive MOF in the solid-state. Finally, different methods of controlled gas release were investigated using CO2-loaded
porous liquid samples, as measured by collecting and measuring the amount of gas released
(Error! Reference source not found.4; Figure S16, Table S4-S5). This included irradiation
at 365 nm while stirring, sonication in a room temperature water bath, heating and stirring at
80 °C, and chemical displacement via the addition of chloroform. Increasing the temperature
of the sample to 80 °C resulted in the highest quantity of CO2 being released, displacing
98.6±3.9 mol/gPL. Both sonication and UV irradiation performed equally well, displacing 8 82.2±7.3 mol/gPL and 79.8±8.7 mol/gPL, respectively. Chemical displacement performed the
least well of the methods tested, displacing 75.9 mol/gPL. In all cases, the amount of gas
released by the porous liquid was significantly higher than that of the neat ionic liquid and
irradiation showed the largest difference, with a 426% increase. The other release methods
ranged from a 115% to 341% increase over the neat ionic liquid. This shows that
photochemical release is almost as effective as traditional methods, such as temperature
swing in terms of the total amount of gas released. Figure 4 Gas displacement experiments for CO2 using different mechanisms of release, including UV,
sonication, heating (80 °C), and chemical displacement (0.4 mL CHCl3), in the 12.5 wt. % porous liquid
(solid) and ionic liquid (dashed lines). Figure 4 Gas displacement experiments for CO2 using different mechanisms of release, including UV,
sonication, heating (80 °C), and chemical displacement (0.4 mL CHCl3), in the 12.5 wt. % porous liquid
(solid) and ionic liquid (dashed lines). Conclusions In conclusion, a photoresponsive Type III porous liquid was formed by dispersing a MOF
containing photoswitchable groups in a size-excluded ionic liquid. Flowable dispersions up to
20 wt. % could be formed, and UV light was successfully used as a gas release mechanism. These porous liquids demonstrate enhanced CO2 uptake over the neat ionic liquid and exhibit
excellent CO2/CH4 selectivity. While the CO2 uptake was found to vary between different
batches of the synthesised MOF, a linear relationship was found between the uptake in the
solid and the resulting uptake in the porous liquids. Taken together, these results suggest
alternative paradigms for selective gas capture and release using material compositions that
can flow. 9 9 14
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4, 1–12. 12 Acknowledgements We thank the Engineering and Physical Sciences Research Council (EPSRC) under the
Grants EP/R005710/1 and EP/W01601X/1, and the Leverhulme Trust via the Leverhulme
Research Centre for Functional Materials Design for financial support. R.L.G. thanks the Royal
Society for a University Research Fellowship. For the purpose of open access, the author has
applied a Creative Commons Attribution (CC BY) licence to any Author Accepted Manuscript
version arising. Corresponding Author Correspondence to R. L. Greenaway Correspondence to R. L. Greenaway Competing Interests The authors declare no competing interests. 13
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Here or There? How Configuration of Transnational Teams Impacts Social Capital
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hainesj, jsolson, gary.olson}@uci.edu {hainesj, jsolson, gary.olson}@uci.edu Abstract. The many challenges of distributed communication and the many
challenges of intercultural collaboration have been researched and discussed at
length in the literature. What is lacking is a combined approach that looks at
both issues of distance and diversity in collaboration. We conducted research in
a large, multinational technology company to better understand team configura-
tional factors in transnational work. In this case study, we found that the devel-
opment of social capital is impacted by whether a person is in their home con-
text or transplanted and their expectations based on that context. This has impli-
cations for the development of intellectual capital in the team. We highlight fac-
tors in the creation of social capital as well as some mechanisms that may miti-
gate cultural difference. In addition to bringing into focus the challenges that
arise in various configurations, this study contributes to the transnational litera-
ture by highlighting the importance of local context in diverse collaborations. Keywords: Transnational collaboration, team configuration, social capital Keywords: Transnational collaboration, team configuration, social capital 1. Introduction Many organizational structures today are truly global. Companies find talent all over
the world, place employees in all sorts of geographic locations, and coordinate teams,
both distributed and co-located [1]. This sort of transnational work requires crossing
all sorts of important boundaries, including temporal, spatial, and cultural. Much has
been written about the multitude of issues in working across spatial and temporal
distances [2-6]. And a great deal of research has focused on transnational or cross-
cultural differences as they related to the workplace [7-9]. Yet, given the prevalence
and significance of transnational work in today’s globalized world, there appears to be
a lack of empirical studies that focus on the variety of ways such collaboration can be
structured and the consequences of different configurations [10-13]. Global organiza-
tions can choose to configure and structure their teams in a wide variety of ways, and
it is important to look at the costs and benefits associated with these choices. First, why might you want to have diverse cultural configurations? Research that
has focused on the value of diversity in groups illustrates that, for many tasks, diver-
sity trumps homogeneity [14] by allowing for a diversity of perspectives, which aid in a number of ways, such as problem solving, information aggregation, and prediction. Other studies have indicated that cultural diversity leads to increased creativity and
satisfaction of experience [15], and with the right mechanisms in place, can result in
better cohesion, trust, and innovation [16]. Most work in this area suggests, however,
that the net benefits of such diversity must account for the costs related to differences
in cognitive, behavioral, and affective aspects [13]. Diversity can most certainly be an
asset, but it can also a liability if not structured and managed well. Second, from an organization’s perspective, why might you choose to have a team
distributed over continents and time zones as opposed to co-located in one area? As
Castells and others have suggested, today’s knowledge-based workforces are defined
by their flexibility and adaptability [17], and it is quite possible to configure teams in
many ways [18]. Benefits to globally distributed collaboration include garnering ex-
pertise in local markets, cost reductions, and a 24-hour development cycle [19, 20]. But, there are still many challenges for distributed teams in terms of processes, tools,
and structuring [1, 21]. And, there are particular difficulties for distributed teams that
cross cultures [13, 22]. 1. Introduction With all the permutations possible, it is important to understand the implications of
various transnational team configurations. In our research, we found that the most
salient implications are related to interpersonal relationships. Thus, we use the theory
of social capital in framing our findings. This theory highlights the importance of
interpersonal relationships, which serve as a valuable resource for social action within
a network [23]. Nahapiet and Ghoshal’s work on social capital in the organization
shows that social relationships within a firm, which are the foundations of social capi-
tal, are intricately tied to the firm’s ability to create new intellectual capital, or knowl-
edge [23]. This theory serves well as a lens to investigate the transnational configura-
tions within firms, enabling insight into interpersonal relationships at the individual
level as well as insight into overall team and organizational functioning. In this paper, we contribute to the transnational literature by examining how the
configuration of global teams and the context of an individual’s location within that
team impact social capital. We seek to examine how distributed and co-located con-
figurations of transnational teams are similar and different in terms of the social rela-
tionships that are developed, and in turn, how that impacts the individual and the
team. We recognize, as others do [11, 24], that one’s culture is not static, and that
although culture influences one’s behavior, behavior is also influenced by the situa-
tion. Over time people adapt to their context, either converging toward the local cul-
ture or diverging. Thus, we focus on these central ideas of location and configuration. 2. Method We conducted research within a large, multinational high-technology company, which
we will refer to by the pseudonym PrimaTech. PrimaTech is headquartered in the US,
but has offices in more than 40 countries worldwide. The structure of the organization
is complex and dynamic, with many teams distributed all over the globe. 2.2 Interviews Prior to data collection, we conducted extensive background research to better under-
stand common cultural characteristics amongst the various national cultures we con-
sidered for the study. In all, we created a bibliography of 88 sources. From this meta-
analysis, we created an informational table that included aspects of culture in the US,
China, India, Japan, Korea, and Singapore along 29 dimensions, including behavioral
elements, cognitive and perception differences, and macro-level social conventions. This table was used to inform the interview script. We conducted pilot interviews
with contacts of Chinese and Indian origin and refined the scripts according to their
feedback, which included areas for further inquiry as well as suggestions on how to
approach certain delicate topics. The interviews themselves were conducted in a semi-structured format to encour-
age participants to openly discuss their perspectives and reflect on experiences. We
collected information on participants’ backgrounds and their work at PrimaTech,
including team collaboration methods and tools, their cross-cultural experiences, and
perspectives on differences in communication and social conventions. We visited the
company’s US headquarters and conducted a portion of the recruiting there, but all
interviews were conducted remotely via video-conferencing. Interviews lasted from
around 45 minutes to over an hour. Participants were not compensated. 2.1
Participants We specifically chose to focus on participants from India and China, as a significant
portion of PrimaTech’s employees come from these two countries. Participants were
independently recruited at multiple work sites using a purposive sampling methodol-
ogy [25]: we broadcast our study requirements on various listservs, and when people
responded, we selected them according to their background, work situation, and
availability for interview. Among the 22 we selected, nine of whom were female, all
were of Indian (13) or Chinese (9) national origin currently working in either the US
or offices in their home country, and all participants had previously or were currently
collaborating in a transnational team. Thirteen were based currently in the US, nine in
offices abroad (Bangalore, Beijing, and Hyderabad). Six had spent two or more years
in the US offices, eight less than two years, and eight spent no time at US offices. Table 1 shows these participant aspects in more detail. 2.3 Teams Teams at PrimaTech are often comprised of people of many nationalities, so partici-
pants were not necessarily working primarily with American-born colleagues, but
truly transnational teams. We use the term transnational here to indicate that the teams
were composed of two or more nationalities, indicating diversity that goes beyond just
different cultures, which can occur within nations [13]. In this research, we look both
at transnational teams with participants in their home countries of India and China
working remotely with those in the US and other countries and transnational teams in Table 1. Characteristics of Participants T/R*
Gender
Nation
Loc. Yrs
at
Co. 2.3 Teams Yrs
US
Office
Role
#
Offices
work
with
Time
on
team
(yrs)
Team
size**
Some
Members
Co-
located
T1
M
India
US
3
0.5
Manager
4
< 1
L
Y
T2
F
China
US
8
8
Infrastructure
7
> 1
L
Y
T3
M
China
US
6
6
Engineer
1
> 1
S
Y
T4
F
China
US
6
0.25
Consumer
Operations
5
< 1
L
Y
T5
M
China
US
6
6
Engineer
2
< 1
L
Y
T6
M
India
US
5
2
Engineer
3
> 1
M
Y
T7
M
China
US
6.5
6.5
Analyst
2
< 1
S
Y
T8
M
India
US
4
0.25
Engineer
3
< 1
L
Y
T9
M
India
US
6
1.5
Manager
6
> 1
L
Y
T10
F
China
US
1.25
1.25
Engineer
5
> 1
M
Y
T11
F
India
US
6.5
3.5
Manager
5
> 1
L
Y
T12
M
India
US
3.5
0.1
Manager
5
< 1
L
Y
T13
M
China
US
4
1.25
UX
10+
< 1
L
Y
R1
F
India
India
1
0
Planner
2
< 1
S
N
R2
F
India
India
6.5
0
Optimizer
2
< 1
S
N
R3
M
India
India
1.6
0
Planner
3
< 1
S
N
R4
F
India
India
1
0
Optimizer
3
< 1
S
N
R5
M
India
India
0.75
0
Planner
3
< 1
M
N
R6
M
India
India
5.5
0
Manager
4
< 1
L
Y
R7
M
India
India
1
0
Consumer
Operations
3
< 1
S
Y
R8
F
China
China
6
0.25
Analyst
2
1
M
Y
R9
F
China
China
2.5
0
Engineer
2
< 1
S
Y
* T=Transplant, R=Remote; **Team size (L = 20+, M = 11-20, S = 4-10)
Table 1. Characteristics of Participants * T=Transplant, R=Remote; **Team size (L = 20+, M = 11-20, S = 4-10) which Indian and Chinese participants moved to the US to be co-located with their
teammates. We label these participants in two groups. Since the terminology of mi-
gration is often problematic and inaccurate [26], we use the term “Transplants” for the
latter. Borrowed from the language of ecology, this term indicates a move from one
environment to another, making it more contextually appropriate. By contrast, “Re-
motes” refer to participants working from their home country, in these cases, based in
Bangalore, Beijing, or Hyderabad. 3. Social Capital and Configuration Many points of interest emerged from our interviews, but most salient were interper-
sonal relationship differences among Transplants and Remotes. In our findings, we
discuss the elements of social capital, which Nahapiet and Ghoshal decompose into
three dimensions: the structural, the cognitive, and the relational dimensions. These
dimensions of social capital in turn impact the conditions for exchange and combina-
tion of intellectual capital [23]. It was out of the scope of this research to investigate
the structural dimension within PrimaTech. The structural dimension is concerned
with network connections between actors, which would require social network analy-
sis. Thus, we focus primarily on the cognitive and relational dimensions. First, we
touch on the differences in expectation between participants in the two configurations. Then we highlight the factors that play a role in shaping cognitive and relational so-
cial capital, respectively, and discuss how these impact the ability of the team to cre-
ate new intellectual capital. We also discuss implications of social capital at an indi-
vidual, team, and organizational level. Finally, we introduce some mechanisms that
appear to mitigate these challenges. 2.4
Analysis Although we were interested in comparing Transplants with Remotes, we did not
have strong hypotheses about what differences would emerge. Therefore, we used
grounded methods [28] in our initial analyses. We used the software Dedoose to aid in
data analysis while engaging in an abductive, iterative process of comparing data and
relevant research literature and theory. We began with open-coding to discover pat-
terns and recurring themes in our initial round of interviews, then created memos to
refine our ideas. This guided data collection as well as our targeted sampling. We then
conducted closed coding, applying lower-level codes to categories to identify emer-
gent concepts. We found that much of what emerged related to differences in expecta-
tions involving social relationships, and thus we used Nahapiet and Ghoshal’s theory
of social and intellectual capital in framing our findings [23]. 2.3 Teams Some structural differences between these two groups should be noted at the ouset. First, the average number of team members was somewhat different between the two
groups. Remotes reported around ten members being involved in their current team,
whereas Transplants frequently reported much larger team sizes. However, there was
not a discernible difference in the number of team members with whom participants
worked directly. Most worked closely with five or fewer colleagues. Second, Trans-
plants reported quite varied tenures with their current teams, ranging from two months
to over five years, while Remotes had all worked with their current team for a year or
less. This is representative of recent growth in PrimaTech’s workforce. The number
of offices with which they regularly collaborated was also variable. Transplants typi-
cally worked across more than four offices, but all had some co-located colleagues. Remotes, on the other hand, worked across three or fewer offices on average. Moreo-
ver, they were often the only member of their team based in their office. So, while
Transplants were mostly part of the hub in a hub-and-spoke or hub-to-hub model,
Remote participants were often spokes in this sort of model [27]. These structural
differences further distinguish the two configurations, but we focus primarily on the
context of location, as it had a more important impact on social relationships. 3.1
Configurations and Expectations Transplants to the US noted very different experiences with team colleagues than
Remotes. The ways in which the two different groups of participants perceived their
interpersonal relationships with colleagues were, in part, predicated upon their expec-
tations. In turn, those expectations were based upon their location and the team con-
figuration. Those who had moved to the US generally noted disappointment in their
lack of social bonds with teammates. On the other hand, remotes approached these
relationships with a pragmatism that showed reserved expectations. Transplants. Those who moved to the US came for a variety of reasons, personal and
professional, but amongst the various expectations, social relationships were a prior-
ity. But as other narratives of migration [29] have shown, lived experiences often do not meet expectations, especially when it comes to a sense of social belonging. One
reason for this is that social relationships at work are often strong in India and China,
but are much more compartmentalized in the US [30]. Thus Transplants often experi-
ence moving from a situation with close relationships and strong social capital to one
in which there seems to be a void. One participant, who had worked first in her home
country of India before coming to the US highlights this differentiation: “When I used
to work in India, work relationships were much more friendly. The demarcation be-
tween work colleagues and friends is a lot fuzzier…Here, it’s just that they are not
friends- we don’t meet up as families and don’t socialize.” (T11) While this is typi-
cally recognized beforehand, as one prepares to move, it still serves as a shock to
many Transplants. They move from having quite rich social relationships with co-
workers to having much weaker social capital, regardless of their tenure, as we dis-
cuss below. Though this difference in work/life compartmentalization is usually
known before a Transplant arrives, there is still an expectation of building work-based
social relationships. Expectations may be dimmed a bit, but they are still there. The
transplants we interviewed did work to cultivate relationships, yet many noted tribula-
tions with creating social relationships, even after years at the same location. The
troubles of expatriates integrating into host communities is not a new concept [31]. 3.1
Configurations and Expectations But it is worthwhile to note that even with the knowledge of work/life difference and
even after attempts at trying to create stronger social relations, expectations still led to
disappointments. pp
Transplants desired stronger relationships, but were unsure of how to do it: “It’s
difficult to get along with the rest of the team as well as I could.” (T11) And often,
this led to feelings of distress or regret: “I should do more. I go out and have dinner,
but sometimes it feels like it’s really hard to fit in, no matter how hard I try.” (T2) In
many instances, this led to feelings beyond a desire for social engagement, into a
deeper level of identity. One participant, who had Anglicized his Chinese name, felt
as if colleagues did not know who he is: “They only see the way I work. They see
‘working [name].’ My Chinese name is hard to pronounce… So, they don’t know my
name. They don’t see my social aspect, so they are not comprehending me. Work is
just one slice of me.” (T13) For those who have just moved, this struggle to create
interpersonal relationships is especially salient. But, notably, this lack of social capital
does not appear to improve with time spent at the US office. One participant, who has
been in the US for over 20 years and working at PrimaTech’s US headquarters for
over eight (T11), indicated a high level of frustration with this aspect of her work. Remotes. In contrast, Remote participants indicated that while they realize they can-
not fully socialize with distributed colleagues, they felt that they do have close
enough relationships. This highlights an adjustment in expectations; distance tem-
pered expectations of social relationships. Employees working on distributed teams at
PrimaTech typically meet all colleagues with whom they work closely within the first
three to four months of being on a project, so most Remote participants had had the
opportunity to meet their colleagues, either through travel to other offices, or through
their colleagues visiting the office in India or China. Participants cited that these visits
gave them a better sense of their colleagues. Beyond the bonds created through visits,
information and communication technologies (ICTs) served as the way through which
they maintained social connections. 3.2 Social Capital Factors and Intellectual Capital in Transnational Teams When working across cultures, some behaviors will converge, some will diverge. As
Hinds et al. note, there must be some degree of convergence for collaboration to work
[11]. In our research, a few factors stood out as limiting convergence, particularly for
Transplants. We highlight these factors through the framework of social capital, first
focusing on the cognitive dimension, then the relational, though both are interrelated
to an extent. In delineating these factors, we investigate how they impact conditions
necessary for exchange and combination of intellectual capital in the organization. Cognitive Social Capital. Nahapiet and Ghoshal describe the cognitive dimension of
social capital as shared cognition that is facilitated through shared codes and language
as well as shared narratives [23]. Shared codes and language are important for the
establishment of social relations and thus the ability to exchange information, but
beyond that, they also influence perception, meaning the perceived value in sharing
information and the ability to combine it meaningfully. Similarly, shared narratives
impact interpretability of information. Small Talk/Topics of Interest. “Small talk,” as Goffman explains, is a bracket to larger
social affairs that has bearing on the overall relation of participants in an interaction
[32]. Thus, it is an important mechanism for establishing stronger capital. Engaging in
small talk is a ritualistic interaction that quite easily occurs within groups that have a
shared cognitive base. However, engaging in small talk can be quite difficult across
cultures due to lack of shared codes and narratives. In our interviews with PrimaTech
employees, many Transplants reported difficulties with small talk, noting that they
often don’t understand the topics at hand. One participant explained: “People go out
to lunch every day and have a lot to talk about. It’s hard to get it all if it’s not work
related.” (T10) She went on to explain she realizes that her different background in-
fluences the perception of what is interesting, so she finds it easier to chat with other
Chinese, rather than struggle to find interesting topics to discuss with her American
colleagues. For this participant, the lack of common topics was so salient that it influ-
enced her to end lunchtime socializing, further isolating her from her teammates. In contrast, many Remotes reported not only engaging in small talk in the begin-
ning moments of a video conference, but finding enjoyment in it. 3.1
Configurations and Expectations Several participants said they talk informally with distributed colleagues over collaborative technologies such as chat or in the first min-
utes of a video conference: “We do a lot of that- talking about movies, what we’ve
been doing over the weekend. I feel like I know them as colleagues.” (R3) These par-
ticipants generally felt that their distributed colleagues had accurate perceptions of
them as colleagues, though nothing approaching deep connections as “friends.” In sum, expectations based on team configuration and location within that configu-
ration played a role in perceptions of social relationship. Transplants expected deeper
friendships than they got; Remotes found a shallower but sufficient sense of friend-
ship. Next, we take a look at factors that play a role in whether social capital is devel-
oped and how that impacts intellectual capital. 3.2 Social Capital Factors and Intellectual Capital in Transnational Teams So, while Trans-
plants reported finding topics of conversation and small talk difficult, Remotes gener-
ally reported small talk as a great way to engage with colleagues for a few moments at the beginning or end of a meeting. This difference relates back to expectation issues,
particularly with regard to the length of time one must socialize; having a short chat at
the beginning of a video conference does not create a burden, whereas sustaining
small talk for an hour of lunch may appear daunting. Similarly, Remotes frequently
noted that they talked about movies, travel, and weekend plans as topics of conversa-
tion, which are fairly general and not highly location-specific, whereas Transplants
had higher expectations placed upon them. Transplants noted pressure to know more
about local sports or politics, both of which require more of a shared cognitive
framework. It may also be the case that Transplants have to engage in this talk with
many more people, potentially from many various backgrounds, feeling more pres-
sure yet. One Transplant (T12) noted that so many people ask how his weekend was
on Mondays that he plans out what he wants to say in advance, on his way to work. Thus, it appears that whether one only occasionally chats with teammates versus hav-
ing regular, extended contact with them affects one’s perception of whether small talk
is a problem or a benefit in building social capital. Cultural References. Whether part of small talk or part of general work discussions,
culturally-specific references can impact one’s ability to understand or engage in con-
versation. This can cause disengagement from social interactions, and, worse, can
impact the ability to participate fully in meetings. Cultural references can create mis-
understandings of the message itself, but beyond that, they can create confusion about
tone. Several Transplants noted that technical conversations were typically fine until
cultural references were applied within them, such as a person referring to an idea
being “out in left field.” This participant elaborates: “I think they tend to use exam-
ples from games or football references, so sometimes can’t tell if they’re being sarcas-
tic or not. It becomes difficult.” (T12) On the other hand, Remotes generally used cultural references as a kicking-off
point for understanding their colleagues better. One participant explained: “What is
nice and what is fun for us —watching cricket here—is like baseball, basketball there. So watching sports connects people. 3.2 Social Capital Factors and Intellectual Capital in Transnational Teams When someone talks passionately about a match,
I understand because I go equally bonkers over cricket.” (R2) These Remotes reported
that if they do not understand a cultural reference, this allows them to then ask ques-
tions to get to know each other better. One possibility for these different attitudes
toward the use of cultural references is that there may be an implicit assumption that
people will understand most references if they are living in the same location. On the
other hand, when talking with someone who lives halfway around the world, indi-
viduals may hold fewer assumptions of what others do or do not know about an-
other’s culture. Remotes, it seems, are expected to not know everything; Transplants,
however, are co-present and expected to know what other locals know. This may be intensified by the fact that those in the US headquarters typically work
with a higher number of distributed offices. So, as one participant points out, you
must know about many more cultures: “I didn’t socialize so much this past year just
because I myself haven’t fully plugged in to American culture. And my work and my
mind’s resources have limited me from doing that. I have to be distributed in many
places with all the offices we work with, so I didn’t spend much time to figure out
local things here.” (T13) This highlights that, beyond transnational context, the con-
figuration of the team and the participant within the team really does matter in terms of creating social capital. Expectations from others and from oneself are shaped by
location, and in turn, location influences the development of social capital. Lack of
shared language, codes, or narrative can serve as a kicking-off point for sharing,
which enable conditions for exchange, while assuming these are shared closes off the
possibility for exchange. Humor. Humor is an intensely social experience that provokes strong emotions [33]. As such, it can serve as a building block for social capital. But, humor is notoriously
difficult to understand across cultures. One Transplant explained: “The most difficult
thing here is to make a joke... It connects to many things out of the workplace…But a
joke is very important for people’s emotional connection. And I cannot connect that
way. That is most difficult.” (T13) Both Transplants and Remotes reported difficulties
with humor, but Remotes had a more generally positive attitude about its impact. 3.2 Social Capital Factors and Intellectual Capital in Transnational Teams Several Remotes explained that joking around became easier for them to handle once
they had met their colleagues in person: “Sometimes I don’t know how to respond if
someone is sarcastic… but everything is easier once you meet- like jokes. Because
I’ve met him and know he’s doing it for fun, that barrier gets broken.” (R7) Thus, this
lack of shared cognition impairs both configurations, but to a different degree. While
humor is still difficult for both, it appears that Transplants, being located together,
may attempt to understand humor on a deeper level, while Remotes seem satisfied to
identify only when humor is being used, as it helps to understand context and tone. Relational Social Capital. The relational dimension of social capital centers on the
interactional and behavioral elements between parties. Nahapiet and Ghoshal describe
this dimension in terms of trust, norms, obligations, and identification. They illustrate
how these impact intellectual capital by influencing access to parties, perceived value
and the motivation to exchange and combine information [23]. Trust. There has been much research on basic trust differences across cultures [34,
35]. Core values play a significant role in base levels of trust as a cognitive factor, but
trust is also a dynamic property of relationships in context. Rocco et al., for instance,
found a positive relationship between non-work communication and trust [36]. So,
trust both shapes and is shaped by interpersonal relationships; when ties with work
colleagues are not strong, a strong level of trust is not manifest and vice-versa. Thus,
we found trust to be an issue both before and after a relationship has been established. Despite trust being an oft-cited component of PrimaTech’s organizational culture,
trust among colleagues was complicated. One Transplant explained the differences in
levels of trust with colleagues in the US versus India in the following way: “The
meaning of a relationship here is different… In India you don’t distinguish between
social friends and professional ones. You trust all friends equally. Here it is very pro-
fessional… I won’t trust any friend here as I would in the Indian office.” (T12) From
this perspective, it appears trust can be gauged a priori. But others mentioned that
trust requires rapport, thus it is also impacted a posteriori. These complexities of trust fundamentally affect group work, whether co-located
or distributed. However, trust issues were mentioned more frequently among Trans-
plants than Remotes. 3.2 Social Capital Factors and Intellectual Capital in Transnational Teams One school of thought indicates that this might be due to “swift
trust.” Jarvenpaa and Leidner found a lack of cultural effects in looking at trust in
global virtual teams [37], and they suggested these teams can rely upon trust that is based more on categorical expectations than interpersonal relationships, known as
swift trust [38]. For Transplants, other factors impact interpersonal relationships,
which then impacts trust a posteriori. For Remotes, though, trust is based more on
team members’ work effort and less on personal factors and is only expected to be
sufficient, not deep. This is enhanced by visits in which brief contact allows for a
sense of good rapport. While this does not mean that trust from Remotes is necessar-
ily stronger in any sense, it may serve as less of an impediment as compared to co-
located teams that may struggle with interpersonal issues. based more on categorical expectations than interpersonal relationships, known as
swift trust [38]. For Transplants, other factors impact interpersonal relationships,
which then impacts trust a posteriori. For Remotes, though, trust is based more on
team members’ work effort and less on personal factors and is only expected to be
sufficient, not deep. This is enhanced by visits in which brief contact allows for a
sense of good rapport. While this does not mean that trust from Remotes is necessar-
ily stronger in any sense, it may serve as less of an impediment as compared to co-
located teams that may struggle with interpersonal issues. Speaking Up: Norms of Openness. PrimaTech’s organizational culture is often de-
scribed as being very “bottom-up.” Being “bottom-up” means that change is often
initiated in a very democratic, communal way. As such, being involved in discussions
and decisions means embracing the norms of openness and speaking up. This social
norm of openness is noted as a key feature needed in knowledge-intensive firms [39]. p
y
g
[
]
“Speaking up” was the most frequently self-cited focus of behavioral change for
participants in both groups and a repeated piece of advice they would give to others. One participant, coming from India noted: “It’s difficult to get your head around it
because if you’ve never worked in place like [PrimaTech], it’s engrained into you to
pay attention to social strata or not to speak up.” (T11) This general lack of “speaking
up” in meetings creates a disparity in being involved. 3.2 Social Capital Factors and Intellectual Capital in Transnational Teams While this happens for both co-
located and remote due to different macro-level social conventions, Transplants have
the opportunity to speak up more often. But, being in a new location and not having
strong social relations in other ways impacts that. One Transplant even noted her
move had a negative effect on speaking up, despite it being a norm at PrimaTech: “I
have been less outspoken, more quiet since coming to [US office]. Part of that is be-
cause I’m transferring to a new location and I don’t know people that well, so my
confidence level isn’t as high. I think more before I speak...” (T4) For Remotes, speaking up is also an issue, but due to constraints, teams often struc-
ture formal conversations differently. For instance, in video conferences, distributed
teams focus more on rotating participation and feedback from the various parties, as
large video conferences with spontaneous input from simultaneous audio streams can
become chaotic. In the same vein, there is an expectation in remote meetings that
people may hold back a bit more; one manager noted that he makes a point of specifi-
cally asking what others think in making decisions, noting that often that person
would have “a brilliant point of view that was missed.” (R6) Remotes tended to rec-
ognize speaking up as a potential issue in advance and address, while Transplants’ co-
located teams perceived no barriers to speaking up, and thus didn’t address it. Notably, outside of formal conversations, those who are co-located also have the
opportunity to communicate through a variety of informal channels that are beneficial
to being involved in decisions, such as side conversations, hall talk, and “meetings
over the water cooler” [8]. But when social relationships with colleagues are not
strong, Transplants will miss out on such opportunities to hear and to have their opin-
ions heard informally, which leads to less knowledge exchange. Feedback. Within these transnational teams, lack of communication sometimes led to
less access and motivation to exchange and combine knowledge. In particular, many
participants noted how they often do not know when they have made mistakes be-
cause colleagues are hesitant to provide negative feedback. These behaviors are in contrast to the literature, which indicates that Americans are direct, while Chinese and
Indians use indirect speech in order to promote harmony [40]. 3.2 Social Capital Factors and Intellectual Capital in Transnational Teams An Indian participant
explained how this is impacted through social relationships, not just hierarchy issues:
“This is confounded based on how close you are with colleagues. People get more
direct negative feedback in India. Here, with a lot mistakes, you don’t get direct feed-
back. I’ve had to seek it out. But one reason might be that I’ve only been here going
on four months, so I don’t have those relationships yet perhaps.” (T1) Additionally, some participants expressed an inability to understand when their
colleagues needed help and feedback. Weak social relations with colleagues made it
difficult to know when a team member needed help, unless it was directly requested:
“If you want to help a coworker, it’s not easy…I don’t know how much they need
help. . . I wish I had the conception to see when help can be appreciated or accepted.”
(T13) Again in this instance, there is not necessarily an advantage for Remotes, but
communication among Remotes and their colleagues was more explicit, so there were
fewer issues and less confusion related to implicit communication norms. Meals. Breaking bread together is a meaningful social bonding activity. Yet this is a
difference for those in the US offices in two ways. First, those who had worked in
their home country before moving to the US cited that lunch is quite social in India or
China, so it was a surprise to see colleagues in the US eating at their desks: “A lot of
people eat lunch alone and keep working here. In China, I would always go out of the
way to meet other people for lunch. It’s a much more social experience there. You
never eat at your desk.” (T4) This highlights one of the unmet expectations of Trans-
plants, who sought more social engagement with teammates, but did not always find
it. Moreover, when these participants did go out to share meals with colleagues, some
were disappointed with the food choices as well as the focus on drinking, which dis-
couraged them from joining. One participant explained that team dinners were un-
comfortable because he did not eat meat or drink alcohol. Yet, teammates would often
choose restaurants for team dinners based on a good wine list. 3.3 Impact on the Individual When transnational teams work together, they face many issues that may impede
success. As we have seen so far, lack of social capital impacts a team’s information
exchange and knowledge creation. But importantly, it also impacts the individual
dramatically, which in turn can also affect the team. Career Development. PrimaTech has a promotion process that is heavily influenced
by one’s peers, which is a unique aspect of the company that all employees encounter. Though most people appreciated the fundamental idea of this process, many partici-
pants expressed some level of concern about the importance of relationships. The idea
of colleagues, rather than just managers, having a significant impact on career ad-
vancement is of much greater concern when one feels he or she does not have strong
social capital with colleagues. This was understandably more of a concern for Trans-
plants, who had difficulties building these relationships. One participant remarked:
“Making friends is relevant to career growth. It’s very difficult to do. You have to talk
about children’s soccer games and golfing, and I’m not interested… but in order to
get people to help and evaluate you, you have to.” (T2) This policy is employed company-wide, so those at international offices review
and are reviewed by not only their co-located peers, but their distributed teammates as
well. Interestingly, Remotes did not indicate strong concerns with this process, indi-
cating that anxiety about this process is perhaps induced by the local context of
Transplants. Remotes may not have developed very strong social capital with col-
leagues abroad, but expectations for this remain low, and thus they feel their work,
not their relationships, is what is being evaluated. Moreover, Remotes straddle differ-
ent social worlds and arenas and thus have strong social capital in their home location. Even though their US-based colleagues may have more weight in reviews, these local
relationships perhaps provide some reassurance in terms of feeling social cohesion,
and thus less anxiety about the process. Transplants, in contrast, have major ties with
their US-based colleagues, which lead to different expectations and different things
expected of them. General Satisfaction. Ostensibly, participants’ enjoyment of their job was also influ-
enced by local contexts. Previous research has shown mixed results in terms of satis-
faction in diverse teams [15], but in general supports the claim that distributed, virtual
teams have lower satisfaction [41]. 3.2 Social Capital Factors and Intellectual Capital in Transnational Teams He noted this limited
his participation in team dinners: “You want to give them company and build a rela-
tionship, build your network, but you’re hungry.” (T12) By comparison, dinners are always part of the agenda when Remotes visit their
teammates, or when teammates visit them. While less frequent, everyone typically
attends, precisely since opportunities to do so are limited, and several Remotes cited
good experiences getting to know their distributed counterparts this way. Families. Finally, getting to know colleagues’ families was a significant difference. Most Transplants cited their US-based colleagues’ strong work/family divide, noting
how different this was from their home culture. This supports the literature on this
topic, which references how familial collectivism is more common in Asian cultures
and is expressed in business relationships through socializing with families, among
other things [11]. Participants based in the offices in India and China often mentioned
socializing with colleagues’ families. While it is not possible for this to be much of a
part of distributed teammate bonding, several Remotes did cite introducing visiting
colleagues to their own families, a point of difference based on distance limitations. 3.3 Impact on the Individual Despite this, Remotes seemed more satisfied in
certain ways, likely due to better social circumstances. Remotes, who unanimously
noted close friendships with colleagues in their home country office, mentioned that
they would hang out and socialize more in the office. They were more willing and
interested in staying around the office for a while, even though they often worked
long hours to accommodate distributed teammates on other continents. In contrast, Transplants often cited the challenges of their local context. One par-
ticipant gave this report of dissatisfaction through this solemn advice: “Be prepared
that people will misunderstand you. And though you’re in [PrimaTech] for five years
in India, you have to prove yourself again over here… You don’t think of how hard it
can be. If you are not used to staying alone or fighting alone, don’t join.” (T12) 4. Mechanisms that May Aid in Convergence What emerges from the findings of this study is the importance of focusing on the
local in global work. Team configuration and the context of location play important
roles in transnational collaboration and have significant implications for teams and
individuals, particularly in how a team maintains social cohesion and a sense of
community. This in turn impacts the conditions necessary for the exchange of intel-
lectual capital. But, as Hinds et al. note, there must be some degree of convergence
for collaboration to work at all [11]. Despite the many points of difference, there are
mechanisms that did help teams collaborate. Here, we present ideas as to what might
help develop social capital in these transnational collaborations. Emerging from our
interviews, we find that an organizational “cultura franca” may help create stronger
bonds, and that hiring practices may influence the possibility of convergence. In other
words, organizational culture may mitigate some effects of different national cultures. 4.2 Selection in Hiring The development of this sort of organizational culture is in part predicated upon the
population that is initiated into it. A factor that clearly emerged from our interviews
was how the organization selects for those who embody the qualities that PrimaTech
promotes. This is a fairly obvious factor in mediating intercultural difference—
selecting people who are similar in certain ways—but from our research, the impor-
tance of this process was quite distinctive at PrimaTech, enabling the organization to
more readily mitigate intercultural challenges. While most participants did not indi-
cate that they considered the organization to be culturally American or Western, one
participant explained that the company chooses people who are more culturally
aligned to certain values, and that this enables easier collaboration: “At [PrimaTech],
it is very different from other Indian companies. I have friends in [other companies],
… and they have a lot more issues. I think this is due to [PrimaTech’s] interview
process. Each person is specifically selected for how they will match...” (R3) While
this selection may or may not be explicit—or true on a broader scale—the process of
choosing certain qualities is quite explicit, at least internally. One participant ex-
plained how there were “unwritten rules” in the hiring process, noting the importance
of the social: “Can you sustain a two-hour layover at an airport and not get bored and
want to talk to this person?” (R6) He noted that this creates a basis –“the common
denominator”—for having somewhat similar employees throughout PrimaTech’s
global locations. Thus, while not necessarily being specific, there is a template for
what is sought out socially in an employee. And since this process is held worldwide,
it contributes to cultivating a workforce that reflects the values of the company, irre-
spective of national culture or other cultural traits. 4. 1 A Cultura Franca The idea of a “cultura franca” is an inchoate idea that emerged in this study. It is
based on the concept of a lingua franca, or bridge language, used to communicate
between parties with different mother tongues. We use the term cultura franca, rather
than organizational culture, to indicate that the strong, distinctive culture of Pri-
maTech serves as a bridge across diverse global cultures and creates a space for
common goals and values. Like a lingua franca, it serves as a vehicle for mutual intel-
ligibility-- in this case, of practices and priorities, rather than language. Additionally,
just as pidgin languages based on a lingua franca evolve in certain locales, the term
cultura franca indicates that the culture is not completely uniform across all the geog-
raphies; it becomes imbued with attributes of the location. Throughout PrimaTech, the cultural values of the organization are evident. All par-
ticipants had very similar ideas about the organizational culture and frequently used
the same, or similar, terms to describe it. The most oft-cited descriptors were “open”
or “transparent,” followed by “democratic,” “independent,” and “trusting.” Many also
described the culture in terms of things being “data-driven” and people being “smart”
and “friendly.” While these were considered to be universal qualities of the organiza-
tion, there were mixed indications as to how similar the culture is across offices. Be-
cause PrimaTech’s offices are very similar in physical environment, structure, and
character across the globe, many referenced these as indications of uniformity. So,
many believed that the culture was quite uniform: “They have done an amazing job of
spreading the culture all over the globe…It’s pretty much the same.” (T9) But on the other hand, many others indicated that, while the culture is promoted the
same way across the globe, the way it is actually exhibited in different offices is not
necessarily the same: “From people I’ve met, the [PrimaTech] culture is the same. Although the way we go about it is completely different.” (R7) It appears that, though
the values that permeate PrimaTech are fairly universal, the way in which they impact
work relationships depends more on the location and local culture therein. The social
environment is often adapted to fit better with the local culture. For instance, partici-
pant R2 noted that, unlike in the US, alcohol is not involved in office activities in India. 4. 1 A Cultura Franca One participant describes these variations: “The basic things are similar across
the geographies, but it’s a bit different because of other cultural differences… But in
work ethic, it is the same... ” (R4) The cultura franca, then, serves as a mechanism to
bridge what might otherwise be major differences in values. It seems that the cultura
franca of the organization may perhaps ease some factors that negatively impact col-
laboration, while still enabling diverse perspectives, but to what extent it enables con-
vergence remains a question. 5. Discussion and Conclusions We conducted research within a large multinational technology company to better
understand the mechanisms by which people conduct transnational work. While
common sense says that presumably co-located work is easier and better for teams,
the issues are more complex, especially in intercultural collaborations. From our in-
terviews we found that social capital is, in part, impacted by whether the person is in
their home context or transplanted, which in turn impacts the individual, the team, and
the organization. g
As our results show, location and configuration played a significant role in creating
expectations, so it is also important to discuss how these expectations relate to lived
experiences. Stronger and richer social capital were expected by Transplants; this led to disappointment when those expectations weren’t fulfilled. This brings up issues of
adaptation. In the literature on migration, it seems that findings generally indicate that
the adaptations are made to accommodate the host culture. This was only supported to
some extent in our interviews. Transplants adapt at some surface level, but not neces-
sarily at a deeper level: “For myself, I have changed a bit over time. When I came to
US, I still had the typical way Chinese do things. But now it is probably more mixed. To do many things, I do things closer to the Western way. But in thinking, I am still
pretty Chinese.” (T5) While we found only minor evidence of adaptation in terms of
social bonding, it remains unclear to what extent participants’ colleagues adapted to
their behavior, clouding the issue further. Previous research on immigrants has shown that surface-level adaptations may be
made through the use of “relational templates” and that people may maintain multiple
templates in engaging in intercultural work. [42] This concept works fairly well in
explaining Remotes’ interactions with distributed teammates: they maintain their
capital with colleagues in their home office and colleagues in distributed offices in
systematically different ways. So, you work with your colleagues in Bangalore in one
way, but when you call the London office, for instance, you use a different behavioral
pattern. While participants did not heavily cite cultural training provided by the orga-
nization, it seems that this may be another mediating factor—being able to clearly
select and apply appropriate relational templates. Templates seem to be a useful con-
cept in thinking about distributed work. 5. Discussion and Conclusions Put another
way: consider, for a moment, PrimaTech’s headquarters to be Rome, and how it
might be easier to act like a Roman for short chunks of time than to try to become a
Roman. But is this better for the team and the organization? If diversity is important,
we want to embrace diverse perspectives. Maybe everyone should not have to act like
a Roman. Or, maybe Romans should be expected to act differently. These factors
would best be addressed through organizational culture and human resources training. g
g
g
Finally, what are implications here for ICTs? It seems that, for Remotes, technol-
ogy provided a solution that went “beyond being there” [43] in certain ways. The use
of ICTs helped mitigate issues and supported the development of sufficient social
capital. In this sense, perhaps what is needed is a return to Hollan and Stornetta’s
focus on creating technologies that meet needs that cannot so easily be met “in the
medium of physical proximity.” The realm of social capital is comprised of structural,
cognitive, and relational dimensions. Ostensibly, ICTs can aid in structural social
capital through the development of networks that connect people, but it seems they
can aid in less obvious ways. From this work it is clear that ICTs can also play a role
in cognitive and relational aspects of social capital. Moving forward, research in this
area would benefit by considering the ways in which technologies can and cannot
support development of social capital—distributed or co-located. In being sensitive to
these possibilities and limitations, designers, individuals, teams, and organizations can
seek more creative ways to build and nurture social bonds in transnational work. Limitations and Future Work. Several limitations of this work should be noted. This study focused on Indian and Chinese participants in transnational teams; we did
not include other nationalities nor the teammates with whom these participants work. Also, while we did visit the US headquarters, we did not visit international sites, and
all interviews were conducted via video conferencing. Thus, our findings may be
limited by interviewees not talking as openly as they might in person. By relying on
interviews alone, we may also be assessing perceived, rather than actual, social capi-
tal. Including other teammates (non-transplant, non-remote) and conducting fieldwork
that goes beyond a case study of single firm would certainly aid in further investigat-
ing these social capital phenomena. 5. Discussion and Conclusions However, with the case of Transplants, it is
difficult to see how a template structure would hold, making adaptation more diffi-
cult. As indicated in the interviews, the US offices are quite multicultural, unlike of-
fices such as Bangalore or Beijing, which tend to be quite nationally homogenous. When constantly surrounded by a local, heterogeneous team, it is impossible to have a
single template that is appropriate for everyone. It is beyond the scope of this study to
evaluate the degree of adaptability or co-adaptability of the team; however it seems
the field of transnational research would greatly benefit from a more micro-level,
longitudinal analysis of changes in behaviors based on the context of location. Implications. Our results raise interesting questions for the configuration of teams. Are the social costs of moving around the globe more than the coordination costs of
working at a distance? This has interesting implications for organizational structure,
individual choices, and seemingly, technology. A team is not merely a division of labor, so it is important to be reflective about
organizational structure. Distance issues in teams are well known, but with the highly-
systematic methods of interaction in distributed team, one wonders whether Remotes
and these teams are reaching their full potential in such configurations. On the other
hand, you have situations where workers cross borders to be close to their team. This
may ameliorate many of the issues that arise with distance, but it does not mean that
conditions for the exchange of intellectual capital will necessarily improve. As we
have seen, the interactions that create strong social capital within a team do not neces-
sarily change with proximity. Solid social relationships are the substrate-- the basis on
which a team can thrive. When looking at issues of dispersion—temporal, spatial,
configurational—it is necessary to also heavily consider how this interacts with cul-
tural differences. Beyond this, though, it seems more emphasis could be placed on organizational
culture and human resources practices. On a practical level for the individual, it could
be that, despite all its challenges, distributed collaboration provides a more optimal,
structured format for intercultural work in that it requires less adaptation and concern
for divergent cultural challenges. The limited contact inherent in distributed collabo-
ration means that workers in their home country may experience more satisfying so-
cial interactions than those who move across borders to other offices. 5. Discussion and Conclusions This could be a useful starting point for develop-
ment of a survey instrument that can operationalize and validate some of these con-
structs, particularly in teasing apart perceived social capital and actual social capital. Future research should focus on creating a better framework for looking at behavioral
dynamics in transnational teams, taking the context of location into account. This line
of research should also look at whether these effects hold when the dominant culture
is non-Western. On a broader level, such research could have broad implications for
individuals, team structuring, and organizational mechanisms for co-adaptability. Acknowledgements. We thank all the participants in this study for their time and
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Mutant polycystin-2 induces proliferation in primary rat tubular epithelial cells in a STAT-1/p21-independent fashion accompanied instead by alterations in expression of p57KIP2and Cdk2
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BioMed Central BioMed Central BioMed Central Open Acc
Research article
Mutant polycystin-2 induces proliferation in primary rat tubular
epithelial cells in a STAT-1/p21-independent fashion accompanied
instead by alterations in expression of p57KIP2 and Cdk2
Kyriacos N Felekkis†1, Panayiota Koupepidou†1, Evdokia Kastanos1,
Ralph Witzgall2, Chang-Xi Bai3, Li Li4, Leonidas Tsiokas3, Norbert Gretz4 and
Constantinos Deltas*1 Address: 1Department of Biological Sciences, University of Cyprus, Cyprus, 2Institute for Molecular Anatomy, University of Regensburg, Germany,
3Department of Cell Biology, University of Oklahoma Health Sciences Center, OK, USA and 4Medical Research Center, University of Heidelberg,
Mannheim, Germany Email: Kyriacos N Felekkis - felekkis@ucy.ac.cy; Panayiota Koupepidou - p.koupepidou@ucy.ac.cy; Evdokia Kastanos - kastanos@ucy.ac.cy;
Ralph Witzgall - Ralph.Witzgall@vkl.uni-regensburg.de; Chang-Xi Bai - chang-bai@ouhsc.edu; Li Li - li.li@zmf.ma.uni-heidelberg.de;
Leonidas Tsiokas - leonidas-tsiokas@ouhsc.edu; Norbert Gretz - norbert.gretz@zmf.ma.uni-heidelberg.de;
Constantinos Deltas* - deltas@ucy.ac.cy * Corresponding author †Equal contributors Published: 25 August 2008
BMC Nephrology 2008, 9:10
doi:10.1186/1471-2369-9-10
Received: 29 January 2008
Accepted: 25 August 2008
This article is available from: http://www.biomedcentral.com/1471-2369/9/10
© 2008 Felekkis 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. Received: 29 January 2008
Accepted: 25 August 2008 Received: 29 January 2008
Accepted: 25 August 2008 This article is available from: http://www.biomedcentral.com/1471-2369/9/10 This article is available from: http://www.biomedcentral.com/1471-2369/9/10 © 2008 Felekkis 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. BMC Nephrology Open Access Research article Open Acc
Research article
Mutant polycystin-2 induces proliferation in primary rat tubular
epithelial cells in a STAT-1/p21-independent fashion accompanied
instead by alterations in expression of p57KIP2 and Cdk2
Kyriacos N Felekkis†1, Panayiota Koupepidou†1, Evdokia Kastanos1,
Ralph Witzgall2, Chang-Xi Bai3, Li Li4, Leonidas Tsiokas3, Norbert Gretz4 and
Constantinos Deltas*1 Methods
C ll
l Cell culture and isolation of renal primary epithelial cells
Human embryonic kidney 293 cells, PC-2 overexpressing
cells and the rat epithelial cell line NRK-52E were main-
tained in DMEM medium supplemented with 10%
(HEK293) or 5% (NRK-52E) fetal bovine serum (FBS). Renal primary epithelial cells were isolated from a 7.5
week-old PKD2 mutant trangenic rat (TGR (CMV-hPKD2/
1–703)), abbreviated in the text as: PKD2(1–703) [19]. There were two transgenic rat lines created initially, 111
and 247, expressing a truncated PC-2, owing to a STOP
codon at postion 704. Of the two models 247 was chosen
for further work owing to a more severe phenotype. Line
111 is not maintained at the moment [19]. The primary
cells were isolated by a sequential filtration method as fol-
lows: Normal Spraque-Dawley rats and PKD2(1–703) rats
were sacrificed following standard procedures; kidneys
were extracted and minced under sterile conditions. The
cell mixture was passed through a 180 μm metal sieve
(Retsch, Germany) followed by filtration through a 40 μm
nylon cell strainer (BD Biosciences). The retained cells
were collected and passed through a second 100 μm cell
strainer. The filtrate of this step comprises the tubular epi-
thelial fraction of the kidney homogenate. Tubular epithe-
lial cells were cultured on laminin-coated tissue culture
plates and maintained in Endothelial Cell Growth
Medium (PromoCell, Germany) supplemented with
5%FBS, ECGS, EGF, Hydrocortison, Amphotericin B and
Gentamycin. Under these conditions the cells maintained
their epithelial phenotype for at least 4 passages. Polycystin-2 has been implicated in cell cycle regulation
mainly through its calcium channel activity and its ability
to activate transcription factor AP-1 [14-16]. However,
there was little direct evidence linking polycystin-2 to cel-
lular proliferation. Recently, PC-2 was directly tied to cell
cycle regulation through direct interaction with Id2, a
member of the helix-loop-helix (HLH) proteins that are
known to regulate cell proliferation and differentiation. Overexpression of wild-type PC-2 in kidney cell lines
induced cell cycle arrest at G0/G1, through upregulation
of p21 and subsequent inhibition of Cdk2 kinase activity. This process was dependent on both PC-2-Id2 interaction
and PC-1-dependent phosphorylation of PC-2. Although
inhibition of Id2 expression corrected the hyperprolifera-
tive phenotype of mutant cells, the contribution of p21/
Cdk2 pathway on the abnormal cell proliferation was not
clearly addressed [17]. Antibodies The following antibodies were used in this study: mouse
anti-HA, goat anti-p21, goat anti-Cdk2 (Santa Cruz, Bio-
technology), rabbit-anti phospho STAT1 (Cell Signaling),
rabbit anti-p57 (Santa Cruz). The rabbit polyclonal anti-
PC2 (epitope corresponds to amino acids 679–742 of
PC2) was previously described[20] http://www.biomedcentral.com/1471-2369/9/10 http://www.biomedcentral.com/1471-2369/9/10 http://www.biomedcentral.com/1471-2369/9/10 Methods
C ll
l In an independent study, PC-2 was
shown to regulate proliferation and differentiation of kid-
ney epithelial cells and suggested that its calcium channel
activity may play an important role in this process [18]. Background and NRK-52E expressing wild-type and mutant PC-2 as
well as primary tubular epithelial cells from a PKD2-
mutant transgenic rat [19]. Interestingly, expression of
mutant PC-2 had an effect on the aforementioned path-
way only in the primary epithelial cells expressing mutant
PKD2, but this was independent of p21. On the contrary
multiple approaches provided unequivocal evidence that
a different cyclin-dependent kinase inhibitor, p57, is
reduced in these cells. These results suggest that p57 might
be the end-point of an alternative pathway that regulates
PC-2-induced proliferation in ADPKD. g
Autosomal Dominant Polycystic Kidney Disease is the
most common hereditary renal disorder with a prevalence
of at least 1:1000 and accounts for 8%–10% for all end-
stage renal failure [1]. The disease is characterized by the
formation of large fluid-containing renal cysts that grow
over time and destroy the renal parenchyma. It is believed that cysts originate from tubular epithelial
cells that exhibit increased proliferation and reduced dif-
ferentiation. This may happen after a second somatic hit
occurs that inactivates the PKD1 or the PKD2 allele inher-
ited from the healthy parent [2-4]. Microdissection of
cystic kidneys revealed that cyst growth is due to an
increase in cell number and not to the stretching of the
cyst wall. In addition, tubular epithelial cells cultured
from ADPKD cysts display augmented levels of prolifera-
tion and upregulation of proliferation-associated genes
such as c-Myc, Ki-67 and PCNA [5-8]. The role of polycys-
tin-1 (PC-1), the protein product of PKD1, in the prolifer-
ation of tubular epithelium has been documented. Polycystin-1 has been implicated in a variety of pathways
tied to proliferation, including G-protein signaling, Wnt
signaling and AP-1. [9-12]. Direct evidence about the
involvement of PC-1 in cell cycle regulation was demon-
strated by the observation that PC-1 overexpression acti-
vates the JAK2/STAT-1 pathway, thereby up regulating
p21waf1 and inducing cell cycle arrest in G0/G1 in a proc-
ess requiring functional polycystin-2 (PC-2). Based on
these results it was postulated that mutations in either
gene could result in deregulated growth [13]. Abstract Background: Autosomal Dominant Polycystic Kidney Disease (ADPKD) is characterized by the
formation of multiple fluid-filled cysts that destroy the kidney architecture resulting in end-stage
renal failure. Mutations in genes PKD1 and PKD2 account for nearly all cases of ADPKD. Increased
cell proliferation is one of the key features of the disease. Several studies indicated that polycystin-
1 regulates cellular proliferation through various signaling pathways, but little is known about the
role played by polycystin-2, the product of PKD2. Recently, it was reported that as with polycystin-
1, polycystin-2 can act as a negative regulator of cell growth by modulating the levels of the cyclin-
dependent kinase inhibitor, p21 and the activity of the cyclin-dependent kinase 2, Cdk2. Methods: Here we utilized different kidney cell-lines expressing wild-type and mutant PKD2 as
well as primary tubular epithelial cells isolated from a PKD transgenic rat to further explore the
contribution of the p21/Cdk2 pathway in ADPKD proliferation. Results: Surprisingly, over-expression of wild-type PKD2 in renal cell lines failed to inactivate Cdk2
and consequently had no effect on cell proliferation. On the other hand, expression of mutated
PKD2 augmented proliferation only in the primary tubular epithelial cells of a rat model but this was
independent of the STAT-1/p21 pathway. On the contrary, multiple approaches revealed
unequivocally that expression of the cyclin-dependent kinase inhibitor, p57KIP2, is downregulated,
while p21 remains unchanged. This p57 reduction is accompanied by an increase in Cdk2 levels. Conclusion: Our results indicate the probable involvement of p57KIP2 on epithelial cell
proliferation in ADPKD implicating a new mechanism for mutant polycystin-2 induced proliferation. Most importantly, contrary to previous studies, abnormal proliferation in cells expressing mutant
polycystin-2 appears to be independent of STAT-1/p21. Page 1 of 13
(page number not for citation purposes) (page number not for citation purposes) BMC Nephrology 2008, 9:10 Transient transfection and Western blot analysis Transient transfection and Western blot analysis
Plasmids were transfected into HEK293 and NRK-52E
cells using Lipofectamine 2000 (Invitrogen) reagent
according to the manufacture's instructions. Western blot
analysis was performed as mentioned before [21]. Briefly,
cells were lysed in Nonipet P-40 (NP40) buffer (0.1%
NP40, 200 mM NaCl, 50 mM TrisCl (pH 7.4) and pro-
tease inhibitors). After centrifugation at 14,000 rpm for 5
minutes, the supernatants were collected. Total amount of
protein was determined using the BCA kit (Pierce). Equal
amount of protein was denatured by addition of equal
volume of 2 × SDS loading buffer and heating for 30 min
at 50°C. Proteins were separated on an SDS-PAGE gel. After transfer to a PVDF membrane, Western blots were
developed by ECL following the vendor's protocol (Amer-
sham). Cdk2 Kinase Assay Cdk2 assay was performed as previously described [22]. Briefly, 250–500 μg of the total cellular protein was
immunoprecipitated with 1 μg of Cdk2 antibody for 2
hours at 4°C. After extensive washing, the precipitate was
subjected to the kinase assay in the presence of 50 mM
HEPES, 7.5 mM MgCl2, 0.5 mM EDTA, 20 mM β-glycero-
phosphate, 1 mM NaF, 5 mM dithiothreitol, 100 μM ATP
and 10 μCi of [γ-32P] ATP in a total volume of 30 μl. As a
substrate, 2 μg of histone H1 (Calbiochem) were added to
the reaction. The reaction was carried out at 30°C for 30
min. After elution, the supernatant was fractionated by
SDS-PAGE, transferred onto a PVDF membrane and auto-
radiographed. Gene Expression Profiling with Microarrays p
f
g
y
Gene expression profiles of primary tubular epithelial
cells (TECs) isolated from PKD2(1–703) rats and SD rats
were compared. RNA isolation, cDNA and cRNA synthesis
and hybridization to arrays of type Rae230A from Affyme-
trix (Santa Clara, CA, USA) were performed according to
the recommendations of the manufacturer. Microarray
data was analysed based on a mixed model analysis using
JMP Genomics, version 3.0 (SAS Institute, Cary, NC,
USA). Standard settings were used, except the following
specifications[23]: log-linear mixed models [24], were fit-
ted for values of perfect-matches, with probe and rat
group considered to be constant and the array-id random. Custom CDF, [25] with Unigene based gene/transcript
definitions different from the original Affymetrix probe
set definitions was used to annotate the arrays. Microarray
data
were
submitted
to
NCBI
GEO
http://
www.ncbi.nlm.nih.gov/geo/query,
sample
number
[GSE11500]. Cell Cycle Analysis Cells were seeded in six-well plates in triplicates. Upon
attachment, cells were synchronized by serum starvation
for 24 h followed by addition of 10% serum-containing
medium for the HEK293 or 2% serum-containing
medium for the primary cells, for 24 hours. Cells were
harvested, fixed with 80% cold ethanol followed by treat-
ment with 25 μg/ml Ribonuclease A (SIGMA) and 50 μg/
ml propidium iodide (SIGMA) for 30 min at 37°C. After
incubation the cells were analyzed by FACS. Plasmids In this study, we examined the contribution of the JAK2/
STAT-1/p21/Cdk2 pathway on PC-2-dependent kidney
epithelial cell proliferation. We utilized cell lines HEK293 HA-PKD2 (WT) was generated by cloning wild-type
human PKD2 in pcDNA 3 (Invitrogen) plasmid. HA-
PKD2/1–702 contains almost the entire of PKD2 (aa 1– Page 2 of 13
(page number not for citation purposes) BMC Nephrology 2008, 9:10 http://www.biomedcentral.com/1471-2369/9/10 http://www.biomedcentral.com/1471-2369/9/10 tained (in mM): 100 K-aspartate, 30 KCl, 0.3 Mg-ATP, 10
HEPES, 10 EGTA, and 0.03 GTP (pH 7.2). The extracellu-
lar solution contained (in mM): 135 NaCl, 5.4 KCl, 0.33
NaH2PO4, 1 MgCl2, 1.8 CaCl2, 5 HEPES, 5.5 glucose (pH
7.4) or 130 KCl, 1 MgCl2, 10 HEPES, 0.1 CaCl2, and 5 glu-
cose (pH 7.4). 702) and was constructed by the addition of a stop linker
in the PKD2 sequence. Finally, HA-PKD2/R742X contains
amino acids 1–742 of PKD2. Both, HA-PKD2/1–702 and
HA-PKD2/R742X were cloned in pcDNA3 vector. Page 3 of 13
(page number not for citation purposes) http://www.biomedcentral.com/1471-2369/9/10 http://www.biomedcentral.com/1471-2369/9/10 http://www.biomedcentral.com/1471-2369/9/10 BMC Nephrology 2008, 9:10 the second-derivative-maximum method of the LightCy-
cler Data Analysis software (Roche Molecular Biochemi-
cals). Relative quantification was performed using serial
dilutions of the Calibrator cDNA to provide a standard
curve for each run. For all experiments, the standard curve
had an error of below 5% and extended over the relative
quantities of all individual samples. HA antibody, showed good expression of HA-tagged WT
PKD2 and HA-tagged R742X PKD2 (Figure 1A). The same
lysates were immunoblotted with anti-PC-2 antibody to
demonstrate that we indeed have PC-2 overexpression in
these clones. As seen in figure 1A, endogenous PC-2 is
barely detectable by Western blot analysis in vector-only
and R742X PKD2 transfectants. The lower molecular
weight band detected most likely represents a non-specific
band detected with the anti-PC-2 antibody, since it is
detected on vector-only transfectants and untransfected
cells (Figure 1A and data not shown). HA antibody, showed good expression of HA-tagged WT
PKD2 and HA-tagged R742X PKD2 (Figure 1A). The same
lysates were immunoblotted with anti-PC-2 antibody to
demonstrate that we indeed have PC-2 overexpression in
these clones. As seen in figure 1A, endogenous PC-2 is
barely detectable by Western blot analysis in vector-only
and R742X PKD2 transfectants. The lower molecular
weight band detected most likely represents a non-specific
band detected with the anti-PC-2 antibody, since it is
detected on vector-only transfectants and untransfected
cells (Figure 1A and data not shown). Genes whose differential expression was tested by gene-
specific qRT-PCR analysis were rat p57 (forward primer:
TGATGAGCTGGGAGCTGAG
and
reverse
primer:
TGGCGAAGAAGTCAGAGATG)
and
Cdk2
(forward
primer: TGTGGCGCTTAAGAAAATCC and reverse primer:
CCAGCAGCTTGACGATGTTA). Differences in the quan-
tity of starting material were compensated by normaliza-
tion with the housekeeping genes HPRT (forward primer:
CTCATGGACTGATTATGGACAGGAC and reverse primer:
GCAGGTCAGCAAAGAACTTATAGCC) and GAPDH (for-
ward primer: GTATTGGGCGCCTGGTCACC and reverse
primer: CGCTCCTGGAAGATGGTGATGG). Normalized
fold-changes between mutant and normal samples were
calculated by the REST XL software. Expression of wild-type or mutant PC-2 does not affect pro-
liferation or STAT-1/p21/Cdk2 activity in HEK293 cells
Figure 1
Expression of wild-type or mutant PC-2 does not
affect proliferation or STAT-1/p21/Cdk2 activity in
HEK293 cells. (A) Whole cell lysates containing equal
amounts of protein from three stable individual clones of
each transfectant (Vector-only, HA-WT PKD2 and HA-
R742X PKD2) were analyzed by Western blotting for expres-
sion of p21, phosphorylated STAT-1, PCNA, β-tubulin, HA
and PC-2. http://www.biomedcentral.com/1471-2369/9/10 (B) Cdk2 immunoprecipitates from two clones of
each transfectant were subjected into an in vitro Cdk2 kinase
assay using Histone 1A as substrate. Equal amount of Cdk2
was confirmed by immunoblotting the precipitates with anti-
Cdk2 antibody. Data are representative of five independent
experiments. Data Analysis and Statistics Data are reported as means ± SEM. Comparisons between
multiple groups were performed using single-factor
ANOVA, and secondary comparisons were performed
using the Tukey test. Statistical analysis was performed
using the SPSS statistical software package. For electro-
physiology experiments, statistical analysis was employed
with the SigmaStat (Chicago, IL) software. Data were
reported as means ± SEM. Due to high variability in cells
transfected with wild type PKD2, statistical significance
was determined by the Mann-Whitey Rank Sum test. Dif-
ferences were considered significant at p < 0.05 if not
stated otherwise (patch clamp and gene expression profil-
ing. Gene expression statistical analysis is described
above. Quantitative RT-PCR The conventional whole-cell voltage-clamp configuration
was used to measure transmembrane currents in single
cells as described previously [20]. Briefly, patch-clamp
recordings were obtained from single cells at room tem-
perature using a Warner PC-505B amplifier (Warner
Instrument Corp., Hamden, CT) and pClamp 8 software
(Axon Instrument, Foster City, CA). Glass pipettes (plain,
Fisher Scientific, Pittsburgh, PA) with resistances of 5–8
MΩ were prepared with a pipette puller and polisher (PP-
830 and MF-830, respectively, Narishige, Tokyo, Japan). After the whole-cell configuration was achieved, cell
capacitance and series resistance were compensated
(~70%) before each recording period. From a holding
potential of -60 mV, voltage steps were applied from -100
to 100 mV in 20 mV increments with 200 ms duration at
3 s intervals. Current traces were filtered at 1 kHz and ana-
lyzed off-line with pClamp 8. The pipette solution con- Q
Total RNA (1 μg) was isolated from cultured cells using
the Rneasy Mini kit (Qiagen) and was reverse transcribed
with the Protoscript reverse transcription kit (New Eng-
land Biolabs) using the VN(dT)23 primer as recommended
by the manufacturer. As a standard for relative RNA quan-
tification (Calibrator), 1 μg of all sample RNAs was
pooled together and reverse transcribed as mentioned
above. Quantitative RT-PCR (qRT-PCR) amplifications
were performed with a LightCycler (Roche Molecular Bio-
chemicals) using the same starting amount and LightCy-
cler® FastStart DNA MasterPLUS SYBR Green I reagents in
a standard volume of 20 μl. Real-time detection of fluori-
metric intensity of SYBR Green I, indicating the amount of
PCR product formed, was measured at the end of each
elongation phase. Fluorescence values measured in the
log-linear phase of amplification were considered using Page 3 of 13
(page number not for citation purposes) Page 3 of 13
(page number not for citation purposes) cycle profile of HEK293 clones is unaffected by expres-
of wild-type or mutant PC-2
re 2
cycle profile of HEK293 clones is unaffected by ycle profile of HEK293 clones is unaffected by expres-
f wild-type or mutant PC-2
e 2
cycle profile of HEK293 clones is unaffected by
ession of wild-type or mutant PC-2. Three differ- We used these tools to test the effect of wild-type and
mutant PC-2 expression on the JAK2/STAT-1/p21/Cdk2
pathway, as it was previously implicated in its regulation
by showing that overexpression of wild type PKD1 acti-
vates JAK2 kinase, which in turn phosphorylates STAT-1
[13]. Lysates from synchronized clones were immunob-
lotted with an anti-phospho-STAT-1 antibody, which
detects the expression of serine phosphorylated STAT-1,
and an anti-p21 to detect endogenous p21 expression. As
shown in figure 1A, p21 levels and STAT-1 phosphoryla-
tion were unaffected by wild-type or mutant PKD2 expres-
sion. Equal loading was confirmed by re-probing the
same membrane with anti-β-tubulin. Similarly, endogenous Cdk2 activity was equivalent
among the different clones as judged by the kinase assay
performed on Cdk2 immunoprecipitates from two
selected clones of each transfectant. Western blot analysis
demonstrated that similar amount of Cdk2 was precipi-
tated from each clone (Figure 1B). Cell cycle analysis per-
formed by propidium iodide (PI) staining revealed that
expression of wild-type or mutant PC-2 does not alter the
cell cycle profile of these cells (Figure 2). Furthermore,
proliferating cell nuclear antigen (PCNA) levels were
equal among the different clones (Figure 1A). Collec-
tively, the results suggest that expression of wild-type and
mutant PKD2 has no effect on the proliferation of
HEK293 cells. To determine whether mislocalization of exogenous WT
and R742X PC-2 is responsible for their inability to regu-
late cellular proliferation, we compared the sub-cellular
localization of HA-tagged WT or R742X PC-2 with endog-
enous PC-2 by immunofluoresence. Both HA-tagged WT
and R742X PC-2 were detected at the same subcellular
compartments (endoplasmic reticulum and plasma mem-
brane) as the endogenous PC-2 (data not shown). ER-localized PC-2 is known to function as a Ca2+-acti-
vated intracellular Ca2+ release channel while plasma
membrane-associated PC-2 is believed to function as a
nonselective cation channel [29-31]. Previous work has
demonstrated that PKD2 overexpression augmented the
amplitude of whole cell currents in renal epithelial cells
[20]. To test the effectiveness of the expressed WT PKD2 in
HEK293 cells we performed whole cell current measure-
ments in vector-only, WT PKD2 and R742X PKD2 clones. Functional expression of transfected wild type PKD2 in
HEK cells has been shown [32]. cycle profile of HEK293 clones is unaffected by expres-
of wild-type or mutant PC-2
re 2
cycle profile of HEK293 clones is unaffected by Figure 3 shows that stable
expression of wild type PKD2 in HEK cells resulted in a
significant increase in the current amplitude of whole cell
inward currents recorded either in normal extracellular
tyrode solution or symmetrical K+ (Figure 3). Outward
currents were larger in WT PKD2 expressing cells com-
pared to untransfected, mock-transfected, or R742X Generation of stable clones expressing wild-type and
mutant PKD2 in HEK293 cells Expression of wild-type or mutant PC-2 does not affect pro-
liferation or STAT-1/p21/Cdk2 activity in HEK293 cells
Figure 1
Expression of wild-type or mutant PC-2 does not
affect proliferation or STAT-1/p21/Cdk2 activity in
HEK293 cells. (A) Whole cell lysates containing equal
amounts of protein from three stable individual clones of
each transfectant (Vector-only, HA-WT PKD2 and HA-
R742X PKD2) were analyzed by Western blotting for expres-
sion of p21, phosphorylated STAT-1, PCNA, β-tubulin, HA
and PC-2. (B) Cdk2 immunoprecipitates from two clones of
each transfectant were subjected into an in vitro Cdk2 kinase
assay using Histone 1A as substrate. Equal amount of Cdk2
was confirmed by immunoblotting the precipitates with anti-
Cdk2 antibody. Data are representative of five independent
experiments. To test the role of PKD2 in renal cell proliferation and spe-
cifically on the p21/Cdk2 pathway, we generated a series
of HEK293 cell lines with stable expression of hemagluti-
nin (HA)-tagged wild-type human PKD2 (WT PKD2), HA-
tagged mutant PKD2 (R742X PKD2) and a selectable
marker (Vector). The R742X PKD2 encodes for a truncated
PC-2 lacking the polycystin-1 (PC-1) interacting region at
the carboxy-terminal of the protein. R742X, is a disease-
causing PC-2 mutant firstly identified in a Greek-Cypriot
family with Polycystic Kidney Disease type 2 [26-28]. Three individual clones were isolated from each transfect-
ant and used for further experimentation. Immunoblot-
ting of whole cell lysates from the selected clones with an Page 4 of 13
(page number not for citation purposes) Page 4 of 13
(page number not for citation purposes) Page 4 of 13
(page number not for citation purposes) BMC Nephrology 2008, 9:10 BMC Nephrology 2008, 9:10 http://www.biomedcentral.com/1471-2369/9/10 Cell cycle
sion of wil
Figure 2 y
p
y
p
yp
g
Cell cycle profile of HEK293 clones is unaffected by
expression of wild-type or mutant PC-2. Three differ-
ent clones of each transfectant were synchronized and sub-
jected to propidium iodide cell cycle analysis by flow
cytometry. The percentage of cells in each phase of the cell
cycle was determined. The results are presented as mean of
triplicate counts for each clone ± SEM. No statistically signifi-
cant difference was detected. Analysis of serum-starved cells
demonstrated that more than 90% of the live cells were
arrested at G0/G1 phase confirming successful synchroniza-
tion of the cells cultured (data not shown). y
p
y
p
yp
g
Cell cycle profile of HEK293 clones is unaffected by
expression of wild-type or mutant PC-2. Three differ-
ent clones of each transfectant were synchronized and sub-
jected to propidium iodide cell cycle analysis by flow
cytometry. The percentage of cells in each phase of the cell
cycle was determined. The results are presented as mean of
triplicate counts for each clone ± SEM. No statistically signifi-
cant difference was detected. Analysis of serum-starved cells
demonstrated that more than 90% of the live cells were
arrested at G0/G1 phase confirming successful synchroniza-
tion of the cells cultured (data not shown). Page 5 of 13
(page number not for citation purposes) Page 5 of 13
(page number not for citation purposes) Page 5 of 13
(page number not for citation purposes) BMC Nephrology 2008, 9:10 http://www.biomedcentral.com/1471-2369/9/10 Functional expression of PKD2 in HEK293 cells
Figure 3
Functional expression of PKD2 in HEK293 cells. Whole cell step currents in vector-only (A and F), wild type PKD2- (B
and G), or PKD2(742X)-stably transfected HEK293 cells (C and H) in normal extracellular tyrode solution (130K+:135Na+/2
Ca2+/5.4 K+) (A-C) or symmetrical K+ (130K+:130K+) (F-H). Current-voltage (I–V) curves derived from a step protocol in
untransfected (black squares), mock-transfected (vector control) (black circles), WT PKD2- (red circles), or PKD2 (R742X)-
stably transfected HEK293 cells (green circles) in normal extracellular tyrode solution (D) or symmetrical K+ (I). ''*'': p < 0.05. Functional
Figure 3 Functional expression of PKD2 in HEK293 cells
Figure 3
Functional expression of PKD2 in HEK293 cells. Whole cell step currents in vector-only (A and F), wild type PKD2- (B
and G), or PKD2(742X)-stably transfected HEK293 cells (C and H) in normal extracellular tyrode solution (130K+:135Na+/2
Ca2+/5.4 K+) (A-C) or symmetrical K+ (130K+:130K+) (F-H). Cell cycle
sion of wil
Figure 2 Current-voltage (I–V) curves derived from a step protocol in
untransfected (black squares), mock-transfected (vector control) (black circles), WT PKD2- (red circles), or PKD2 (R742X)-
stably transfected HEK293 cells (green circles) in normal extracellular tyrode solution (D) or symmetrical K+ (I). ''*'': p < 0.05. closely resembling mature kidney epithelial cells, NRK-
52E. PKD2-transfected cells in symmetrical K+. PKD2-mediated
K+ currents were larger compared to Na+/Ca2+ currents as
was expected for PKD2 which shows higher permeability
to K+ compared to Na+ or Ca2+ [16,20]. Overexpression of
R742X PKD2 did not have a significant effect on whole
cell inward or outward currents in HEK293. Collectively,
the electrophysiology data show that transfection of wild
type PKD2 resulted in functional expression in HEK293
cells. However, PKD2 has no effect on the STAT-1/p21/
Cdk2 pathway or on the proliferation status of these cells. The rat kidney epithelial cell line, NRK-52E was tran-
siently transfected with vector-only (CT), WT PKD2,
R742X PKD2 and 1–702 PKD2 (a PKD2 mutant lacking
the entire carboxy-terminal region of the protein). At 48
hours after transfection, cells were synchronized by serum
starvation. Whole cell lysates were immunoblotted with
anti-p21 and anti-phospho-STAT-1 antibodies. Neither
p21 levels nor STAT-1 phosphorylation is affected by
expression of wild-type or mutant PKD2 (Figure 4A). Sim-
ilarly, the levels of active Cdk2 were comparable among
the four transfectants. In addition to the JAK2/STAT-1/
p21/Cdk2 pathway, the proliferation capacity of NRK-52E
transfected with WT, R742X and 1–702 PKD2 appeared
unaltered compared to vector only transfectants as judged
by PCNA Western blot analysis. Good expression of the
wild-type PC-2 and of the two truncated proteins was
achieved as judged by anti-HA and anti-PC2 blotting. In
summary, these results duplicate the observation in
HEK293 that wild-type or mutant PKD2 expression do not Page 6 of 13
(page number not for citation purposes) Renal tubular epithelial cells from PKD2(1–703) transgenic
rat display augmented proliferation independent of the
JAK2/STAT-1/p21/Cdk2 pathway The unexpected but significant results above, prompted us
to utilize primary renal epithelial cells obtained from a 7.5
week-old mutant PKD2 transgenic rat (1–703) [abbrevi-
ated PKD2(1–703)], expressing a truncated form of PC-2
lacking the C-terminal region of the protein. The trans-
genic animals manifest a cystic phenotype characterized
by the formation of multiple cysts in the kidneys [19]. Tubular renal epithelial cells were isolated by sequential
filtration of renal cells and cultured in low serum-contain-
ing medium. The epithelial character of the isolated cells
and the absence of contaminating fibroblasts were con-
firmed by cadherin and vimentin expression respectively
(Figure 5A). In contrast to the cell lines examined, primary tubular epi-
thelial cells (TECs) isolated from PKD2(1–703) transgenic
rat, demonstrated an increase in cellular proliferation
compared with their normal counterparts. Specifically,
Western blot analysis on whole cell lysates demonstrated
that TECs isolated from the PKD2(1–703) rat have signif-
icantly higher levels of PCNA than TECs isolated from
normal Sprague-Dawley rats (Figure 5A). In addition, the
percentage of cells in the G0/G1 phase of the cell cycle was
lower in the mutant cells than in normal cells as judged by
cell cycle analysis (90.6 ± 0.93 to 84.1 ± 1.28). In concert,
the percentage of G2/M-phase mutant cells was higher
than G2/M-phase normal cells (5.06 ± 0.31 to 12.9 ±
1.37) (Figure 5B). Despite the higher proliferative activity
of mutant cells, p21 levels and STAT-1 phosphorylation
remain unaltered (Figure 5A), suggesting that PKD2-
induced proliferation is STAT-1/p21-independent. We then hypothesized that alternative pathways might be
responsible for PKD2-induced proliferation in this sys-
tem. To this end, we performed a genome-wide gene
expression analysis on TECs isolated from two normal
Sprague Dawley rats and three PKD2 (1–703) rats. Differ-
entially expressed genes were identified with ANOVA. We
concentrated only on genes involved in the cell cycle reg-
ulation (Figure 6A). From all the cell cycle genes listed in
figure 6A, only two differ significantly in expression
between normal and mutant cells, those being Cdk2 and
cyclin-dependent kinase inhibitor 1C or p57KIP2. On the
contrary, p21 did not show any significant difference,
confirming the Western blot results (Figure 6A and 5A). Figure 4
Expression of wild-type or mutant PC-2 does not
affect proliferation or STAT-1/p21/Cdk2 activity in
NRK-52E cells. (A). Examination of the effect of wild-type and mutant PKD2
on the JAK2/STAT-1/p21/Cdk2 pathway in NRK-52E cells Examination of the effect of wild-type and mutant PKD2
on the JAK2/STAT-1/p21/Cdk2 pathway in NRK-52E cells
HEK293 cells were generated by transformation of human
embryonic kidney cell cultures with sheared adenovirus 5
DNA [33]. The cell line has an epithelial morphology and
is widely used as a kidney epithelial model. Nevertheless,
there is controversy as to whether these cells are of true
kidney origin, since expression studies have demonstrated
that HEK293 cells have an unexpected relationship with
neurons [34]. For these reasons we decided to perform the
same experiments in a different cell line system more Page 6 of 13
(page number not for citation purposes) Page 6 of 13
(page number not for citation purposes) http://www.biomedcentral.com/1471-2369/9/10 BMC Nephrology 2008, 9:10 Figure 4
Expression of wild-type or mutant PC-2 does not
affect proliferation or STAT-1/p21/Cdk2 activity in
NRK-52E cells. (A). Whole cell lysates containing equal
amounts of protein from NRK-52E cells trasnsiently trans-
fected with vector-only, HA-WT PKD2, HA-R742X PKD2
and HA-1–702 PKD2 were analyzed by Western blotting fo
expression of p21, phosphorylated STAT-1, PCNA, tubulin,
HA and PC-2. A non-specific band is detected in vector-onl
and WT PKD2 lanes in the HA blot and co-migrates with
mutated PC-2 in this cell line. (B) Cdk2 immunoprecipitates
from each transfectant were subjected into an in vitro Cdk2
kinase assay using Histone 1A as substrate. Equal amount o
Cdk2 was confirmed by immunoblotting the precipitates wit
anti-Cdk2 antibody. Data are representative of three inde-
pendent experiments performed. No statistically significant
difference was detected modify the activity of the JAK2/STAT-1/p21/Cdk2 path-
way. Renal tubular epithelial cells from PKD2(1–703) transgenic
rat display augmented proliferation independent of the
JAK2/STAT-1/p21/Cdk2 pathway Discussion Various studies on renal cystic tissues and cell lines dem-
onstrated that altered regulation of tubular epithelial cell
proliferation is a key factor in the pathogenesis of ADPKD. What remains unclear is the timing of the misregulated
growth as well as the pathways involved. Recently, in an
attempt to answer these questions a number of groups
provided evidence for the involvement of Cdk2 in the
process of cystogenesis. Progression through the cell cycle
is regulated by a family of cyclin-dependent kinases (CKs)
whose activities are controlled by the relative ratio of cyc-
lins and Cdk inhibitors (CKIs) [36,37]. There are two
classes of CKIs in mammals, the p21CIP1 and p16INK4 fam-
ilies. Members of the p16NK4 family bind and inhibit only
Cdk4 and Cdk6 kinases [38]. In contrast, members of the
p21CIP1 family (p21CIP1, p27KIP1 and p57KIP2) inhibit all
G1/S phase CDKs. The transition of cells from the G0/G1
into the S phase of the cell cycle involves the activities of
Cdk2, Cdk4 and Cdk6 [37]. Primary tubular epithelial cells (TECs) isolated from the kid
neys of a PKD transgenic rat expressing a truncated PC 2
(PKD2(1 703)) display higher proliferative activity compared
with TECs isolated from normal Sprague Dawley rats
Figure 5
Primary tubular epithelial cells (TECs) isolated from
the kidneys of a PKD transgenic rat expressing a
truncated PC-2 (PKD2(1–703)) display higher prolif-
erative activity compared with TECs isolated from
normal Sprague-Dawley rats. (A). Whole cell lysates
containing equal amounts of protein from TECs isolated from
normal Sprague-Dawley rat (SD) and TECs isolated from
PKD transgenic rat (Mut) were analyzed by Western blotting
for expression of p21, phosphorylated STAT-1, PCNA, tubu-
lin, PC-2, cadherin, vimentin and megalin. All blots are repre-
sentative of experiments performed on at least two different
transgenic animals. Endogenous PC-2 can be seen on long
exposures that also bring out high background (not shown)
(B). Cell cycle profile of normal (SD) or mutants (Mut) TECs. The results are presented as mean of triplicate counts (three
independent cultures) for each animal. ± SEM (ANOVA p <
0.01. * = significant difference). The data are representative
of two experiments performed using two different pairs of
animals. Bhunia et. al. were the first to address the role of CDKs in
PKD-induced proliferation. Specifically, they demon-
strated that one of the functions of the polycystin-1/2
complex is to regulate the JAK/STAT pathway and conse-
quently control cellular proliferation. http://www.biomedcentral.com/1471-2369/9/10 Whole cell lysates
containing equal amounts of protein from TECs isolated from
normal Sprague-Dawley rat (SD) and TECs isolated from
PKD transgenic rat (Mut) were analyzed by Western blotting
for expression of p21, phosphorylated STAT-1, PCNA, tubu-
lin, PC-2, cadherin, vimentin and megalin. All blots are repre-
sentative of experiments performed on at least two different
transgenic animals. Endogenous PC-2 can be seen on long
exposures that also bring out high background (not shown)
(B). Cell cycle profile of normal (SD) or mutants (Mut) TECs. The results are presented as mean of triplicate counts (three
independent cultures) for each animal. ± SEM (ANOVA p <
0.01. * = significant difference). The data are representative
of two experiments performed using two different pairs of
animals. http://www.biomedcentral.com/1471-2369/9/10 BMC Nephrology 2008, 9:10 Primary tubular epithelial cells (TECs) isolated from the kid-
neys of a PKD transgenic rat expressing a truncated PC-2
(PKD2(1–703)) display higher proliferative activity compared
with TECs isolated from normal Sprague-Dawley rats
Figure 5
Primary tubular epithelial cells (TECs) isolated from
the kidneys of a PKD transgenic rat expressing a
truncated PC-2 (PKD2(1–703)) display higher prolif-
erative activity compared with TECs isolated from
normal Sprague-Dawley rats. (A). Whole cell lysates
containing equal amounts of protein from TECs isolated from
normal Sprague-Dawley rat (SD) and TECs isolated from
PKD transgenic rat (Mut) were analyzed by Western blotting
for expression of p21, phosphorylated STAT-1, PCNA, tubu- mRNA levels were augmented in the mutant cells (nor-
malized fold change 1.2 ± 0.015) (Figure 7A). Cdk2 pro-
tein upregulation and p57 protein downregulation were
also verified by immunoblotting. Consistent with the
microarray data, Cdk2 protein levels were significantly
elevated in mutant primary cultures (normalized fold
change 2.2 ± 0.06). Similarly, p57 levels were downregu-
lated in mutant TECs (normalized fold change 1.9 ± 0.2)
(Figure 7B). On the contrary, Western blot analysis dem-
onstrated, as expected, that p57 protein levels remain
unchanged in HEK293 stable clones and NRK-52E trans-
fectants (Figure 7C). It should be noted that p57 levels in
the cell lines examined is expressed at very low levels and
it was barely detectable by Western blot. Given that in the
PKD2(1–703) transgenic rat the cysts originate predomi-
nantly from the proximal tubule segment of the nephron,
we wanted to exclude the possibility that proximal tubule
cells are overrepresented in the primary mutant TECs cul-
ture, thus confounding the interpretation of the results. In
order to do that, lysates from normal and mutant TECs
were immunobloted with anti-Megalin antibody, a proxi-
mal tubule marker [35]. As shown on figure 5A, Megalin
protein levels were equivalent among normal and mutant
TECs suggesting that the proportion of cells of proximal
origin was comparable among the different cultures and
did not create a sampling bias. Primary tubular epithelial cells (TECs) isolated from the kid-
neys of a PKD transgenic rat expressing a truncated PC-2
(PKD2(1–703)) display higher proliferative activity compared
with TECs isolated from normal Sprague-Dawley rats
Figure 5
Primary tubular epithelial cells (TECs) isolated from
the kidneys of a PKD transgenic rat expressing a
truncated PC-2 (PKD2(1–703)) display higher prolif-
erative activity compared with TECs isolated from
normal Sprague-Dawley rats. (A). Renal tubular epithelial cells from PKD2(1–703) transgenic
rat display augmented proliferation independent of the
JAK2/STAT-1/p21/Cdk2 pathway Whole cell lysates containing equal
amounts of protein from NRK-52E cells trasnsiently trans-
fected with vector-only, HA-WT PKD2, HA-R742X PKD2
and HA-1–702 PKD2 were analyzed by Western blotting for
expression of p21, phosphorylated STAT-1, PCNA, tubulin,
HA and PC-2. A non-specific band is detected in vector-only
and WT PKD2 lanes in the HA blot and co-migrates with
mutated PC-2 in this cell line. (B) Cdk2 immunoprecipitates
from each transfectant were subjected into an in vitro Cdk2
kinase assay using Histone 1A as substrate. Equal amount of
Cdk2 was confirmed by immunoblotting the precipitates with
anti-Cdk2 antibody. Data are representative of three inde-
pendent experiments performed. No statistically significant
difference was detected. The chip data were verified by quantitative real-time PCR
analysis after normalization using two housekeeping
genes, HPRT and GAPDH. In agreement with the chip Page 7 of 13
(page number not for citation purposes) http://www.biomedcentral.com/1471-2369/9/10 Page 8 of 13
(page number not for citation purposes) Discussion They showed that
overexpression of wild-type polycystin-1 can activate
JAK2/STAT-1, a process that resulted in upregulation of
the CKI p21waf1. As expected, increase in p21 levels led to data, p57 mRNA levels were downregulated in the mutant
animals as compared with their normal counterparts
(normalized fold change 4.7 ± 0.19). Similarly, Cdk2 Page 8 of 13
(page number not for citation purposes) Page 8 of 13
(page number not for citation purposes) BMC Nephrology 2008, 9:10 http://www.biomedcentral.com/1471-2369/9/10 Tubular epithelial cells (TECs) isolated from the PKD2(1–
703) rat (Mut) have reduced p57 and augmented Cdk2
mRNA and protein compared with TECs isolated from nor-
mal rat (SD)
Figure 7
Tubular epithelial cells (TECs) isolated from the
PKD2(1–703) rat (Mut) have reduced p57 and aug-
mented Cdk2 mRNA and protein compared with
TECs isolated from normal rat (SD). (A). Real-time
PCR of p57 and Cdk2 in isolated TECs. Data represent the
mean of normalized fold change from three independent
samples ± SEM (ANOVA p < 0.01. * = significant difference). Data were normalized using two housekeeping genes, HPRT
and GAPDH. (B). Protein levels of p57 and Cdk2 repre-
sented as the mean of normalized fold change of two inde-
pendent Western blot experiments ± SEM (ANOVA p <
0.05. * = significant difference). Data were normalized by β-
tubulin expression. (C). p57 protein levels were determined
by western blot analysis in HEK293 stable clones and in
NRK-52E transfectants. As expected, protein level of p57 is
not perceptibly altered. Tubular ep
703) rat (M
mRNA and
mal rat (SD
Figure 7 Tubular epithelial cells (TECs) isolated from the PKD2(1–
703) rat (Mut) have reduced p57 and augmented Cdk2
mRNA and protein compared with TECs isolated from no
mal rat (SD)
Figure 7 Tubular epithelial cells (TECs) isolated from the PKD2(1
703) rat (Mut) have reduced p57 and augmented Cdk2
mRNA and protein compared with TECs isolated from nor
mal rat (SD)
Figure 7
Tubular epithelial cells (TECs) isolated from the
PKD2(1–703) rat (Mut) have reduced p57 and aug-
mented Cdk2 mRNA and protein compared with
TECs isolated from normal rat (SD). (A). Real-time
PCR of p57 and Cdk2 in isolated TECs. Data represent the
mean of normalized fold change from three independent
samples ± SEM (ANOVA p < 0.01. * = significant difference). Data were normalized using two housekeeping genes, HPRT
and GAPDH. (B). Discussion Protein levels of p57 and Cdk2 repre-
sented as the mean of normalized fold change of two inde-
pendent Western blot experiments ± SEM (ANOVA p <
0.05. * = significant difference). Data were normalized by β-
tubulin expression. (C). p57 protein levels were determined
by western blot analysis in HEK293 stable clones and in
NRK-52E transfectants. As expected, protein level of p57 is
not perceptibly altered. Page 9 of 13
(page number not for citation purposes) Genome-w
sion of Cd
(PKD2(1–7
Figure 6 In support
of this, a recent report focused on the dynamics of cyst for-
mation by utilizing an inducible Pkd1 mouse model,
demonstrated that proliferation was not appreciably
higher in cystic specimens than in aged matched controls. Based on their results, the authors suggested that the rela-
tionship between cellular proliferation and cyst formation
may be indirect [41]. Similar data were obtained from
Zebrafish studies where it was shown that increased cell
number in cyctic phenotype is a secondary consequence
of tubule dilation rather than the leading cause of cyst for-
mation [42]. In our study, it appears that mutated PC-2-
induced proliferation in primary cells proceeds independ-
ently of the STAT-1/p21 pathway since there is no change
in the levels of p21 or on STAT-1 phosphorylation. Based
on these results it is clear that in the rat system we investi-
gated, PC-2-induced proliferation proceeds through an
alternative pathway other than STAT-1/p21. In this study we attempted to examine further this hypoth-
esis. We generated stable clones expressing either wild-
type or mutant R742X PKD2 in HEK293. To our surprise,
overexpression of wild-type PC2 did not affect prolifera-
tion of these cells. Cell cycle profile analysis, PCNA, p21
expression levels and Cdk2 activity remained unchanged
among different transfectants. The reason for this discrep-
ancy remains unclear given that the same cell line and
similar experimental conditions were used in the previous
studies [13,17]. In order to eliminate the possibility that
the exogenously expressed wild-type PKD2 was not func-
tional, we performed whole cell current measurements in
vector-only, WT PKD2 and R742X PKD2 clones. As
expected, HEK293 clones expressing wild-type PKD2 dis-
played an increase in the current amplitude of whole cell
inward and outward currents recorded either in normal
extracellular tyrode solution or symmetrical K+. Such
result excludes the possibility that an inactive PC-2 was
expressed in HEK293 cells. In addition, absence of pheno-
type could not be attributed to the mislocalization of the
expressed protein as determined by immunofluorescent
analysis. In an attempt to clarify these contradictory results we uti-
lized a different cell line system. The NRK-52E cells are
"normal" rat tubular epithelial cells, thus we hypothe-
sized that this is a more appropriate system to study PC-2-
induced proliferation and STAT-1/p21/Cdk2 activation. Nevertheless, similar results were obtained with the NRK-
52E cells (Figure 4). The disparity of our results compared
to previous studies is puzzling. Li et. Genome-w
sion of Cd
(PKD2(1–7
Figure 6 Genome-w
sion of Cd
(PKD2(1–7
Figure 6 p
y
p
p
g
(
(
))
g
Genome-wide expression analysis reveals differential
expression of Cdk2 and p57 in TECs isolated from
transgenic rat (PKD2(1–703)). (A). List of cell cycle-
related genes examined by microarray analysis. The (*)
denotes statistical significance after Bonferroni correction. (B). Each data point on the volcano plot stands for one gene. The cutoff of p-value after Bonferroni correction is shown by
the red line. Only the two significantly differentially
expressed genes are labeled with their gene symbol. p
y
p
p
g
(
(
))
g
Genome-wide expression analysis reveals differential
expression of Cdk2 and p57 in TECs isolated from
transgenic rat (PKD2(1–703)). (A). List of cell cycle-
related genes examined by microarray analysis. The (*)
denotes statistical significance after Bonferroni correction. (B). Each data point on the volcano plot stands for one gene. The cutoff of p-value after Bonferroni correction is shown by
the red line. Only the two significantly differentially
expressed genes are labeled with their gene symbol. polycystin-2. These results implied that compromised
polycystin-1 activity is expected to have the opposite
effect, thus explaining the abnormal proliferation
observed in ADPKD cystic cells. An independent study addressed directly the role of PC2
in cell cycle regulation and Cdk2 activity. It was demon-
strated that PC2 could directly interact with Id2, a mem-
ber of the HLH family that is known to control cell
proliferation and differentiation. The direct association of
PC2 with Id2 was shown to regulate the nuclear transloca-
tion of Id2 and thus modulate the cell cycle through the inhibition of Cdk2 and cell cycle arrest. The ability of
polycystin-1 to modulate Cdk2 activity was dependent on inhibition of Cdk2 and cell cycle arrest. The ability of
polycystin-1 to modulate Cdk2 activity was dependent on Page 9 of 13
(page number not for citation purposes) Page 9 of 13
(page number not for citation purposes) BMC Nephrology 2008, 9:10 http://www.biomedcentral.com/1471-2369/9/10 http://www.biomedcentral.com/1471-2369/9/10 http://www.biomedcentral.com/1471-2369/9/10 discrepancy given that high expression of wild-type and
mutated PC-2 was achieved in our HEK293 clones (Figure
1) and in NRK-52E cells (Figure 4) after transient transfec-
tion. Id2/p21/Cdk2 pathway [17]. Based on these results a
model was proposed according to which PC1 can increase
PC2 phosphorylation leading to enhanced Id2/PC2 inter-
action and reduced Id2 nuclear import. Genome-w
sion of Cd
(PKD2(1–7
Figure 6 This in turn, pre-
vents Id2 repression of E-box-dependent activation of
transcription of genes such as p21. Increased p21 will
inhibit Cdk2 activity and arrest the cells at G0/G1 phase
of the cell cycle. At the same time PC1 can lead to Cdk2
inhibition independent of Id2 through the JAK/STAT
pathway. Based on this model mutations in either PC1 or
PC2 can disrupt these pathways leading to abnormal cell
proliferation [17]. A recent report also demonstrated
reduced levels of p21 in human and animal PKD tissues
as well as in affected cell lines implying a role of p21/
Cdk2 in cystogenesis [39]. Id2/p21/Cdk2 pathway [17]. Based on these results a
model was proposed according to which PC1 can increase
PC2 phosphorylation leading to enhanced Id2/PC2 inter-
action and reduced Id2 nuclear import. This in turn, pre-
vents Id2 repression of E-box-dependent activation of
transcription of genes such as p21. Increased p21 will
inhibit Cdk2 activity and arrest the cells at G0/G1 phase
of the cell cycle. At the same time PC1 can lead to Cdk2
inhibition independent of Id2 through the JAK/STAT
pathway. Based on this model mutations in either PC1 or
PC2 can disrupt these pathways leading to abnormal cell
proliferation [17]. A recent report also demonstrated
reduced levels of p21 in human and animal PKD tissues
as well as in affected cell lines implying a role of p21/
Cdk2 in cystogenesis [39]. One of the unwanted side effects of cellular immortaliza-
tion might be the alteration of basal proliferation rate in
cells. This can be highly significant in proliferation stud-
ies. As a result we decided to switch to a primary cell cul-
ture system. We examined the ability of mutated PC-2 (1–
703) to activate the STAT-1/p21/Cdk2 pathway in pri-
mary renal epithelial cells isolated from PKD2(1–703)
transgenic rat [19]. Isolation of TECs from the transgenic animals was per-
formed using a sequential filtration method. Using this
method we avoided any potential activation of surface
receptors taking place during antibody-based isolation
techniques. Purified tubular epithelial cells were cultured
in low serum medium and on laminin-coated plates to
avoid differentiation. The epithelial character of the cells
was regularly evaluated by measuring epithelial (cad-
herin) and fibroblastic (vimentin) markers. TECs isolated
from different animals showed augmented PCNA levels, a
decrease of the G0/G1 phase cells and increase of the G2/
M phase cells. Genome-w
sion of Cd
(PKD2(1–7
Figure 6 This was the first time in our hands that we
observed a higher proliferation activity in cells overex-
pressing a mutated PC-2. These results indicated that
indeed PC-2 can alter cellular proliferation in renal epi-
thelial cells, but it also suggests that such process is com-
plicated and possibly multifactorial and can not be easily
recapitulated in in vitro cell line systems [40]. In support
of this, a recent report focused on the dynamics of cyst for-
mation by utilizing an inducible Pkd1 mouse model,
demonstrated that proliferation was not appreciably
higher in cystic specimens than in aged matched controls. Based on their results, the authors suggested that the rela-
tionship between cellular proliferation and cyst formation
may be indirect [41]. Similar data were obtained from
Zebrafish studies where it was shown that increased cell
number in cyctic phenotype is a secondary consequence
of tubule dilation rather than the leading cause of cyst for-
mation [42]. In our study, it appears that mutated PC-2-
induced proliferation in primary cells proceeds independ-
ently of the STAT-1/p21 pathway since there is no change
in the levels of p21 or on STAT-1 phosphorylation. Based
on these results it is clear that in the rat system we investi-
gated, PC-2-induced proliferation proceeds through an
alternative pathway other than STAT-1/p21. Isolation of TECs from the transgenic animals was per-
formed using a sequential filtration method. Using this
method we avoided any potential activation of surface
receptors taking place during antibody-based isolation
techniques. Purified tubular epithelial cells were cultured
in low serum medium and on laminin-coated plates to
avoid differentiation. The epithelial character of the cells
was regularly evaluated by measuring epithelial (cad-
herin) and fibroblastic (vimentin) markers. TECs isolated
from different animals showed augmented PCNA levels, a
decrease of the G0/G1 phase cells and increase of the G2/
M phase cells. This was the first time in our hands that we
observed a higher proliferation activity in cells overex-
pressing a mutated PC-2. These results indicated that
indeed PC-2 can alter cellular proliferation in renal epi-
thelial cells, but it also suggests that such process is com-
plicated and possibly multifactorial and can not be easily
recapitulated in in vitro cell line systems [40]. Authors' contributions All authors have read and approved the manuscript. KNF
and PK performed most of the experiments. KNF also
helped in the conception of the experimental plan and in
the writing of this manuscript. EK helped in the establish-
ment of HEK293 stable clones. RW and NG created and
maintained the transgenic PKD rat. NG provided the PKD
transgenic rats. LT and C–XB provided the cDNA con-
structs and performed the electrophysiology experiments. NG and LL designed and performed the microarray exper-
iments. Finally CD conceived the study, supervised the
work and helped in the writing of this manuscript. Genome-w
sion of Cd
(PKD2(1–7
Figure 6 al [17], observed cell
cycle arrest and Cdk2 inhibition in HEK293T cells after
expression of wild-type PC-2, and not in HEK293 cells
used in our study. HEK293T cell line is a derivative of
HEK293 that stably expresses the large T-antigen of SV40. In these cells transfected plasmids that contain the SV40
origin are replicated to a copy number of 400–1000 plas-
mids/cell and therefore express the transgene at higher
levels. However, this is unlikely to be the reason for the Using gene expression profiling we were able to identify a
candidate that may mediate the PC2-induced prolifera-
tion in PKD2(1–703) rat. Among all the cell cycle related
genes, only two showed misregulation in TECs isolated
from diseased rats, cyclin-dependent kinase inhibitor 1C Page 10 of 13
(page number not for citation purposes) BMC Nephrology 2008, 9:10 http://www.biomedcentral.com/1471-2369/9/10 lines, which are far from representing the complexity of
whole organs or organisms. (p57kip2) and Cdk2. The p57 kip2 belongs to the p21WAF/
Cip1 family. Studies have shown that p57 binds tightly to
the G1 and S phase kinases, cyclin E/Cdk2, cyclin D2/
Cdk4, cyclin A/Cdk2 and to a lesser extent to cyclin B/
Cdc2 and effectively inhibits their activity [43]. An impor-
tant difference between p57 and the other members of the
family, is that p57 is not regulated by p53 but by p73 [44-
46]. (p57kip2) and Cdk2. The p57 kip2 belongs to the p21WAF/
Cip1 family. Studies have shown that p57 binds tightly to
the G1 and S phase kinases, cyclin E/Cdk2, cyclin D2/
Cdk4, cyclin A/Cdk2 and to a lesser extent to cyclin B/
Cdc2 and effectively inhibits their activity [43]. An impor-
tant difference between p57 and the other members of the
family, is that p57 is not regulated by p53 but by p73 [44-
46]. Acknowledgements We thank Dr Bettina Kränzlin and Dr Sigrid Hoffmann for their assistance
with the transgenic rats and Maria Saile for the microarray experiments. We thank Dr Petros Petrou for critical discussions and suggestions during
the course of this work. This work was funded mainly through an internal
competitive grant from the University of Cyprus and a grant from the
Cyprus Research Promotion Foundation ENISX/0504/12. In conclusion, the level of p57 contribution in the PC-2-
induced proliferation in renal epithelial cells is still
unclear. Future experiments will focus on identifying the
pathways leading to p57 reduction and whether this
decrease is necessary for PC-2-induced proliferation in
renal tubular epithelial cells. We consider it of particular
significance that no matter how these experiments pan-
out, our study introduces a new pathway in ADPKD,
through which PC-2 might lead to Cdk2 activation and
increase in cellular proliferation, which is independent of
STAT-1/p21. Also, once again it should be emphasized
that biological systems are unpredictably complex and
may exert similar effects and end results through more
than one pathway. Finally a word of caution should be
expressed as regards the interpretation of experiments per-
formed on genetically grossly modified established cells Competing interests The authors declare that they have no competing interests. Conclusion
h
h We have shown that p57KIP2, a cyclin-dependent kinase
inhibitor is downregulated and cyclin-dependent kinase 2
(Cdk2) is upregulated in primary tubular epithelial cells
isolated from a PKD2 transgenic rat. In addition, primary
cells expressing mutant PKD2 exhibit increased prolifera-
tion compared to their normal counterparts. On the con-
trary, expression of mutant PKD2 in two kidney cell lines
failed to alter cellular proliferation and p57KIP2 protein
levels. Most importantly, although exogenous expression
of mutant PKD2 ablated current activity, compared to
wild-type, however in cell lines or primary TECs had no
effect on the STAT-1/p21/Cdk2 pathway. In conclusion
this report highlights the probable involvement of p57KIP2
on epithelial cell proliferation in ADPKD implicating a
new mechanism for mutant polycystin-2 induced prolifer-
ation. We observed a downregulation of p57 at both mRNA and
protein levels in mutant cells with the absence of any
change in p21 levels. This possibly signifies that PC-2
might alter cellular proliferation through p57/Cdk2 in
these cells. It is possible that expression of mutant PC-2
can result in p57 downregulation by augmenting Id2
nuclear import and subsequent inhibition of p57 tran-
scription [17]. This hypothesis is in agreement with exper-
iments in neural cells where it was shown that Id2 could
regulate cell cycle through p57 [47]. In addition to p57 downregulation, we observed an
increase in Cdk2 protein level. This is interesting since it
appears that Cdk2 activity might be augmented simulta-
neously in two different ways (downregulation of the
inhibitor and upregulation of the kinase). Whether Cdk2
increase is part of a positive feedback loop is still not
known. Nevertheless, this simultaneous alteration in p57
and Cdk2 levels might result in a rapid increase in Cdk2
activity and subsequently to higher proliferation rate. A
concern regarding our results might arise from the possi-
bility that the isolated TECs are not equally representative
of the various nephron segments in healthy and mutant
rats, a concern however that cannot easily be addressed
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ORIGINALARBEIT ORIGINALARBEIT lektrotechnik & Informationstechnik (2021) 138/6: 349–354. https://doi.org/10.1007/s00502-021-00903-3 Verteiltes Nachverfolgen von OPC UA Methodenkommunikation. Mit zunehmender Digitalisierung werden Shopfloorarchitekturen zunehmend verteilt und serviceorientiert. Somit wird die Komplexität
der Überwachung der Kommunikation zwischen den teilnehmenden Systemen zu einer immer größeren Herausforderung. Hierbei hilft die verteilte Ablaufverfolgung bei der Ermittlung der Kausalität und unterstützt somit die Analyse von Latenzaspekten,
falsch konfigurierten Kommunikationsendpunkten und Kommunikationsengpässen. In diesem Artikel präsentieren wir eine erste Machbarkeitsstudie, die untersucht, inwieweit es möglich ist, OPC UA-Methodenaufrufe
mithilfe des Zipkin-Frameworks verteilt zu verfolgen. Wir zeigen, wie dieser Standard in Verbindung mit dem Eclipse Milo OPC UA Open
Source Stack verwendet und in unserem Industriedemonstrator “Factory in a Box” integriert werden kann. Schlüsselwörter: verteilte Produktionssysteme; verteiltes Nachverfolgen; OPC UA; Methodenaufruf; Korrelationsweiterleitung Received May 10, 2021, accepted June 29, 2021, published online July 15, 2021
© The Author(s) 2021 1.1 Motivating scenario Mayr-Dorn, Christoph, Johannes Kepler University Linz, Altenbergerstraße 69, 4040
Linz, Austria (E-mail: christoph.mayr-dorn@jku.at); Pereszteghy, Benno, Johannes
Kepler University Linz, Linz, Austria; Holzweber, Jan, Pro2Future GmbH, Linz, Austria;
Mayrhofer, Michael, Pro2Future GmbH, Linz, Austria Distributed tracing of OPC UA method calls C. Mayr-Dorn
, B. Pereszteghy, J. Holzweber, M. Mayrhofer With increasing digitalization, shopfloor architectures transition to service-oriented, distributed layouts in which the complexity of
monitoring communication between systems becomes a major challenge. Distributed tracing assists in establishing causality and hence
supports the analysis of latency aspects, wrongly configured communication endpoints, and bottlenecks. In this paper, we present a
first feasibility study, which investigates to what extent it is possible to trace OPC UA method calls in a distributed manner using the
Zipkin framework. We show how this standard can be used in conjunction with the Eclipse Milo OPC UA open source stack and how
it can be integrated into our industry demonstrator “Factory in a Box”. Keywords: distributed production systems; distributed tracing; OPC UA; method invocation; correlation propagation Keywords: distributed production systems; distributed tracing; OPC UA; method invocation; correlation propagation Oktober 2021 138. Jahrgang 1. Introduction As digitalization advances, shopfloor architectures transition to
service-oriented, distributed layouts. As a communication protocol,
OPC UA is introduced at multiple hierarchy levels, e.g., cell, station,
substation, and even component (see, for example, the VDMA R+A
OPC UA Demonstrator [17]). The two trends together create highly
distributed CPPS, in which the complexity of monitoring communi-
cation between systems becomes a major challenge. Our lab-scale production cell, as part of a university-funded demon-
strator project Factory in a Box1 (FIAB), aims at illustrating the con-
cepts that enable flexible production. Our particular demonstrator
can customize the drawings on a piece of paper at multiple plot-
ting stations. The production cell consists of the following machine
types: input stations that provide pallets with paper, plotters that
load the pallets and draw images in one color each, turntables that
transport the pallets between plotters, and finally, output stations
where the finished product (i.e., paper) is placed. Distributed tracing assists in understanding which communication
and action on the shopfloor is the result of what other action. This,
in turn, helps establish causality and hence supports the analysis of
latency aspects, wrongly configured communication endpoints, and
bottlenecks. Communication between the machines is purely based on OPC
UA. Plotters and output stations are designed and programmed with
the IEC 614992 industry standard using the open source IDE Eclipse
4diac [13] and the respective FORTE runtime environment that builds
on the Open62541 OPC UA [5] server. The turntables (and virtual As a centralized schema for monitoring is typically only feasible
when a single vendor provides an integrated solution, yet with ele-
ments coming from multiple vendors, a more lightweight approach
is more feasible. 1https://github.com/jku-isse/factory-in-a-box.
2http://www.iec61499.de/. In this paper, we present a first feasibility study to what extent it
is possible to trace OPC UA method calls in a distributed manner. To
this end, we propose to use the widely used Zipkin standard for trac-
ing information modeling and trace correlation information propa-
gation. We show how this standard can be used in conjunction with
the Eclipse Milo OPC UA open source stack and integrated into our
industry demonstrator: “Factory in a Box”. We also discuss the vari-
ous ways OPC UA interaction affordances can be instrumented, and
the open issues deriving from a communication environment that
aside from method calls also uses data access, monitored items, and
events. Part of this work has been supported by LIT-2019-8-SEE-118, LIT-ARTI-2019-019, and LIT
Secure and Correct Systems Lab funded by the state of Upper Austria, as well as
Pro2Future (FFG under contract No. 854184). Pro2Future is funded within the Austrian
COMET Program—Competence Centers for Excellent Technologies — under the auspices
of the Austrian Federal Ministry of Transport, Innovation and Technology, the Austrian
Federal Ministry for Digital and Economic Affairs and of the Provinces of Upper Austria and
Styria. COMET is managed by the Austrian Research Promotion Agency FFG. Oktober 2021 138. Jahrgang heft 6.2021 © The Author(s) 349 ORIGINALARBEIT C. Mayr-Dorn et al. Distributed tracing of OPC UA method calls Fig. 1. Example control flow through a distributed system (left), with instrumented and non-instrumented components, using method calls
and events (full lines); displaying component internal control flow with dashed lines. Info icons mark the instrumentation points where trace
information is sent to the central trace collector (e.g., a Zipkin server). The diagram to the right displays the resulting spans and their relations. Span names identify where the tracing information has been captured Fig. 1. Example control flow through a distributed system (left), with instrumented and non-instrumented components, using method calls
and events (full lines); displaying component internal control flow with dashed lines. Info icons mark the instrumentation points where trace
information is sent to the central trace collector (e.g., a Zipkin server). The diagram to the right displays the resulting spans and their relations. Span names identify where the tracing information has been captured Tracepoints by themselves just represent events in a system. 2. Distributed tracing background This section provides only a brief introduction to the concepts used
in distributed tracing. The interested reader finds a more thorough
discussion in [11]. – name: the activity or operation this span represents, e.g., an OPC
UA skill or the OPC UA method name if available. – parentId: empty if this is the root span, otherwise parent span’s
id. The idea of distributed tracing is tracking how a batch, an order,
a single command, or event is processed by a (distributed) produc-
tion system. The ripple effect of further requests or events is then
captured in a “trace”. Figure 1 (left) depicts a simplified control
flow graph through an example system. Different approaches are
possible to know which request/events belong to the same trace
(see also Sect. 5). The state-of-the-art approach to obtain such a
correlation is using metadata propagation of a trace identifier. This
implies the instrumentation of the various system components with
tracepoints. These tracepoints are typically placed where a request or
event enters a component, respectively, where a response or event
is returned. – id: uniquely identifies this span – id: uniquely identifies this span – kind: additional, optional information to allow interpretation of
timestamp, duration, and remoteEndpoint. We discuss the impli-
cations of the possible values (CLIENT, SERVER, PRODUCER, and
CONSUMER) below. – timestamp: an as accurate as possible measure representing the
start of this span. – duration: how long this span lasted. The interpretation depends
on the span’s kind. – localEndpoint: describes the networked component that created
this span in terms of a serviceName, ipv4 or ipv6 address, and port
number. A key aspect of distributed tracing is capturing the causality
amongst requests, i.e., how is the sending of one request was
caused by the reception of another, earlier request. In the exam-
ple flow it would be potentially difficult to assess based only on
timestamps and trace identifier information alone whether request
4 occurred due to request 2 or due to request 3. To this end, trace
metadata propagation typically also includes a parent request iden-
tifier. For example, both requests 2 and 3 will have request 1 as a
parent, request 4 will have request 2 as a parent. – remoteEndpoint: describes the networked component to which a
causal relation exists in the scope of this trace. The span’s kind
determines how to interpret the remoteEndpoint. 1https://github.com/jku-isse/factory-in-a-box.
2http://www.iec61499.de/. Even
with causality relationships among these events (that then build up
an acyclical directed graph), users find it usually hard to easily inter-
pret such trace information. Most modern tracing approaches thus
utilize “spans” to group events that meaningfully describe a compo-
nent’s processing activities. These spans build up a tree where lower
spans describe the more detailed, fine granular processing. Figure 1
(right) depicts the span hierarchy for the depicted control flow on
the left. Note that the uppermost client span (i.e., the root span), is
complete before the end of Consumer D and potentially even Pro-
ducer D span due to their asynchronous execution. twins of the prior introduced machines) are implemented in Java,
using the Eclipse Milo OPC UA framework. Hence, despite the toy
character of the setup, the used software and communication in-
frastructure is industrial grade. We use Lego Mindstorm EV3-based
PLCs as a basis as this enables rapid and cheap machine prototyping
without the need to test individual subcomponents such as motors,
actuators, and sensors. Also, it ensures their seamless integration. In the scope of this article, we are especially interested in track-
ing individual orders through the FIAB shopfloor, and hence, need
to trace how the machines communicate for fulfilling each order. Given the highly dynamic layout, one critical support is quick de-
tection of incomplete or incorrect wiring of communication paths
between machines, production deadlocks, identify the potential for
parallelizing activities, and understanding to what extend the pro-
duction time of each order is determined by the machine one the
one hand and coordination needs among the machines on the other
hand. In short, we need to understand how the behavior of ma-
chines (i.e., method calls, etc.) is causally related to the individual
production orders. Ultimately, span-based distributed tracing such as Zipkin foresees
the sending of spans to the central span collection server (e.g., a
Zipkin server) upon the finishing event of a span (indicated in Fig. 1
with the info icons). 2.1 Zipkin In Zipkin, a span entry defines the following properties which we
briefly introduce. We discuss a more detailed mapping to OPC UA
in Sect. 3. – traceId: same random id for all spans that belong to the same
trace. e&i elektrotechnik und informationstechnik © The Author(s) 2. Distributed tracing background – annotations: capture additional events that occur between the
start and end of this span. Each annotation consists of a times-
tamp and a string describing the event, e.g., for capturing when
an error occurred, perhaps an internal state transition happened,
etc. heft 6.2021 © The Author(s) 350 ORIGINALARBEIT C. Mayr-Dorn et al. Distributed tracing of OPC UA method calls <B3
t r a c e I d ="80f198ee56343ba864fe8b2a57d3 . . . "
spanId =\ e457b5a2e4d86bd1"
parentSpanId ="05e3ac9a4f6e3b90"
/ <B3 – tags: provide additional endpoint or vendor-specific information
such as version information, query string, request parameters, etc. as a set of key-value pairs. – tags: provide additional endpoint or vendor-specific information
such as version information, query string, request parameters, etc. as a set of key-value pairs. 3.2 Implications of OPC UA usage CONSUMER described the span from the view of a component that
receives an event from an event broker (described in the span’s re-
moteEndpoint) without any explicit knowledge of who the sender
of the event is. The span’s timestamp indicates the time of obtain-
ing the event from the broker, the duration, if present, measuring
the time between the reception by the event broker and the start
of being processed by the consumer component. Contemporary web-based systems primarily use a procedure call-
centric architecture where a request is typically expecting a response
with the desired content. In contrast, with OPC UA, method requests
are expected to finish as quickly as possible, thus being most of-
ten used merely as triggers to longer running actions on the server-
side. The result and success of such actions are then inspected using
separate method calls, data access operations, monitored items, or
events. Note that in the case of CLIENT and SERVER the span’s dura-
tion is typically more meaningful as it describes a component’s/ser-
vice’s/skill’s latency as well as network latency, whereas the span’s
duration for PRODUCER and CONSUMER only measures the latency
of the event broker and requires application-specific knowledge to
measure any type of service/skill latency. Consequently, even when instrumenting OPC UA method calls
and creating spans of kind “SERVER”, respectively “CLIENT”, this
will result in a span structure more similar to spans of kind “PRO-
DUCER” and “CONSUMER”. We demonstrate this behavior in
Sect. 4 where we also discuss the opportunity to lift tracing instru-
mentation into the component logic to obtain a more easily under-
standable span hierarchy. 3
t r a c e I d ="80f198ee56343ba864fe8b2a57d3 . . . "
spanId =\ e457b5a2e4d86bd1"
parentSpanId ="05e3ac9a4f6e3b90" Aside from traceId, and id, (and parentId for child spans), no other
properties are strictly mandatory. In order to provide the B3 information for injection into the ac-
tual OPC UA additionalHeader element, we needed to extend a few
classes in the Eclipse Milo stack (v0.3.8) as the header is not suffi-
ciently exposed via the stack’s API. Specifically, on the client-side we
extended the OpcUaClient and the UaStackClient to enable hand-
ing over and insertion of B3 information into the header and the
resulting dispatching of span information to the Zipkin server upon
method call completion. On the server-side, we extended the Server-
SymmetricHandler, StackServer, SessionManager, and Session to ex-
tract the B3 header information from the request, and thereby en-
able any server logic to pass on the B3 information for further prop-
agation, and likewise, the dispatching of span information to the
Zipkin server. Note that the client can send span information to the
Zipkin server also when invoking a method on a non-instrumented
server, as it doesn’t rely on header information in the invocation re-
sponse. We additionally relied on the Brave library5 for the actual
dispatching of the span information to Zipkin. CLIENT describes a span from the view of a component that re-
quests some service from a remote component (described in the
span’s remoteEndpoint), capturing in the span’s timestamp the in-
stant the request was sent and measuring in the span’s duration
the time until a response is received. p
SERVER describes a span from the view of a component that re-
ceives a service request from a remote client (described in the span’s
remoteEndpoint), capturing in the span’s timestamp the instant the
request was received, and measuring in the span’s duration the
time it takes to send back a response. PRODUCER describes the span from the view of a component that
dispatches an event to an event broker (described in the span’s re-
moteEndpoint) without any knowledge of who the receiving con-
sumers will be. The span’s timestamp indicates the time of hand-
ing the event over to the broker. The span’s duration, if present,
measures the time between handing over the event, and the event
being actually sent to consumers. 3. Mapping Zipkin to OPC UA Aside from method invocations, requests to read and write an
OPC UA node’s value via OPC UA Data Access can be equally in-
strumented with B3 header extension information. Here, however,
we have to differentiate the direction of the correlation information
flow for reads and writes. A write can be interpreted as a form of re-
quest. Note, that this will not be the case for every data item; hence
such instrumentation only makes sense where a write is expected
to trigger some behavior on the server-side that propagates correla-
tion information and is traced. Here, the request header will contain
the relevant B3 correlation information. Upon dispatching the write
request, the OPC UA client may then create a span of kind “PRO-
DUCER”, and the server should create a span of kind “CONSUMER”
whenever that node is next accessed locally. A read, in contrast, can
be used as a mechanism to providing a return value from a long-
running action, or a trigger to execute some next steps on the client
side. In both cases, the B3 header in the response will be meaning-
ful to propagate context from server to client. Creating of spans is
reversed here. Whenever the node value changes at the server, the
server may create it as a span of kind “PRODUCER”, and the OPC
UA client will create a span of kind “CONSUMER” whenever it reads Two aspects need consideration when applying distributed tracing
to OPC UA. First, the propagation of trace meta-data, and second,
the use of spans. 5https://github.com/openzipkin/brave. 3https://github.com/openzipkin/b3-propagation. 4https://reference.opcfoundation.org/v104/Core/docs/Part4/7.28/. 3https://github.com/openzipkin/b3-propagation.
4https://reference.opcfoundation.org/v104/Core/docs/Part4/7.28/. https://github.com/openzipkin/b3-propagation.
4https://reference.opcfoundation.org/v104/Core/docs/Part4/7.28/. 4. Evaluation use case In the Factory in a Box scenario, the primary communication via OPC
UA occurs between the MES and the machines (input station, output
station, turntables, plotters) as well as between machines to coor-
dinate the handover of pallets. In doing so, we can obtain a single
trace from the MES’ transport request(s) to the turntable(s) and the
subsequent handover handshakes. Figure 2 provides a conceptual
architecture overview of the involved components in the Factory in
a Box for transporting a pallet from input station to Turntable 1,
on to Turntable 2, a plotter, and ultimately reaching the output sta-
tion. To this end, turntables offer an OPC UA server endpoint for
receiving transport commands (i.e., method invocations), and then
connect with respective OPC UA clients to the other machines to
coordinate the pallet handover. A turntable also exposes its OPC UA
server to other turntables to coordinate pallet handover between
turntables. Note, that the input station and output station need not
be controlled from the MES as they signal via their state (i.e., acces-
sible as an OPC UA node) whether they are ready to provide a pallet,
respectively, whether they can take on a pallet. The spans are listed in their dispatch order by timestamp. In this
particular case, the MES happened to dispatch the transport re-
quest to Turntable 2 first, and immediately afterward to Turntable 1. Turntable 1 then proceeds to initiate the handshake to the input sta-
tion (the two bottom spans). Only once Turntable 1 has completed
loading the pallet and is in a ready state will Turntable 2 commence
with the handshake to Turntable 1 (the two brown spans), and then
the handshake to the output station. The time before and after the
set of two handshake method calls is spent on turning and con-
veying (internal component behavior that involves no traceable OPC
UA method calls). The attentive reader will notice that the spans’
duration is extremely short. As we outlined in the previous section,
the method calls are merely used as triggers to an action such as
“initiate” or “start” a handshake and trigger a transport request. To
obtain spans of longer duration, it is necessary to push the tracing
instrumentation from the OPC UA stack up into the (sub) compo-
nents that manage logic at a higher abstraction level, for example,
the state machine that governs the handshake. 3.1 Propagating trace correlation information
3 Note that in the former case, the client could also retain
the B3 propagation information from the initial long-running action
trigger, thus not relying on any B3 response header information. For
both read and write, however, there is more instrumentation needed
on the server-side than for method invocations, as the server has to
maintain B3 information for every node that is used for correlation
propagation. The same read-specific aspects hold for propagating
correlation information when using monitored items. Transparent instrumentation of OPC UA events is more difficult,
as, at the time of writing, no standardized mechanism for including
meta-information in an event is available. Hence, any correlation
information needs to be explicitly modeled in the event data model. There are several aspects visible in the trace of Fig. 3 (left). The
root span is created in the MES (indicated by “DEFAULT” spans),
where the transport system coordinator has separate OPC UA clients
to connect to both involved turntables. A transport request is then
dispatched to the two turntables at the same time. This is visible
in the timestamps of the two “/transportrequest” spans that reside
at nesting level two. Figure 3 (right) provides details of one such
request in the span’s tags, here specifically the method invocation
parameters and OPC UA method node identifier. e&i elektrotechnik und informationstechnik 3.1 Propagating trace correlation information
3 Zipkin uses the B3 header specification3 for which mappings to HTTP
headers, Apache Kafka record header, gRPC ASCII headers, or JMS
headers already exist. We propose to encode the B3 equivalent information in an OPC
UA method call’s additionalHeader element defined in the OPC UA
standard.4 At the moment, no standardization exists yet that would manage
how multiple, independent header extensions can coexist in the ad-
ditional header element. Hence, the exact injection and extraction
procedure of the B3 header from OPC UA method calls is very likely
to change; the encoding of the B3 information itself, however, will
not be affected by this. Oktober 2021 138. Jahrgang heft 6.2021 © The Author(s) 351 ORIGINALARBEIT C. Mayr-Dorn et al. Distributed tracing of OPC UA method calls Fig. 2. Conceptual architecture excerpt of the Factory in a Box showing OPC UA method calls (full lines), and machine internal communication
paths (dashed lines) Fig. 2. Conceptual architecture excerpt of the Factory in a Box showing OPC UA method calls (full lines), and machine internal communication
paths (dashed lines) Fig. 2. Conceptual architecture excerpt of the Factory in a Box showing OPC UA method calls (full lines), and machine interna
paths (dashed lines) ponent which coordinates the handover. This requires explicit ex-
traction of the B3 header information from the OPC UA method call
and thus is specific to the communication means used within the
machines (here IEC 61499 for plotters, and Akka actor framework
for turntables). The exact mechanism involved is outside the scope
of this article. In FIAB, we limited the instrumentation to sending
spans from the client-side as not all server-side OPC UA stacks in-
volved in FIAB are yet instrumented (i.e., currently only Eclipse Milo,
but not yet the open62514 server used in the Forte framework on
the plotters and input and output stations). the value. Note that in the former case, the client could also retain
the B3 propagation information from the initial long-running action
trigger, thus not relying on any B3 response header information. For
both read and write, however, there is more instrumentation needed
on the server-side than for method invocations, as the server has to
maintain B3 information for every node that is used for correlation
propagation. The same read-specific aspects hold for propagating
correlation information when using monitored items. the value. © The Author(s) heft 6.2021 6https://opentracing.io/. Oktober 2021 138. Jahrgang 6. Conclusions and outlook Nevertheless, already at the current level of trace instrumenta-
tion, the available trace information allows to inspect the timing of
requests, here for example, between initiate and start, and analyze
any deviations thereof over time, or the context of requests in case
one of them fails. In this article, we motivated the potential for distributed tracing to
better understand causality in highly distributed and dynamic CPPS. We introduced how to integrate Zipkin with OPC UA method calls
and discussed that additional instrumentation is also useful when
reading and writing to nodes via OPC UA Data Access. We also high-
lighted that due to the event-centric nature of CPPS, method call-
based spans are not ideally suited to quickly and easily understand
span structures. To this end, span generation based on component
internal models such as state machines seems very promising. As we
outlined in Sect. 5, Zipkin is just one applicable framework, with the
OpenTracing APIs6 aiming for interoperability. However, all similar
approaches that have their origin in the web and cloud domain face
similar challenges concerning passing correlation information and,
most importantly, the event-centric nature of OPC UA communica-
tion. 4. Evaluation use case There, a compo-
nent could then start a span upon receiving an internal “start” re- Figure 3 displays the resulting trace information as displayed in the
Zipkin UI for an exemplary transport scenario where a pallet is moved
from input station to output station via the turntables without, for
the sake of clarity, involving a stop at a plotter. Note that a turntable
needs to internally forward the trace correlation information ob-
tained when receiving the transport request to the handshake com- heft 6.2021 352 ORIGINALARBEIT C. Mayr-Dorn et al. Distributed tracing of OPC UA method calls Fig. 3. Example trace of a transport request from the MES to two turntables, as well as the handshake communication to an input station, among
the turntables, and an output station. Span details of one request displayed on the right Fig. 3. Example trace of a transport request from the MES to two turntables, as well as the handshake communication to an input station, among
the turntables, and an output station. Span details of one request displayed on the right only derive insights from analyzing vast amounts of heterogeneous
log entries (e.g., [2]) or schema-based approaches (e.g., [1]) that de-
fine a model for each component on how to interpret logged system
calls. Such approaches, however, have significant limits in their abil-
ity to enable inspection of the causal relationships of requests (and
their properties) for individual traces. quest, capture in span annotations the events triggered by receiv-
ing of state updates via monitored items, and eventually close the
span upon transitioning into the handshake state machine’s “Com-
pleted” state. Any OPC UA-level spans would then be captured as
they currently are but only as sub spans of a higher-level handshake
span. Similarly, the MES transport system coordinator would have to
manage separate spans for each end-to-end transport request. References 11. Shkuro, Y. (2019): Mastering distributed tracing: analyzing performance in microser-
vices and complex systems. Birmingham: Packt Publishing Ltd. 12. Sigelman, B. H., Barroso, L. A., Burrows, M., Stephenson, P., Plakal, M., Beaver, D.,
Jaspan, S., Shanbhag, C. (2010). Dapper, a large-scale distributed systems tracing
infrastructure. 1. Barham, P., Donnelly, A., Isaacs, R., Mortier, R. (2004): Using magpie for request ex-
traction and workload modelling. In 6th {USENIX} symposium on operating systems
design and implementation ({OSDI}’4) (Vol. 4, pp. 18). 13. Strasser, T., Rooker, M., Ebenhofer, G., Zoitl, A., Sunder, C., Valentini, A., Martel, A. (2008): Framework for distributed industrial automation and control (4diac). In 2008
6th IEEE international conference on industrial informatics (pp. 283–288). 2. Chow, M., Meisner, D., Flinn, J., Peek, D., Wenisch, T. F. (2014): The mystery ma-
chine: end-to-end performance analysis of large-scale Internet services. In 11th
{USENIX} symposium on operating systems design and implementation ({OSDI} 14)
(pp. 217–231). 14. Sun, X., Dai, J., Liu, P., Singhal, A., Yen, J. (2018): Using Bayesian networks for proba-
bilistic identification of zero-day attack paths. IEEE Trans. Inf. Forensics Secur., 13(10),
2506–2521. 3. Kaldor, J., Mace, J., Bejda, M., Gao, E., Kuropatwa, W., O’Neill, J., Ong, K. W., Schaller,
B., Shan, P., Viscomi, B., et al. (2017): Canopy: an end-to-end performance tracing and
analysis system. In Proceedings of the 26th symposium on operating systems principles
(pp. 34–50). 15. Van Hoorn, A., Waller, J., Hasselbring, W. (2012): Kieker: a framework for application
performance monitoring and dynamic software analysis. In Proceedings of the 3rd
ACM/SPEC international conference on performance engineering (pp. 247–248). 4. Mace, J., Fonseca, R. (2018): Universal context propagation for distributed system
instrumentation. In Proceedings of the thirteenth EuroSys conference (pp. 1–18). 4. Mace, J., Fonseca, R. (2018): Universal context propagation for distributed system
instrumentation. In Proceedings of the thirteenth EuroSys conference (pp. 1–18). 16. Vierhauser, M., Rabiser, R., Grünbacher, P., Seyerlehner, K., Wallner, S., Zeisel, H. (2016): Reminds: a flexible runtime monitoring framework for systems of systems. J. Syst. Softw., 112, 123–136. 5. Mahnke, W., Leitner, S. H., Damm, M. (2009): OPC unified architecture. Berlin:
Springer. 6. Mazak, A., Lüder, A., Wolny, S., Wimmer, M., Winkler, D., Kirchheim, K., Rosendahl, R.,
Bayanifar, H. Biffl, S. (2018): Model-based generation of run-time data collection sys-
tems exploiting automationML. Automa, 66(10), 819–833. https://doi.org/10.1515/
auto-2018-0022. 17. Zimmermann, P., Axmann, E., Brandenbourger, B., Dorofeev, K., Mankowski, A.,
Zanini, P. 5. Related work A plethora of runtime monitoring approaches [8] focus on the mon-
itoring of resources, e.g., for fault detection or resource usage [15],
often supported by model-driven techniques to simplify instrumen-
tation [6]. In this category, approaches that address distributed sys-
tems, mostly focus on aspects and events of the individual dis-
tributed components but not their interaction. Here passing of cor-
relation metadata is less of a requirement, or implicitly provided in
the identifier of the instrumented components [16]. As production systems become more flexible and need to sup-
port frequent reconfiguration on the move to lot-size one, per or-
der, batch, or request tracing becomes important. To this end, meta-
data propagation based techniques such as Zipkin (originally devel-
oped by Twitter) are suitable candidates to support these existing ap-
proaches. Zipkin is not the only distributed tracing technology with
multiple frameworks emerging over the past decade (e.g., Dapper
[12] at Google, Jaeger [10] at Uber, or Canopy [3] at Facebook). Funding Note
Open access funding provided by Johannes Kepler Univer-
sity Linz. Publisher’s Note
Springer Nature remains neutral with regard to jurisdic-
tional claims in published maps and institutional affiliations. Open Access
Dieser Artikel wird unter der Creative Commons Na-
mensnennung 4.0 International Lizenz veröffentlicht, welche die Nutzung,
Vervielfältigung, Bearbeitung, Verbreitung und Wiedergabe in jeglichem
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und die Quelle ordnungsgemäß nennen, einen Link zur Creative Commons
Lizenz beifügen und angeben, ob Änderungen vorgenommen wurden. Die
in diesem Artikel enthaltenen Bilder und sonstiges Drittmaterial unterliegen
ebenfalls der genannten Creative Commons Lizenz, sofern sich aus der Ab-
bildungslegende nichts anderes ergibt. Sofern das betreffende Material nicht These tracing infrastructures are then the basis for sophisticated
use cases such as anomaly detection [7] or attack path detection
[14]. Additional use cases and their support by distributed tracing
frameworks are discussed in depth by Sambasivan et al. [9]. As the
number of different use cases extends to debugging, taint prop-
agation, auditing, or provenance, a generic multipurpose context
propagation mechanism becomes sensible [4]. Alternative approaches to metadata propagation-based monitor-
ing include treating the distributed system as a black box and thus © The Author(s) heft 6.2021 353 ORIGINALARBEIT C. Mayr-Dorn et al. Distributed tracing of OPC UA method calls C. Mayr-Dorn et al. Distributed tracing of OPC UA method calls 8. Rabiser, R., Guinea, S., Vierhauser, M., Baresi, L., Grünbacher, P. (2017): A comparison
framework for runtime monitoring approaches. J. Syst. Softw., 125, 309–321. unter der genannten Creative Commons Lizenz steht und die betreffende
Handlung nicht nach gesetzlichen Vorschriften erlaubt ist, ist für die oben
aufgeführten Weiterverwendungen des Materials die Einwilligung des jew-
eiligen Rechteinhabers einzuholen. Weitere Details zur Lizenz entnehmen Sie
bitte der Lizenzinformation auf http://creativecommons.org/licenses/by/4.0/
deed.de. 9. Sambasivan, R. R., Fonseca, R., Shafer, I., Ganger, G. R. (2014): So, you want to trace
your distributed system? key design insights from years of practical experience. Parallel
Data Lab., Carnegie Mellon Univ., Pittsburgh, PA, USA. Tech Rep CMU-PDL 14. Data Lab., Carnegie Mellon Univ., Pittsburgh, PA, USA. Tech Rep CMU-PDL 14 10. Shkuro, Y. (2017): Evolving distributed tracing at uber engineering. Uber Engineering
Blog. References (2019): Skill-based engineering and control on field-device-level with OPC
UA. In 24th IEEE international conference on emerging technologies and factory au-
tomation, ETFA 2019, Zaragoza, Spain, September 10-13, 2019 (pp. 1101–1108). New York: IEEE. https://doi.org/10.1109/ETFA.2019.8869473. 7. Nedelkoski, S., Cardoso, J., Kao, O. (2019): Anomaly detection and classification using
distributed tracing and deep learning. In 2019 19th IEEE/ACM international sympo-
sium on cluster, cloud and grid computing (CCGRID) (pp. 241–250). New York: IEEE. Jan Holzweber is a Bachelor Student at the Johannes Kepler
University Linz, Austria and programmer at
the Pro2Future competence center. is a senior researcher at the Institute for Soft-
ware Systems Engineering at the Johannes
Kepler University Linz, Austria. He holds a
Ph.D. in Computer Science from the Techni-
cal University Vienna. His current research in-
terests include software process monitoring
and mining, change impact assessment, and
software engineering of cyber-physical pro-
duction systems. e&i elektrotechnik und informationstechnik is a Bachelor Student at the Johannes Kepler
University Linz, Austria and programmer at
the Pro2Future competence center. Benno Pereszteghy is Area Manager for “Cognitive Robotics and
Shopfloors” at the Pro2Future competence
center and completing his PhD at the Jo-
hannes Kepler University Linz, Austria. recently completed his Bachelors Programm
at the Johannes Kepler University Linz, Aus-
tria. © The Author(s) heft 6.2021 e&i elektrotechnik und informationstechnik 354
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IoT and Smart Home Data Breach Risks from the Perspective of Data Protection and Information Security Law
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Business systems research
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Abstract Background: IoT and smart devices have become extremely popular in the last few
years. With their capabilities to collect data, it is reasonable to have concerns about
the protection of users’ personal information and privacy in general. Objectives:
Comparing existing regulations on data protection and information security rules with
the new capabilities provided by IoT and smart devices. Methods/approach: This
paper will analyse information on data collected by IoT and smart devices and the
corresponding legal framework to explore whether the legal framework also covers
these new devices and their functionalities. Results: Various IoT and smart devices
pose a high risk to an individual's privacy. The General Data Protection Regulation,
although a relatively recent law, may not adequately regulate all instances and uses
of this technology. Also, due to inadequate technological protection, abuse of such
devices by unauthorized persons is possible and even likely. Conclusions: The number
of IoT and smart devices is rapidly increasing. The number of IoT and smart home
device security incidents is on the rise. The regulatory framework to ensure data
controller and processor compliance needs to be improved in order to create a safer
environment for new innovative IoT services and products without jeopardizing the
rights and freedoms of data subjects. Also, it is important to increase awareness of
homeowners about potential security threats when using IoT and smart devices and
services. Keywords: IoT, smart homes, security, data protection, personal data protection
JEL classification: K22
Paper type: Research paper
Received: Dec 17, 2019
Accepted: Jul 16, 2020
Citation: Vojkovic, G., Milenkovic, M., Katulic, T., (2020), “IoT and Smart Home Data
Breach Risks from the Perspective of Data Protection and Information Security Law”,
Business Systems Research, Vol. 11, No. 3, pp. 167-185. DOI: https://doi.org/10.2478/bsrj-2020-0033 Keywords: IoT, smart homes, security, data protection, personal data protection
JEL classification: K22
P
t
R
h Paper type: Research paper Received: Dec 17, 2019
Accepted: Jul 16, 2020 Citation: Vojkovic, G., Milenkovic, M., Katulic, T., (2020), “IoT and Smart Home Data
Breach Risks from the Perspective of Data Protection and Information Security Law”,
Business Systems Research, Vol. 11, No. 3, pp. 167-185. DOI: https://doi.org/10.2478/bsrj-2020-0033 IoT and Smart Home Data Breach Risks from
the Perspective of Data Protection and
Information Security Law Goran Vojković, Melita Milenković
University of Zagreb, Faculty of Transport and Traffic Sciences
Tihomir Katulić University of Zagreb, Faculty of Law, Croatia Business Systems Research | Vol. 11 No. 3 |2020 Business Systems Research | Vol. 11 No. 3 |2020 Introduction A current buzzword in the field of information technology – IoT (Internet of Things) -is
expected to create a whole new sector of products and services. Recent Cisco 167 Business Systems Research | Vol. 11 No. 3 |2020 Business Systems Research | Vol. 11 No. 3 |2020 research estimates predicted a 43% year on year growth of IoT connections
(Bhattacharjya et al., 2018). Unfortunately, it is also increasingly connected with the
rise of information security incidents and potential personal data breaches with
significant impact on the privacy of individuals around the world (Lin et al. 2016). Legal
science, especially in the field of data protection has only recently started to
acknowledge the scope of the problem (Bu-Pasha, 2020; Edwards, 2016; Yang et al.,
2019). Sometimes referred to as the internet of everything or industrial network, IoT is a
broad term that encompasses various technologies. Most commonly it is understood
as an adaptable technology allowing machines and devices to interact with each
other over the Internet. It allows for remote control and access through existing
network infrastructure, provide opportunities for more direct integration of the physical
world into computer-based systems, and resulting in improved efficiency, accuracy
and economic benefit in addition to reduced human intervention (Balamurugan et
al., 2018). It however creates an increasingly complex environment to ensure the
recognized fundamental rights such as privacy and data protection (Geneiatakis et
al., 2017). While there is a particular research into the effect smart home and smart
city development has had on the protection of rights and freedoms of individuals,
there are many issues and aspects that need to be visited and considered when
considering the mechanisms applicable from the current legal framework. The current
state of legal science explores unequivocally legal issues of transborder transfer of
data (Sullivan, 2019), trustworthiness of personal digital assistants (Furey & Blue, 2019),
efforts in establishment of user centric privacy (Skarmeta et al., 2019) to ideological
resistance to smart home platforms as strongholds of digital capitalism (Goulden,
2019). Therefore, remarkably underlying the conflict between the struggling efforts of
lawmakers and the indominable march of information technology development. With
that in mind, the focus here is to analyse whether current provisions can be used to
approach data protection and information security issues and serve as adequate
compliance mechanisms. p
The security threats posed by IoT devices have already been described in the
literature (Jurcut et al., 2020). Introduction When we include more data collection capabilities
enabled by smart meters (Iskraemeco, 2019) we come to a new situation that is not
foreseen by the existing legal framework. Nor does the General Data Protection
Regulation (GDPR, European Commission 2016a), as a new regulation, and consumer
protection regulations, adequately regulate these new areas of possible violations of
personal privacy. In this paper, using technical knowledge and comparing it with the
legal framework, our intention to show to what extent the legal framework can be
applied, and where amendments are needed. We will use the provisions, and recitals
of regulations that speak about the purpose of regulations. This explanation is
important because targeted interpretation. Targeted or teleological interpretation or
interpretation from the status of only one of the many methods of interpretation or
interpretation came to the most important, decisive method (Kačer & Ivančić-Kačer,
2019, p. 400). p
)
IoT devices provide services to all kinds of applications through data gathering,
identification, processing and communication capabilities. IoT connects infrastructure
components and services for smart homes and offices, smart city infrastructure and is
applicable in various fields of work – from general industry to real estate, healthcare,
public safety. In principle, IoT generally allows for more interactive and efficient
transportation and power utilities, etc. (Tzafestas, 2018). Developing IoT infrastructure
can be expected to contain massive numbers of different sensors that collect, process
and transfer data in addition to already widespread personal information processing 168 Business Systems Research | Vol. 11 No. 3 |2020 Business Systems Research | Vol. 11 No. 3 |2020 devices that are omnipresent in daily use such as personal computers, smartphones,
television sets, game consoles and digital media reproduction devices. Because of these characteristics, ΙοΤ is a technology of interest for modern hackers
and cybercriminals. "While IoT deployments have been receiving much hype, their
unique characteristics coupled with their interconnected nature indeed present new
security challenges. Various technical difficulties, such as limited storage, power, and
computational capabilities hinder addressing IoT security requirements, enabling a
myriad of vulnerable IoT devices to reside in the Internet-space. Indeed, unnecessarily
open ports, weak programming practices coupled with improper software update
capabilities serve as entry points for attackers by allowing malicious re-programming
of the devices, causing their malfunction and abuse" (Neshenko et al., 2020). Another
point to consider is that the data that are generated around a single device may not
be sensitive in itself. Introduction However, Iot networks usually consist of a large number of
interconnected devices and when data is combined with data generated from other
devices, it can reveal information such as the consumer habits, patterns of behaviour,
and other data which may present significant risk for rights and freedoms of data
subjects. New functionality brings new data security and privacy challenge, experts
noticed that the weakest links of IoT can be driven by the low-cost devices with low
security and cryptography technologies (Vongsingthong & Smanchat, 2015). Obviously, as IoT as a broad term encompasses many potential uses, it is not possible
to cover all the issues and questions in the space available. In this paper we deal
mostly with risks and challenges facing IoT smart home devices and not the IoT in
industrial environment (smart containers and similar) in general, although it might not
be possible to draw an exact line between such uses judging from the way these
devices are incorporated into wide area networks. The General Data Protection
Regulation has been in force only two years, from 25th May 2018. Meanwhile, the
replacement of many measuring devices with smart meters has begun. Such smart
meters that provide the utility provider with a significantly better insight into the life and
habits of the user, require precise regulation. Users need to be aware of what kind of
data is being collected on them. Furthermore, a large number of other smart and IoT
devices do not have quality protection. Consumers need to be aware of this, which is
not within the scope of the GDPR, but consumer protection regulations. In this paper,
we would like to raise awareness of the need to apply and improve the legal
framework. Therefore, we prove why IoT devices represent a higher risk for personal data than
previous monitoring technologies. After all, we put into perspective that technology
and GDPR, including recitals, show us the purpose of Regulation. Since the GDPR
includes information security requirements, and security is also important for devices
that are not directly connected to service providers, we analyse the IoT and
information security regulation with typical cases of IoT security breaches. As many
metering devices do not collect user names directly (e.g. a three-tenant apartment),
we analyse whether the data of such devices is still personal data. . Introduction Finally, concluding
that the GDPR does not provide all the answers related to the safety, we also analyse
the legal framework for consumer protection and make proposals for new regulations. IoT personal data processing as a source of risk While obviously far more efficient than previous monitoring devices, the IoT based
devices equipped with sensors that can obtain personal data present a potentially
considerable risk for personal data breaches and consequently rights and freedoms
of data subjects. For example, electricity suppliers change the existing electricity
consumption meters in households to the new digital meters which, as they are 169 Business Systems Research | Vol. 11 No. 3 |2020 Business Systems Research | Vol. 11 No. 3 |2020 connected to the Internet and allow various levels of internet access, represent a
significant fraction of total IoT devices currently in use. connected to the Internet and allow various levels of internet access, represent a
significant fraction of total IoT devices currently in use. In a corridor of a residential building, on a standard electricity meter, the
consumption in the accounting period, and day/night consumption are stored locally. The data on consumption is available only to a person physically in front of the meter,
and that person can usually see the actual current consumption per rotation speed. On the other hand, a question whether a person is currently present in the household
and is using an electrical appliance can usually only be ascertained by directly
observing the meter, in the SE Europe the meter is usually positioned in the corridor of
the building. These appliances are now being replaced by smart meters running as IoT
devices. As a local example, let us consider the function of a typical meter such as the
"Iskraemeco AM550" electricity meter which is extensively being installed with users in
the Republic of Croatia (further: Croatia). The device offers several ways of
communication that exclude a need for manual readings such as Full DLMS-COSEM
and IEC 1107 compliance; four independent communication interfaces: Optical port,
RJ11, for in-house display, M-bus, wired and wireless, WAN/NAN Communication
modules – PLC G2/G3, and point-to-point 2G/3G/4G. The manufacturer itself states
that specific user applications for "Smart Grid" features are new levels of ability to
customize the meter (Iskraemeco, 2019). The device is capable of measuring much
more than electricity consumption itself, it also provides two-way ‘’energy’’
measurements, active energy and power, 4Q reactive energy & power, apparent
energy & power, instantaneous value of voltage, current, power factor, frequency
and power and an absolute measurement of active energy & power (Iskraemeco,
2019). Business Systems Research | Vol. 11 No. 3 |2020 Communication standard is widespread and documented: BS EN 13757-4:2005
Communication systems for meters and remote reading of meters. Wireless meter
readout (radio meter reading for operation in the 868-870 MHz SRD band). Water
meter does not collect an extensive data set like electricity meter, but one who has
access and collects data daily or every few days can collect sensitive data, for
example when households is empty. Experts agree that using Internet of Things
technologies leave many of the safety concerns ultimately with and in the hands of
the consumer. (Tzafestas, 2018). (
)
Should an unauthorized and potentially malicious user obtain access to the data
of the electricity meter, there could be unintended but potentially very serious
consequences for the data subject. For example, if data on power consumption
could be established, with knowledge of the energy and consumption of individual
devices, the malicious users could easily obtain real time and historical data about
data subjects habits and behaviour such as when the resident comes home from work
or other functions, which is the optimal room temperature preferred or even when
energy intensive appliances such as ovens or vacuum cleaners are being used
indicating activity or habits. Additionally, it could be ascertained when the
occupant/data subject is showering, whether there is more than one person in the
apartment, when and how long they watch television or spend time in various places
in the household. What once was a simple record of electricity consumption now is
representing a detailed record of personal life and habits. Speaking from the legal perspective, the current situation with the lack of security
of connected products has been somewhat helped by the fact that manufacturers
have not been held responsible with respect minimum information security levels of
their product. Since the consumer awareness is still low, there have not yet been
enough cases to establish the clear frame of their liability. Since there is no regulatory
nor economic incentive, the market fails to provide appropriate measures. (Opinion
Consumers and IoT security, 2019). This makes the devices extremely vulnerable, when
there is no obligation to install updates or apply similar measures, even middle skilled
hacker can jeopardize device. j
IoT devices can also be misuse for DDoS attacks. "Consumer products that fall in the
IoT category are connected products. Their connectivity will typically be to a service
accessible over wide-range (e.g. Business Systems Research | Vol. 11 No. 3 |2020 Internet) protocols and optionally to a smartphone
application accessible over short-range protocols (e.g. Bluetooth, WLAN, etc.). In
scenarios where IoT products are compromised and used in a distributed Denial-of-
Service (DDoS) attack, it is the former type of connection that is exploited by malicious
actors" (Opinion Consumers and IoT security, 2019). IoT personal data processing as a source of risk This device has been singled out as an example due to the fact it has been installed
in many households in Croatia and the region, however there are many similar devices
that can be found in the market elsewhere. It is obvious that a device that has so far
only measured the power consumption has been replaced by a much more capable
one, one that now also measures several other values that can directly or indirectly
be used to follow and profile the user behaviour through time, presenting a potential
new risk for the data subjects. Another example can a be smart water meter. One of these products readily
available in the European market is "Apator smart +" class of water meter. For this
example, we can use S SMART+ - VANE-WHEEL SINGLE-JET DRY WATER METERS (DN15-
20). It is described thus: " For measuring the flow and volume of water with a
temperature up to 30 °C or 50 °C, or warm water with a temperature up to 90 °C in
the closed-circuit system with the full flow of the flux, and on the maximum working
pressure up to 16 bar (PN16)." Features of device include: " Pre equipped for installation
of radio module for communication in the Wireless M-Bus, impulse module and M-Bus
module" (APATOR, 2019). (
)
If that module is installed reading the collected data is quite simple: "Wireless M-bus
is a complete solution for wireless reading of various scales. Communication takes
place at 868 MHz according to the wireless M-bus protocol (standard EN 13757-4:
2005). The ‘’Walk-By-Read mode’’ uses a handheld reader or a laptop equipped with
a Bluetooth converter. In most cases, we perform the drive-by reading, meaning that
our smart car equipped with antennas and a Bluetooth converter can come in front
of the building and all internal water meters are read automatically and when we
enter the shaft to read the main water meter, we immediately know if there is any
difference in the reading " (Vodoservis Mate, 2019). 170 Business Systems Research | Vol. 11 No. 3 |2020 IoT and General Data Protection Regulation While the new European general framework of data protection, the General Data
Protection Regulation (GDPR), does not specifically mention IoT devices, nor do the
national application laws such as the Croatian General Data Protection Regulation
Application Act, it does feature a number of recitals and provisions applicable to data
collection through smart home devices and IoT. g
In preamble, the Regulation in Recital 6 of the GDPR acknowledges that: “Rapid
technological developments and globalization have brought new challenges for the
protection of personal data. The scale of the collection and sharing of personal data
has increased significantly. Technology allows both private companies and public
authorities to make use of personal data on an unprecedented scale in order to
pursue their activities. Natural persons increasingly make personal information
available publicly and globally. Technology has transformed both the economy and 171 Business Systems Research | Vol. 11 No. 3 |2020 Business Systems Research | Vol. 11 No. 3 |2020 social life and should further facilitate the free flow of personal data within the Union
and the transfer to third countries and international organizations, while ensuring a
high level of protection of personal data” (European Commission, 2016b). The legislative response to these developments from the perspective of data
protection, apart from the General Data Protection Regulation and associated
Directives and Regulations such as the Directive 2016/680 and the Directive 2016/680
and the Regulation 2018/1725, has been mostly through the national implementations
acts dealing with the position and competences of independent national supervisory
bodies and regulating the issues left by the GDPR to the national legal systems, as well
as updating national laws to comply with data protection principles set forth by the
Regulation. According to a study by the Global Privacy Enforcement Network in 2016, most of
the connected devices fail to adequately explain to customers how their personal
data is processed which is now a direct violations of the transparency principle and
applicable provisions of the General Data Protection Regulation. Failure to inform
data subjects on the details of processing is perhaps not surprising given the extent to
which IoT services involve significantly more parties than traditional services, for
example, sensor manufacturers, hardware manufacturers, IoT operating systems
vendors, IoT software vendors, mobile operators, device manufacturers, third party
app developers, however it does represent a significant breach of data subjects
rights.(Borelli et al., 2015). IoT and General Data Protection Regulation 11 No. 3 |2020 Business Systems Research | Vol. 11 No. 3 |2020 of consent which is unsuited to many IoT situations), struggle of IoT service operators
with data protection principles enshrined by the Regulation such as principles of
transparent, fair and legal processing, data minimisation and data security –
confidentiality and integrity (Bastos et al., 2018). of consent which is unsuited to many IoT situations), struggle of IoT service operators
with data protection principles enshrined by the Regulation such as principles of
transparent, fair and legal processing, data minimisation and data security –
confidentiality and integrity (Bastos et al., 2018). These authors correctly suggest that data breach reporting provisions may present,
at least at this point in development, a challenging requisite for IoT service and
product providers as both identifying risk and determining consequences of a data
breach in IoT environment that may number thousands or hundreds of thousands of
distinct devices that may work largely independently or predominantly collaborate in
a network which is a potentially daunting exercise with no clear cut forensic protocols
or assessment schemes. Another interesting point these authors raise, on which we agree is the requirement
of data protection by design and by default as stipulated by the Article 25 of the
GDPR. Taking into account the conditions set forth by the GDPR may not be an easy
task in development of new innovative IoT devices and services. Cost of
implementation of appropriate measures may be prohibitively high considering the
relatively simple nature of sensors and monitoring devices that limit the ability of
manufacturers to effectively implement efficient technical and organisational
measures to help comply with GDPR requirements. p
p y
q
Similar observations were made previously by other authors, notably Wachter who
conducted a systematic analysis of available literature in 2018 and identified a
number of key issues, some going back to papers published years before GDPR which
was adopted in 2016, especially including security and consent. To previously outlined
GDPR requirements, Wachter also adds provisions regarding certain data subjects'
rights, general security requirements for data controllers and data protection impact
assessment provisions (Wachter 2018a). Most IoT devices have a physical aspect that demands ensuring the physical safety
regarding the device as well as the user and the environment that the device is
deployed in. IoT and General Data Protection Regulation g
(
)
Naturally, IoT products and services allow for collecting large amounts of data,
some of which may be of potentially of sensitive nature. As far as the idea of a device
that communicates with people or generally exchanges information with the
environment in order to facilitate certain tasks, e.g., a washing machine that
automatically orders detergents is a concern. It is obvious that certain safeguards
need to be undertaken as not to jeopardize the privacy of the users. The Regulation
mandates that the service provider ensures the safety and security of processing. The Regulation takes this further in Recital 39 elaborating on the principles of data
protection, especially the principles of purpose limitation, storage limitation and data
minimization: “Natural persons should be made aware of risks, rules, safeguards and
rights in relation to the processing of personal data and how to exercise their rights in
relation to such processing. In particular, the specific purposes for which personal data
are processed should be explicit and legitimate and determined at the time of the
collection of the personal data. The personal data should be adequate, relevant and
limited to what is necessary for the purposes for which they are processed. This
requires, in particular, ensuring that the period for which the personal data are stored
is limited to a strict minimum. Personal data should be processed only if the purpose
of the processing could not reasonably be fulfilled by other means. In order to ensure
that the personal data are not kept longer than necessary, time limits should be
established by the controller for erasure or for a periodic review.” The volume of data that IoT products and services collect can contain personal
and sensitive data interesting to a wide array of third parties ranging from financial
institutions such as banks and insurance companies, to communications and
entertainment service providers as well as market researchers etc. The integration of home and IoT brings new forms of risks, and residents’ perception
on these new risks is quite important for the development of smart home technologies. IoT devices use wide array of sensors that collect and process staggering amounts of
data. A number of authors have recognized several key points regarding the
application of the GDPR to IoT processing, mostly focusing on issues regarding
identifying appropriate legal basis (such as the prevalent and often inappropriate use 172 Business Systems Research | Vol. IoT and General Data Protection Regulation The data controllers, and by extension their data processors offering Internet of
things devices and services are required, starting with the Article 5 of the GDPR, to
behave in an accountable way towards personal data, processing the data in
accordance with the principles and compliance mechanisms put forth by the
Regulation. g
Through registration process and information channels offered to their users, data
subjects using such devices and services should be notified about the nature, volume
and scope of personal data processing. The controllers should adopt proper technical and organizational protection
measures, assert the level of risk to the rights and freedoms of their data subjects and
regulate their relations with data processors as regulated by the Article 28 of the
Regulation. Needless to say, controllers should carefully examine the processing operations
conducted by their devices and services, establish proper and applicable basis for
personal data processing and rely on contractual and consent basis according to
standards regulated by the new legal framework as well as existing practice as put
forth by the Article 29 Working Party and the European Data Protection Board
guidance documents, national supervisory body guidance and opinions and
established legal practice. Data controllers responsible for IoT infrastructure will need to develop ways to let
users exercise their data protection rights. Some of those rights, such as the right of
data portability are there to prevent unwanted user lock-ins often observable in
different IT industry fields. There is also a question of user control over collection and
processing of data. As before, principles of personal data processing and now firmly
recognized and established rights of data subjects and their firm enforcement should
help mitigate the feeling of the loss of user control and foster a safer environment for
further development of these technologies. The level of control that data subjects enjoy over their data is largely dependent
on how well the manufacturers of IoT based equipment and products manage to
inform their customers about the nature and purpose of data their devices collect. This includes both what the data collected is used for and what the likely
consequences for the users are (Open Rights Group, 2019). IoT and General Data Protection Regulation This integration between cyber-security and physical safety raises the
need for a new approach to these problems and development of sufficient security
controls (Columbus, 2018). Smart household appliances such as electricity meters,
toasters, TVs, refrigerators, and other smart household appliances can also cause
some major issues as they gather a wealth of data and life habits of natural persons
living in these households (e.g. the exchange of data between home appliances). On average, current research shows that the regular smart home ecosystem is one
comprised of about 20 smart devices, including the household gateway or router
(Pascu, 2018). (
)
Also, this may represent potential threat in the event of exploitation of such data,
given that these are personal data of natural persons through which third parties can
monitor and use their habits. In 2014, the European Commission issued a recommendation regarding the
preparations for the roll-out of smart metering strategies (European Commission
(2014)). In this Recommendation, data protection and security considerations are
outlined. It's also quite ironic that mobile phones act as a tracking device so hackers
can use their location information to steal data, hurt or at least find out natural
persons daily routines and behaviour patterns. With respect to IoT in context of smart homes, data protection laws demand several
considerations mandated by the General Data Protection Regulation and potentially
expanded on by national application laws such as privacy by design and by default,
adequate analysis and treatment of risks to rights and freedoms of data subjects,
processing in compliance with the principles of processing and on established and 173 Business Systems Research | Vol. 11 No. 3 |2020 Business Systems Research | Vol. 11 No. 3 |2020 properly evaluated legal basis (especially concerning potential for widespread
unlawful secondary use of collected data). Users who enthusiastically accept IoT or those that are compelled to do so, i.e. through obligatory replacement of electricity meters are usually not aware of the
extent of the processing – the type, nature and volume of personal data collected,
let alone their potential (mis)use. The Regulation now mandates that data controllers
inform data subjects on the purpose, volume and scope of their processing. IoT and General Data Protection Regulation This is the reason why it is
necessary to make amendments to the Regulation at Member States level, according
to which manufacturers must inform consumers with the installation of new IoT devices
in their households on which data can be collected by the particular device. IoT and information security regulations As the structure and organization of the Internet does not take into account the
borders between nations and other established parameters of competence, the
problems concerning the availability and regular service of Internet service providers
may have an obstructive effect on one of the Member States or the EU as a whole. Safety and security of network and information systems and the personal data
processing that underlines so much of the current Internet economy is the key for
development of the internal digital single market as well as increasingly for public 174 Business Systems Research | Vol. 11 No. 3 |2020 Business Systems Research | Vol. 11 No. 3 |2020 Business Systems Research | Vol. 11 No. 3 |2020 safety as more and more communal infrastructure services rely on networked
technology for more efficient and smarter function. As the EU lawmakers adopted the Network and Information Security Directive, its
main objective was to raise the level of Member State cooperation in establishing and
maintaining a high level of network and information security throughout the EU
(European Commission, 2016b). Establishing cooperation bodies, defining responsibilities and designating contact
institutions in Member States, and adopting national information and network security
strategies was a required formal step in this effort, however the regulation of
information security obligations for providers in Member States was required by
transposing the Directive into national legal systems by 2018. In reality, this process took
a while longer but was finally completed in early 2020 with 13 Member States adopting
a single national law as the transposition measure and 15 Member States adopting or
updating two or more national laws which most of the Member States achieved
through one or more national transposition measures. In the case of Croatia, the transposition of the NIS Directive was carried out through
provisions of the Act on Cyber Security of Essential Service Operators and the Digital
Services Providers – The Cyber Security Act of key service providers and digital service
providers (Hrvatski Sabor, 2018a). It contains the relevant definitions of key terms and
concepts (Article 5), applicable criteria for appointing essential service operators and
digital service providers (Articles 6-13, Annex I&II), as well as establishing obligations for
these service providers in order to maintain adequate level of cybersecurity (Articles
14 through 24). IoT and information security regulations g
)
While the essential service providers are recognized and designated directly by the
Act on the basis of NIS Directive Criteria and the comparative practice and
experiences of other Member States, the recognition and designation of digital
services providers is subject to a procedure. This procedure includes, alongside of
criteria as regulated by the Cyber Security Act, designation by the competent body,
in this case the Ministry of Economy, Entrepreneurship and Crafts (Hrvatski Sabor,
2018a, Articles 1-3). )
Foreseeable use of IoT in offering certain essential services such as power and water
distribution, as well as proliferation of smart home devices will trigger recognition and
designation of controllers of these services as essential or digital service providers. These controllers are now obliged to implement technical and organizational
measures to effectively manage risks as well as measures to prevent and mitigate
effects of information security incidents on the security and safety of information
systems (Hrvatski Sabor, 2018a, Article 14). In particular, essential services operators need to implement such technical and
organizational measures to effectively ascertain the incident risk, prevent, discover
and solve information security incidents and mitigate incident effect to the lowest
possible impact level (Hrvatski Sabor, 2018a, Article 15). In turn, digital services providers when implementing required technical and
organizational measures need to ensure safety of systems and installations, incident
discovery and solving, maintain service continuity, adequately monitor, audit and test
implemented measures and follow recognized information security standards in
information security (Hrvatski Sabor, 2018a, Article 16). The provisions of the Cyber Security Act of 2018 were operationalized in the
provisions of the national Regulation on Cyber Security of Essential Service Operators
and Digital Service Providers which was also enacted in 2018 (Hrvatski Sabor, 2018c). As there is no case law to examine following the start of application of the new
regulations, at this moment speculation about their applicability is purely theoretical. 175 Business Systems Research | Vol. 11 No. IoT and information security regulations 3 |2020 While the measures contained in these provisions represent an organisational and
financial burden for the service providers, they do prima facie lead to higher degree
of security and the Regulation demands that essential service operators create and
manage an information security policy that needs to define goals and guidelines how
to preserve the continuity of function of key systems, assess and manage risks, describe
the system of information security management including the auditing of
implementation of cybersecurity measures, provide key operational security
procedures and include organisation and implementation of educational and
awareness raising efforts. The Regulation further regulates the obligation of identified
essential service providers to ensure physical security of the key systems, to ensure the
availability of equipment required for continuing function and maintenance of
essential systems etc. The essential service providers are required to manage
contractual relations with external service providers that may affect the key systems,
including assessing risks before contracting outsourced services and products. Essential service providers are also mandated to control access to essential system
premises, log and note access to essential systems, implement antimalware and anti-
denial-of-service (anti-DOS) controls as well as manage development, research and
continuity of operation of essential services. The operators are required by the
Regulation to conduct vulnerability assessments, and in cases of serious security
incidents to notify competent authorities designated by the Cyber Security Act. Business Systems Research | Vol. 11 No. 3 |2020 Risks of having an increasingly complex information system: the more devices,
people, interactions and interfaces, the more the risk for data security also increases. It means that there is more variety and diversity in the system, so the challenge of
managing all points in the network to maximize security also increases. Some of the
worst case scenarios of IoT attacks that have so far been noted by security researchers
include content streaming platform attacks, massive malware distribution attempts,
hacking of increasingly connected smart vehicles as well as medical devices: g
hacking of increasingly connected smart vehicles as well as medical devices: g
g y
1. In October 2016, the largest DDoS attack ever occurred on service provider
Dyn using an IoT botnet, followed by the crash and unavailability of large
sections of the Internet, including Twitter, Guardian, Netflix, Reddit and CNN. This IoT botnet was enabled by malicious software called Mirai. Once infected
with Mirai, computers constantly searched the Internet for vulnerable IoT
devices, and then used known default usernames and login passwords,
contaminating them with malware. Among the devices that were attacked
were digital cameras and DVR players. g
p
y
2. In 2017, the US Food and Drug Administration (FDA) confirmed that certain
medical devices had vulnerabilities that could allow hackers to access the
devices such as pacemakers and defibrillators used to monitor and control
patients' heart functions and prevent heart attacks. Because of the vulnerability
of the transmitter, hackers could control the shocks, manage the incorrect
pacing, and drain the battery. a) 3. An IBM affiliated security research website reported a possibility of an attack
targeting an Internet connected vehicle. A team of researchers was able to
take total control of a Jeep SUV using the vehicle’s CAN bus (Dunlap, 2017). The Jeep computer needed a firmware update, and it used the Sprint mobile
network, and a team of experts realized they could make the car behave the
way they wanted: make it run off the road, slow down and speed up potentially
endangering the occupants of the vehicle or bystanders. Example cases of IoT security breaches p
y
Practical use of IoT is already riddled with numerous incidents and disclosed or
discovered vulnerabilities that may reveal the incident threat level and risk for users’
rights and freedoms. Some of them were reported on by mainstream news or
technology magazine, such as the case of SAM Seamless Network in 2019 (Narendra,
2019) Smart home appliances offer new venues for attack. An attacker can hack into the
smart home system and unlock the front door, open windows or turn on appliances
causing damage to the home of the victim. These new type of smart home attacks
result in new forms of risks for the residents (Denning et al., 2013). Cameras (security
cameras of baby monitor) can be hacked and used for illegal access or join into a
zombie network with the purpose to commit a distributed denial of service attack
(Wallace, 2018). Garage doors collect data on when you usually arrive home from
work, giving tech-savvy thieves information they need to plan a break in. There are
many similar examples, and new devices and uses are connected into smart home
platforms practically on everyday basis. The fast growth and proliferation of devices and associated risks would benefit from
a systematic overview and classification. One such classification groups risks into the
five categories (Apiumhub, 2018): 1. Risk to personal data and privacy: The Internet of Things presents a higher risk
to personal data processing. 1. Risk to personal data and privacy: The Internet of Things presents a higher risk
to personal data processing. 2. Technical vulnerabilities in authentication: As IoT devices are connected
through the network, they usually employ some sort of cloud interface. Its
vulnerability may lead to compromise of data subject security. 3. Human factor: As IoT is a relatively new technology, the staff at companies
offering these services and products are relatively uninformed which hightens
the risks to information systems and data subject personal data 3. Human factor: As IoT is a relatively new technology, the staff at companies
offering these services and products are relatively uninformed which hightens
the risks to information systems and data subject personal data 4. Inadequate data encryption: IoT networks usually lack sufficient data
encryption capabilities. 4. Inadequate data encryption: IoT networks usually lack sufficient data
encryption capabilities. 176 Business Systems Research | Vol. 11 No. 3 |2020 Business Systems Research | Vol. 11 No. 3 |2020 We can conclude
that IoT affects personal data and generally information about people's’ habits
and their movement, in two (2) ways: a) As smart devices are technologically supported by IoT, they collect
much more data than "dummy" devices, b) IoT devices, on the contrary, are much more vulnerable to hacking or
other forms of abuse than classical devices. These mentioned issues make security issues far more complicated, both legal and
technological. Huawei forecasts 100 billion IoT connections by 2025, and McKinsey
Global Institute suggests that the financial impact of IoT on the global economy may
be as much as $3.9 to $11.1 trillion by 2025 (James, 2015). These mentioned issues make security issues far more complicated, both legal and
technological. Huawei forecasts 100 billion IoT connections by 2025, and McKinsey
Global Institute suggests that the financial impact of IoT on the global economy may
be as much as $3.9 to $11.1 trillion by 2025 (James, 2015). Smart devices obviously have benefits for both consumers and businesses. In 2017
alone, the market for these devices brought in $ 84 billion, almost 16% more than in
2016, according to a report by Ablondi (2018). By 2023, 274M homes worldwide, or 14%
of all households, will have at least one type of smart system installed (Strategy
Analytics, 2018). )
While there are various ways to protect consumers from the IoT security threats –
education about information security basics as an integral part of digital literacy,
changing default privacy and security settings and managing personal access codes
etc. - some age long accepted practices still apply in the digital domain. When
buying an IoT device or home appliance it is important to know that buying a reliable
device from a reputable supplier means greater chance of the supplier satisfying EU
data protection and information security regulations such as naming representatives
or having accountable subsidiaries in the EU. Such suppliers will have conducted data 177 Business Systems Research | Vol. 11 No. 3 |2020 Business Systems Research | Vol. 11 No. 3 |2020 protection impact assessments and other compliance activities for their products and
the companies themselves, have invested into information security standard
certification and have a history of understanding the modern regulatory framework in
contrast to cheap products from mostly unknown suppliers available only through
wholesale Internet commerce sites. Safety wise smart home device suppliers and platforms operators should create
vulnerability management program. Is IoT Data necessarily a Personal Data? Is IoT Data necessarily a Personal Data? y
IoT devices obviously create interesting new legal and policy challenges that
lawmakers have not encountered before. At the same time, IoT technologies add to
many issues that already exist and manifest in data protection practice (Rose et al.,
2015). Utility companies and other similar service providers may claim that the data
they collect on IoT devices is not personal data, that is, they do not fall within the scope
of GDPR. It is also clear why - the theses we cite in this article state that sensitive
personal data may be involved, and the storage and processing of such data must
be conducted in accordance with the rules imposed by Regulation. It is not only a
matter of formally defining information as personal, but also a matter of security. Art. 32 of the Regulation has the following on the issue of data controller obligations
concerning Security of processing: " Taking into account the state -of -the -art, the
costs of implementation and the nature, scope, context and purposes of processing
as well as the risk of varying likelihood and severity for the rights and freedoms of
natural persons, the controller and the processor shall implement appropriate
technical and organisational measures to ensure a level of security appropriate to the
risk, including inter alia as appropriate: (a) the pseudonymisation and encryption of
personal data; (b) the ability to ensure the ongoing confidentiality, integrity,
availability and resilience of processing systems and services; (c) the ability to restore
the availability and access to personal data in a timely manner in the event of a
physical or technical incident; (d) a process for regularly testing, assessing and
evaluating the effectiveness of technical and organisational measures for ensuring
the security of the processing.” Based on the evaluation of the established ECJ and national supervisory body
practice and applicable WP29 and EDPB guidelines, data collected by IoT may
indeed represent personal data if it relates to an identified or identifiable natural
person, and in some cases it may even reveal sensitive data such as data relating
to health, biometric data and data related to aspects of data subject behaviour.. The easiest way to avoid the extra cost coming from obligations to ensure safe and
secure processing of such data is to deny that data in question are personal
information. Business Systems Research | Vol. 11 No. 3 |2020 Such program should identify and fix device
weaknesses that can emerge over time – a common reason would be the use of
outdated operating systems or antivirus software for personal use. “Users’ right to data
protection and right to privacy must be balanced in the design and governance of
identification technologies in the IoT” (Wachter, 2018b). Therefore, the appropriate measures must be taken to make smart homes safer and
more suitable for life. It is also necessary to carry out a careful assessment of safety
risks, which must be preceded by security implementation to ensure that all underlying
problems are detected immediately and that timely protection measures have been
taken. Is IoT Data necessarily a Personal Data? Therefore, we consider it important to establish here that personal 178 Business Systems Research | Vol. 11 No. 3 |2020 Business Systems Research | Vol. 11 No. 3 |2020 information is what is indeed at stake. Such an issue is important not only for IoT meters
but also for other IoT devices which collect data on the activities of natural persons. information is what is indeed at stake. Such an issue is important not only for IoT meters
but also for other IoT devices which collect data on the activities of natural persons. GDPR Art. 4. defines: "‘personal data’ meaning any information relating to an
identified or identifiable natural person; ‘data subject’; an identifiable natural person
is one who can be identified, directly or indirectly, in particular by reference to an
identifier such as a name, an identification number, location data, an online identifier
or to one or more factors specific to the physical, physiological, genetic, mental,
economic, cultural or social identity of that natural person". Following the coming into force of the Lisbon Treaty and the Charter of
Fundamental Rights protection of personal data in the EU has been defined as a
fundamental right separate from the right to privacy. GDPR, Preamble 1 also states
that "The protection of natural persons in relation to the processing of personal data is
a fundamental right. Article 8(1) of the Charter of Fundamental Rights of the European
Union (the ‘Charter’) and Article 16(1) of the Treaty on the Functioning of the European
Union (TFEU) provide that everyone has the right to the protection of personal data
concerning him or her." Also, Preamble 39 quotes: "Any processing of personal data
should be lawful and fair." The question of what belongs to personal data has already been extensively
analysed in legal literature and the judicature of ECJ (i.e. regarding the dynamic ip
addresses in the Case C-582/14: Patrick Breyer v Bundesrepublik Deutschland). One of
the earliest examples is the one published by the UK’s Information Commissioner's
Office (ICO), analysing the provisions of the UK Data Protection Act 1998 (DPA). Is IoT Data necessarily a Personal Data? apartments or
houses; consumption bills are tied to the name of the natural person; telecoms
additionally offer various benefits of "family packages" to gain an accurate family
image), and therefore we can conclude that the data that can be traced on the
habits of householders and guests using IoT devices, whether metric or otherwise are
undoubtedly personal information, even when they are not individualized, e.g., data
on the electricity consumption of an apartment where two people reside. y
p
p
p
p
Comparison of different categories of such data may pose a particular privacy risk,
e.g. a utility company in a local government unit may collect data from different
measuring devices as well as from other sources, e.g., local video surveillance of
parking lots. A negligent or malicious operator comparing such information could get
a very accurate and sensitive image of one's movements and habits, and deeply
invade a person's privacy. IoT and customer protection rules
The European Consumer Protection Framework does not regulate IoT and smart
devices in terms of informing IoT device users of the ability to collect data by such
devices. This is understandable because the legal framework is outdated (Council
Directive 93/13/EEC of 5 April 1993, Directive 98/6/EC of the European Parliament and
of the Council, Directive 2005/29/EC of the European Parliament and of the Council
and Directive 2011/83/EU of the European Parliament and of the Council). National
regulations, such as the Croatan Customer Protection Act provides only a brief
reference in the need to comply with data protection regulations (Hrvatski Sabor,
2018b). The new proposed legal framework partly addresses the issue of data collection
through the introduction of new digital services (EU to modernize Act on Consumer
Protection). Thus, it is proposed that: "In respect of personal data of the consumer, the
trader shall comply with the obligations applicable under Regulation (EU) 2016/679."
Also interesting is the following provision regarding the provision regarding the use of
personal data by the trader, considering the obligations of the trader in the event of
termination(Council of the European Union, 2019). However, there is no obligation on the trader to explicitly inform the user about the
ability to collect data from an individual smart device, e.g., electricity meter. This is in
stark contrast with the provisions of the GDPR, from the basic principles of processing
to the right of data subjects to be informed and to access their data. Is IoT Data necessarily a Personal Data? ICO’s
document “Determining what is Personal Data” states: "It is important to remember
that it is not always necessary to consider ‘biographical significance’ to determine
whether data is personal data." This document also cites an example of a case of collecting water measurement
data: "A utility company may not record the name of the occupier of the house to
which it provides water, but may simply note the address of the property and address
all bills to 'the occupier’. Data concerning the water consumption for a particular
address will be personal data about the occupier where this data determines what
that individual will be charged for. In this last example, even without a name
associated with the water consumption data, this data will be personal data in that it
determines what the occupier will be charged and the occupier is identified, even
without a name, as the person living at the property in question and is therefore
distinguished from other individuals. Also, if necessary, the water company is likely to
be able to easily obtain the name of, if not the occupier, then at least the registered
owner of the property" (Determining what is personal data, 2012). In countries where utility bills typically come labelled with a user’s name, this data
represents personal data. Further let’s consider that the scope of personal data in
practical, if not legal sense, is wider today than it was twenty years ago and on the
basis of which these examples are cited. Recent literature supports this view: "It should
be noted that personal data can relate to more than one person. For example, the
location of a person at any particular time may also constitute the personal data of
other known individuals in the same location. Joint property ownership can amount to
the personal data of two persons. The content of an email sent by a tenant living in
an apartment block complaining about his neighbour may contain information
relating to both the complainer and the person about whom the complaint was
made. A page of text recording a discussion between two individuals, perhaps
including a detailed exchange of views or a disagreement, may be the personal data
of both" (Carey, 2018, pp. 14). 179 Business Systems Research | Vol. 11 No. 3 |2020 Utility providers, telecoms, and other companies can relatively easily collect
information about individuals residing in a residential facility, e.g. Is IoT Data necessarily a Personal Data? Here, we
mention the Croatian practice - the dummy meter is being replaced by a smart
device, where the user only signs a record of the change of the meter, but is not
familiar with the new possibilities of collecting data on the changed device, except in
the case of self-information. Conclusion The focus of this paper was to present the applicable data protection and information
security regulative context to the rise of Internet-of-Things data processing paradigm,
also to outline the activities that data controllers and processors should undertake to
mitigate possible data breaches. Even though personal data protection has been
recognized as a fundamental right of individuals in the EU for almost two decades,
and after almost the same development period, now there is an established
information security regulatory framework with obligations for essential service
operators and digital service providers. There is still much effort required to increase
awareness of both service providers and the homeowners about potential security 180 Business Systems Research | Vol. 11 No. 3 |2020 Business Systems Research | Vol. 11 No. 3 |2020 threats that may be possible when using these devices and services. IoT rapidly
occupies the global market which brings unprecedented benefits mostly from
improved efficiency, accuracy and economic benefits. It changed the world of wi-fi
connected devices; thus, they are becoming more interconnected in a smart grid
and can easily reduce human intervention which was the idea from the beginning of
development. Our research has confirmed that users should be protected and acquainted with
the smart devices implemented into their households according to the GDPR, bearing
in mind that utility service install smart devices into their households priorly without
letting users know what type of device is being installed, and without their proper
consent. Future research should analyse developing comparative law and discuss
best practices, especially on the level of EU Member States. The new laws and
regulations transposing the NIS Directive and accompanying the GDPR present an
opportunity to improve regulatory framework to ensure data controller and processor
compliance and create a safer environment for new innovative IoT services and
products without jeopardizing the rights and freedoms of data subjects. The paper predicts and gives examples of practical implications showing that this can
sometimes be detrimental to utility users because they are not aware of their rights,
and thus do not even recognize potential security threats that cannot be ruled out. In
the end, however, the complexity of smart home infrastructure may very well prove
impossible to apply standard information security solutions to smart devices such as
smart TVs, connected home appliances or connected energy sensors and water
distribution services. Conclusion Therefore, integrated security approach, risk identification, risk
management and ultimately accountable behaviour of data controllers and other
service providers is of foremost importance. In this paper we explained how such devices bring numerous savings nevertheless
on the other hand IoT brings unsuspected risks in the area of personal data protection
and security of infrastructure. As number of IoT and smart home device security
incidents continues to rise, it is going to be quite difficult to ensure safe and secure
processing of user data without empowering users themselves through active
decision-making about the security status of their own IoT home network. References
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(22
May 2020) About the authors Goran Vojković, PhD, Associate Professor, Chair of Transport Law and Economics,
University of Zagreb, Faculty of Transport and Traffic Sciences. He was born in Split in
1971. He has more than twenty years of work experience in the civil service and private
sector, working on different types of jobs; from the Governmental Office Counsellor to
the Head of Governing Council of the Port Authority of the port of Vukovar. For four
years he was a member of Supervisory board of JANAF company, and, for two terms,
he was an external member of the Committee for Legislation of the Croatian
Parliament. Also, he has a great experience working on the EU projects both in Croatia
and Bosnia and Herzegovina. Currently, he works as an Associate Professor at the
Faculty of Traffic and Transport, University of Zagreb, and at the University North, and
he co-operates with the other higher education institutions in Croatia. The author can
be contacted at goran.vojkovic@fpz.hr. Melita Milenković, LL.M., Research and Teaching Assistant, Chair of Transport Law and
Economics. Faculty of Transport and Traffic Sciences, University of Zagreb. She was
born in Slavonski Brod, Croatia, she graduated from the Faculty of Law, University of
Osijek where she obtained her Master’s degree in Law. Currently, she is employed as
a Research and Teaching Assistant at the University of Zagreb, Faculty of Transport
and Traffic Sciences, Chair for Transport Law and Economics. She is at the 3rd year of
doctoral studies programme at the University of Ljubljana, Faculty of Law, presently in
the final phase of drafting and submitting a thesis under the title: ''Comparative
Analysis of Concessions for Managing Airports – the Applicative Croatian Model''. She
has written papers and articles in the field of Administrative Law, Transport Law, ICT
Law
and
Public
Procurement
Law. The
author
can
be
contacted
at
melita.milenkovic@fpz.hr. Tihomir Katulić, PhD, Assistant Professor, ICT Law Department, Faculty of Law, University
of Zagreb. Teaches undergraduate and graduate courses in Information Technology
Law
ranging
from
Personal
Data
Protection
and
Privacy
in
Electronic
Communications, Internet Governance, Introduction to Information Security and
Intellectual Property. Member of the Internet Society, Croatian Academy of Legal
Sciences, Croatian Copyright Society, International Association of Privacy
Professionals and Internet Governance Forum. Certified ISO 27001 and ISO 37000 lead
auditor and an experienced data protection consultant. Business Systems Research | Vol. 11 No. 3 |2020 Business Systems Research | Vol. 11 No. 3 |2020 41. Vongsingthong S., Smanchat, S. (2015), “Review of data management in Internet of
Things”, Asia-Pacific Journal of Science and Technology, Vol. 20 No. 2, pp. 215-240. 2. Wachter, S. (2018b), “The GDPR and the Internet of Things: A three-step transparenc
model”, Law, Innovation and Technology, Vol. 10 No. 2, pp. 266-294. 43. Wachter, S. (201a), "Normative challenges of identification in the Internet of Things:
Privacy, profiling, discrimination, and the GDPR", Computer Law & Security Review Vol. 34 No. 3, pp. 436-449. 44. Wallace, B. (2018), “A look at the security risks of IoT Devices”, available at:
https://hackernoon.com/a-look-at-the-security-risks-of-iot-devices-f0d6ffe1441d
(22
May 2020) y
)
45. Yang, L., Noe, E., Eliot, N. (2019), "Privacy and Security aspects of e-government in smart
cities", in Rawat, D. B., Ghafoor, K. Z. (Eds.), Smart Cities Cybersecurity and Privacy,
Elsevier, Amsterdam, pp. 89-102. 45. Yang, L., Noe, E., Eliot, N. (2019), "Privacy and Security aspects of e-government in smart
cities", in Rawat, D. B., Ghafoor, K. Z. (Eds.), Smart Cities Cybersecurity and Privacy,
Elsevier, Amsterdam, pp. 89-102. 184 Business Systems Research | Vol. 11 No. 3 |2020 Business Systems Research | Vol. 11 No. 3 |2020 About the authors Occasionally writes opinions
and columns for Croatian magazines Bug, Mreža, Banka, Infotrend, Novi Informator,
Poslovni dnevnik, Privredni vjesnik and Sistemac on topics ranging from data
protection, copyright, digital media, information security, computer crime to
electronic commerce and internet governance. The author can be contacted at
tihomir.katulic@pravo.hr. 185
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